Annual Review of Medicine Volume 59 2008

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Annu. Rev. Med. 2008.59:1-12. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

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The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development∗ Janet Woodcock1 and Raymond Woosley2 1

Center for Drug Evaluation and Research, US Food and Drug Administration, Rockville, Maryland 20857; email: [email protected]

2

The Critical Path Institute, Tucson, Arizona 85721; email: [email protected]

Annu. Rev. Med. 2008. 59:1–12

Key Words

The Annual Review of Medicine is online at http://med.annualreviews.org

biomarkers, biomarker qualiﬁcation, clinical trials

This article’s doi: 10.1146/annurev.med.59.090506.155819

Abstract

c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0001$20.00 ∗ The U.S. Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

Societal expectations about drug safety and efﬁcacy are rising while productivity in the pharmaceutical industry is falling. In 2004, the US Food and Drug Administration introduced the Critical Path Initiative with the intent of modernizing drug development by incorporating recent scientiﬁc advances, such as genomics and advanced imaging technologies, into the process. An important part of the initiative is the use of public-private partnerships and consortia to accomplish the needed research. This article explicates the reasoning behind the Critical Path Initiative and discusses examples of successful consortia.

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INTRODUCTION

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In 2004, the US Food and Drug Administration (FDA) launched the Critical Path Initiative, a project that is intended to improve the drug and medical device development processes, the quality of evidence generated during development, and the outcomes of clinical use of these products. Why would a regulatory agency be involved in such a modernization effort? FDA’s mission is to protect and promote the health of the public. With respect to drugs, biological products, and medical devices, this translates into ensuring reasonable product safety while also facilitating the translation of scientiﬁc innovations into commercial products. The ongoing tension between these two objectives results in assertions that FDA requirements are stiﬂing innovation, and simultaneously that FDA standards are too low. The thesis of the Critical Path Initiative is that scientiﬁc advances in the development process are the best way to resolve these conﬂicts to the satisfaction of most parties and to the beneﬁt of the public. Although the initiative concerns all regulated medical products, this review discusses Critical Path in the context of drug development.

BACKGROUND Rising Expectations about Drug Development In 1962, congressional amendments to the Food, Drug, and Cosmetic Act created for the ﬁrst time a requirement that drugs be scientiﬁcally shown to be effective before they could be marketed (a requirement for safety had been in effect since 1938). During the 1960s–1980s, drug developers, the academic community, and regulators worked to develop and reﬁne ways to design, conduct, and analyze randomized controlled clinical trials that could produce the needed evidence. Many important advances in pharmacotherapy (e.g., cardiovascular therapies, psychiatric drugs, anti-infectives, and cancer treatments) 2

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were introduced during this era. However, the evidence generated in drug development programs was still somewhat limited. For example, dose-response information was usually scanty, often few women were studied, data on long-term use (even for chronically administered drugs) were lacking, evaluations of subgroups such as patients with renal or hepatic insufﬁciency were not conducted, and data on drug-drug interactions were not available. From the mid-1980s through the 1990s, as an increasing number of drug therapies became available, the FDA as well as the international regulatory community established the expectation that such information would be obtained during most drug development programs. Therefore, modern development programs usually are much more extensive and contain many more clinical studies and patient exposures than was usual in 1960–1985. Despite these advances, there remains a great deal of uncertainty about the performance of drugs that are new to the market. Data from long-term use are still usually limited. Current drug development programs cannot detect drug-related adverse outcomes that represent a small increase in frequency of a problem that is already common in the treated population (e.g., ischemic cardiovascular events). Technologies to predict the occurrence of rare, catastrophic side effects are not available. Additionally, despite attempts to make the results of clinical trials more generalizable, the patients enrolled in trials do not reﬂect the full range of the population or treatment situations that occur in practice. As a result, new safety issues are often identiﬁed only after drugs enter the market. Nevertheless, in the past decade, aggressive marketing techniques have led to immediate uptake and widespread use of many new drugs, combined with a general expectation that their performance is well understood over a wide range of clinical situations. In particular, many members of the public believe that if prescription products are advertised on television, they must be safe. The increasing

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recognition of this problem has led to calls for larger trials and longer patient exposures prior to drug marketing. Not only drug development but also medical practice has become increasingly complex since the 1962 amendments. For many diseases, multiple subgroups and disease stages have been deﬁned, and numerous therapeutic options exist. Drug development programs are rarely designed to answer the questions posed by evidence-based medicine and by insurers: What therapeutic option has the best outcomes in various patient groups or, similarly, what option provides the best value? If comparative trials are performed premarket, they usually involve a demonstration of “noninferiority” in comparison with a single control drug. Increasingly, members of the health care community, as well as Congress, are calling for more of this information to be developed.

Problems with the Pharmaceutical Pipeline The pharmaceutical industry is facing a productivity crisis. Despite rising investment in pharmaceutical research and development, successful development of novel drugs is slowing (Figure 1). In fact, 2004 represented a 20-year low in introductions of new chemical entities (NMEs) worldwide (1). The same phenomenon has been observed in the United States, where the submission rate of new drug applications for NMEs has shown a downward trend in the past decade (2). Not surprisingly, the investment needed per successful NME has risen to an estimated $800 million or more (3, 4). This cost is driven by the high rate of clinical failure, estimated at 70%– 90% of candidates (5). The rising percentage of late-stage clinical failures, now ∼50% of compounds tested in phase 3 trials, is of particular concern. The high cost of successful drug development may discourage investment in more innovative, risky approaches, as well as in therapeutics for diseases that represent smaller markets. Additionally, the need

Global R&D spend ($bn)

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Figure 1 Comparison of global pharmaceutical industry research and development investment and global output of new molecular entities. Source: Hoekema A. 2007. Sharing risks and rewards—basis for a turnkey pharma-biotech alliance in osteoarthritis. Drug Disc. World Spring:54

to recoup this investment during the period of market exclusivity, prior to the introduction of generic copies, is an incentive for aggressive marketing techniques (6). However, rapid market uptake means that a large number of individuals may have already been exposed by the time a drug problem is discovered after marketing. Thus, rising societal demands for greater certainty about the outcomes of drug therapy are occurring at a time when the pharmaceutical industry is experiencing difﬁculty in sustaining innovation. These concurrent trends are a cause for signiﬁcant concern, given the number of medical conditions that currently have unsatisfactory or no therapeutic options. The FDA, with its dual roles of protecting and promoting health, is charged with implementing policies that ensure that the beneﬁts of new products will outweigh their risks, while simultaneously promoting innovations that can improve health. The challenges inherent to this mission drove the genesis of the Critical Path Initiative. www.annualreviews.org • The FDA Critical Path Initiative

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Expectations have been widespread that 30 years of signiﬁcant public investment in biomedical research would produce an explosion of new therapies for previously untreatable or inadequately treated diseases. The failure of this surge to materialize has prompted extensive speculation on the cause of this “pipeline problem.” Many in the drug development community believe that genomics and other newer technologies are not yet sufﬁciently mature to reliably support drug development. Others blame industry business decisions or regulatory requirements. In 2004, the FDA published a White Paper entitled “Innovation or Stagnation: Challenges and Opportunities on the Critical Path to Medical Product Development” (7). While acknowledging that a combination of factors has likely led to the current drug development situation, this paper called attention to an important and

generally unrecognized problem: the lagging science of drug development. Drug development can be conceptualized as a process leading from basic research through a series of developmental steps to a commercial product (Figure 2). The FDA White Paper identiﬁed the “Critical Path” as a process beginning with identiﬁcation of a drug candidate and culminating in marketing approval. Along the path to marketing, the product is subjected to a series of evaluations to predict its safety and effectiveness and to enable its mass production. Despite extensive investment in basic biomedical science over the past three decades, there has been very little change in the science of the development process. The sophisticated scientiﬁc tools used in drug discovery and lead optimization are generally not utilized in the preclinical and clinical development stages. Instead, traditional empirical evaluation is used in both animal and human testing. We are

Working in Three Dimensions along the Critical Path Prototype Design or Discovery

Preclinical Development

Clinical Development

Material Selection Structure Activity Relationship

In Vitro and Animal Testing

Human and Animal Testing

In Vitro and Computer Model Evaluation

In Vitro and Animal Models

Human Efficacy Evaluation

Physical Design

Characterization Small-Scale Production

Manufacturing Scale-up Refined Specifications

Basic Research

Safety

Dimensions

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FDA’S CRITICAL PATH INITIATIVE

Medical Utility

Industrialization

FDA Filing/ Approval & Launch Preparation Safety Follow Up

Mass Production

Figure 2 The critical path of drug development. First, a candidate drug emerges from a drug discovery program. The candidate must successfully complete a series of evaluations of its potential safety and efﬁcacy and must be amenable to mass production. For each candidate ﬁnishing the pathway, 5000–10,000 are evaluated in the discovery phase. 4

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using the tools of the last century to evaluate this century’s advances. How did this situation come about? The FDA’s analysis, which has been generally accepted, is that “no one is charged” with improving developmental science. The National Institutes of Health (NIH) focus on innovative biomedical science, not the applied science of the development process; as a result, academia also concentrates on basic science. The pharmaceutical industry is concerned with developing innovative products. The FDA, as a regulator, is not charged with— nor is it funded for—improving the process, although it has been involved in such efforts. Additionally, the science needed is generally integrative “big science” that requires contributions from multiple disciplines and sectors and is not within the purview of a single investigator or ﬁrm. How is the evaluative science of development related to “translational science”? Translational science, which is also called “experimental medicine,” or simply “clinical pharmacology” in the case of drug development, involves moving a scientiﬁc innovation from the laboratory into early clinical studies (8). Improvement in this part of the process is an essential step in modernizing drug development.

THE CRITICAL PATH PROGRAM FDA’s 2004 Critical Path White Paper generated considerable discussion and debate among drug and device developers, academics, and patient advocacy groups. Over 100 groups submitted comments on the paper. After extensive consultation with numerous stakeholders, FDA issued the “Critical Path Report and List” in 2006 (9). This report enumerated leading areas for scientiﬁc improvement in the development process: development and utilization of biomarkers; modernizing clinical trial methodologies and processes; the aggressive use of bioinformatics, including disease modeling and trial simulation; and improvement in manufactur-

ing technologies. It also contained the “2006 Critical Path Opportunities List,” 76 discrete projects that, if completed, could improve product development and subsequent use. A number of these projects are now being undertaken, many in partnership with FDA (10).

Development and Qualification of New Biomarkers Development of new biomarkers was identiﬁed as the highest priority for scientiﬁc effort. Genomic, proteomic, and metabolomic technologies, as well as advanced imaging techniques, hold tremendous promise for generating new biomarkers that can reﬂect the state of health or disease at the molecular level (11). Although much prior discussion about the use of biomarkers in drug development has focused on surrogate endpoints for effectiveness, most uses of new biomarkers are not expected to involve surrogacy. For example, prediction of adequate safety is an essential part of drug development. Currently, preclinical safety testing involves traditional animal toxicology studies, as well as in vitro assays such as the Ames test. Animal toxicology tests are very useful for assessing safety for initial human testing; however, they often fail to uncover the types of toxicities seen after widespread human exposure. New technologies, such as gene expression assays in whole cell or animal systems, proteomics, or metabolomics, may provide much greater insight into the whole spectrum of pharmacologic effects of a candidate drug. Such technologies may also be useful in comparing the candidate’s effects (particularly off-target effects) to those of other drugs in its class or other drugs intended for similar uses (12). Drug developers are just beginning to use such technologies in the preclinical safety workup, and the clinical implications of such ﬁndings have not been worked out. The current scheme for clinical safety testing has also failed to incorporate recent scientiﬁc advances. Human safety during drug development is primarily evaluated on an www.annualreviews.org • The FDA Critical Path Initiative

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observational basis from subjects exposed in the various developmental trials. The markers used to assess potential human toxicity are also assays that have been available for decades, e.g., clinical chemistries and hemograms. Few explanatory studies are carried out to determine the mechanism of an observed side effect, and assays to predict rare side effects are not available. Despite premarket exposure of thousands of subjects, serious side effects are frequently uncovered after marketing. New types of biomarkers may provide opportunities for prevention or early detection of these adverse events. The current problems with predicting and evaluating drug efﬁcacy could also be ameliorated by using biomarkers. Many drug efﬁcacy problems stem from the extreme variability of human disease response. New biomarkers can improve diagnosis, deﬁne disease subsets that may differ in response, deﬁne individual variability in the drug’s molecular target, and provide an early readout of response to therapy (11). For example, both in vitro diagnostics and imaging techniques are expected to provide additional information about disease subsets. This is already beginning to happen in cancer, where gene expression assays are being used to supplement histologic and clinical assessments of tumors, e.g., evaluating the likelihood of recurrence and the need for adjuvant therapy. For disorders such as psychiatric conditions that are currently diagnosed by clinical symptoms, it is hoped that genetic or imaging markers may help to distinguish biologically based subsets. A related type of biomarker is one used to predict treatment responsiveness. Many new cancer therapies target a speciﬁc molecule or cellular pathway. Genetic, proteomic, or other molecular assays that assess target status within a tumor may be used to predict responsiveness to a targeted drug. This is the strategy used with the drugs trastuzumab (Herceptin®) and imatinib (Gleevec®). Interindividual drug target heterogeneity due to genetic polymorphisms may be important in diseases other than cancer. Using biomarkers to classify patients by

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disease type or response probability can improve drug development by reducing variability and increasing the size of the treatment effect. If the biomarkers are then incorporated into clinical practice, clinical variability can also be reduced. Decreasing interindividual differences in drug exposure is another strategy to reduce response variability. Recently, FDA has approved a number of assays for genetic polymorphisms in drug-metabolizing enzymes. Many marketed drugs are subject to polymorphic metabolism, leading to a wide range of exposures in the treated population (13). The safety and effectiveness of these drugs, as well as investigational drugs with variable metabolism, could be improved by using dose adjustments directed by genetic tests. The absence of practical processes to establish the clinical signiﬁcance of a given biomarker has severely limited the use of existing biomarkers in drug development and the clinic. The return on investment for diagnostic test manufacturers is seldom sufﬁcient to enable extensive clinical trials, and investigational drugs are rarely developed in concert with new diagnostic tests. To address these issues, FDA and other stakeholders have established the concept of biomarker qualiﬁcation, which means determining the clinical signiﬁcance of the biomarker in a speciﬁc context (14). For example, a genetic test might be qualiﬁed to identify a subset of disease for the purpose of trial enrollment. The quantity of data needed for qualiﬁcation depends on the intended use, and most uses require far less data than would be required to establish a surrogate endpoint for efﬁcacy. Because the development and qualiﬁcation of new biomarkers will beneﬁt many parties, consortia have been formed for this purpose. “The Biomarker Consortium” (http:// www.biomarkersconsortium.org) at the Foundation for NIH (FNIH) is a leading example. Initiated by federal partners NIH, FDA, and Center for Medicare and Medicaid Services (CMS), along with private sector organizations PhRMA (the pharmaceutical

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manufacturers’ trade organization) and BIO (the Biotechnology Industry Organization), the consortium now has multiple industrial, academic, and patient group members. It is funding biomarker qualiﬁcation trials for ﬂuorodeoxyglucose-positron emission tomography (FDG-PET) scanning in non-Hodgkin’s lymphoma and lung cancer and is evaluating a number of additional proposals.

CLINICAL TRIAL MODERNIZATION Other areas in urgent need of improvement are the design, conduct, and analysis of clinical trials during drug development. This part of the Critical Path Initiative includes establishing standards for clinical trial data and its management; fully automating trial process and data management; improving the clinical trial quality management system; and modernizing FDA oversight of the clinical trial process. Signiﬁcant progress in data standardization has been achieved by the clinical trial standards organization, the Clinical Data Interchange Standards Consortium (CDISC) (http://www.cdisc.org). Working with the National Cancer Institute (NCI), FDA has adopted a number of CDISC standards for regulatory submissions. Currently, a CDISC initiative called Clinical Data Acquisitions Standards Harmonization (CDASH), organized to develop standards for case report forms, is ongoing (15). Additionally, FDA is working with the NIH to harmonize and simplify various investigator reporting requirements. Over the past several years, FDA has been modernizing its oversight of clinical trials, has held several public meetings, and has issued guidance and draft regulations. Many parties are interested in improving the consistency, quality, and reliability of clinical trials while reducing the paperwork burdens (16). Discussions about forming a public-private partnership to accomplish these objectives are also ongoing.

BIOINFORMATICS One of the greatest scientiﬁc ﬂaws in the current process of medical product development is its failure to produce generalized knowledge despite a huge investment in data generation. For example, FDA holds the world’s largest collection of animal test data and correlated human trial data, but most of this information is unusable in its current form, except to document a speciﬁc development program. As a result, opportunities for major improvement are missed. Under the Critical Path Initiative, stakeholders are beginning to take advantage of these opportunities. For example, FDA and various partners have created a standard for a digital electrocardiogram (ECG) recording, and FDA requested that ECG data submitted to it be in this format. At the same time, a data warehouse to hold the ECG data was established. Since that time, >500,000 digital ECGs have been added to the warehouse, and a collaboration with Duke University has been established for overall data analysis (17). This resource may help scientists efﬁciently evaluate candidate drugs for adverse cardiac repolarization effects, a concern that is currently addressed (somewhat less than satisfactorily) by extensive clinical testing. As data standards for regulatory submissions are implemented, processes and protocols to utilize the data for research purposes without compromising proprietary interests need to be developed. One important use of such data will be to construct quantitative models of disease processes, incorporating what is known about biomarkers, clinical outcomes, and the effects of various interventions. These models can then be used for trial simulations, to better design appropriate trials and clinical outcome measures (18). Although the FDA has constructed several disease models, this work is in its early stages and will require extensive partnerships. However, there is little doubt that such quantitative approaches constitute the future of product development and assessment.

www.annualreviews.org • The FDA Critical Path Initiative

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DRUG MANUFACTURING

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Perhaps surprisingly, the manufacturing of pharmaceuticals suffers from the problems of drug development in general. Many drug manufacturing processes are characterized by inefﬁciency, waste, and neglect of modern process control technologies. Thus, the pharmaceutical manufacturing sector would also beneﬁt from incorporation of new science and technology. FDA is spearheading these changes through its Pharmaceutical Quality for the 21st Century Initiative (19).

CONSORTIA INVOLVED IN CRITICAL PATH ACTIVITIES After FDA’s 2004 call for public-private collaborations, scientists at several universities created programs to work with FDA to conduct the needed research. Despite limited funding, these programs have been able to make signiﬁcant progress addressing projects called for in the 2006 Critical Path Opportunity List. Investments in programs created by the University of Arizona, Duke University, Massachusetts Institute of Technology (MIT), and the University of California at San Francisco are beginning to produce results (see Sidebar). The ﬁrst to respond, the University of Arizona, offered to create a nonproﬁt research and education institute dedicated solely to work with FDA on the Critical Path

ACADEMIC CONTRIBUTIONS TO DRUG DEVELOPMENT SCIENCE The Center for Biomedical Innovation at MIT (CBI), the Center for Drug Development Sciences (CDDS) at the University of California, San Francisco, and the Duke Clinical Research Institute (DCRI) are examples of university-based centers that are making major contributions to the regulatory sciences. Because of their broad access to outstanding scientists in the academic community, they are important resources for companies that are developing medical products, and they train the innovative leaders.

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Initiative. The Arizona community (state and local governments, business, and philanthropic groups) saw this opportunity to partner with FDA as a way to leverage the state’s $2 billion investment in biotechnology. In early 2004, with a planning grant from the state, the FDA, the University of Arizona, and SRI International (a nonproﬁt corporation, formerly Stanford Research Institute) agreed to create the Critical Path Institute (C-Path). C-Path is envisioned as a neutral third party, without ﬁnancial support from the regulated industry. Because of C-Path’s neutral funding and its mission to focus on process, not products, FDA can actively participate in the work without concerns about conﬂicts of interest. C-Path’s strategy is to invite stakeholders to join consortia in which they can work with the FDA to improve the process of medical product development. The University of Arizona and SRI committed “in kind” support, predominantly the time and effort of their scientists. The FDA agreed to participate under a Memorandum of Understanding. C-Path was incorporated in January 2005 and began initial operations in July 2005 with a $10 million, ﬁve-year commitment from the City of Tucson, Pima County, regional municipalities, and private foundations in Arizona (http://www.C-Path.org). C-Path has approximately 20 employees working with the FDA and industry scientists on the projects listed in Table 1. These projects were selected using three speciﬁc criteria. The ﬁrst and essential requirement is that there be champions for the project within the FDA. Also, there must be two or more companies willing to work together on the project, and there must be a source of external funding that is independent of commercial interests. The projects focus on precompetitive aspects of drug development, e.g., preclinical toxicology. The ﬁrst consortium formed by C-Path, the Predictive Safety Testing Consortium (PSTC), was announced in March 2006 by Secretary of Health and Human Services Michael Leavitt. Since then, the PSTC has

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C-Path Institute projects (http://www.C-Path.org)

Development gap

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C-Path process

C-Path project

Deliverables

Inconsistent technical methods employed across the industry

Create consortium and process for sharing and validation of methods

Predictive Safety Testing Consortium (PSTC)

New FDA guidances to improve and accelerate preclinical safety testing Increased safety of new drugs

Drugs, devices, and diagnostics developed separately. New cancer drugs only 10%–20% effective

Create cross-industry and cross-agency (FDA/NIH/CMS) collaborations to evaluate multiple technologies

Lung cancer diagnostics validation clinical trial with NCI, FDA, and industry

Test to predict lung cancer response Change drug label Model for future drug/diagnostic products

Drugs and diagnostics developed separately. Warfarin side effects cost ∼$1 billion/year and only half (2 million) of patients who need warfarin get treatment

Create cross-industry and cross-agency (FDA/NIH/CMS) collaborations to evaluate multiple technologies

Genomic-based dosing for warfarin, clinical trial with NHLBI, FDA, and industry

Reduce adverse events Increase indicated warfarin treatment Change warfarin label: recommend genetic test Model for future pharmacogenetic clinical trials

High failure rate of clinical trials

Create consortium of orphan disease foundations

Create disease model registries (Nieman-Pick C, valley fever, adrenal cancer)

Template for disease model-based clinical trial design and fewer failed drug development programs

grown from an initial eight to 15 global pharmaceutical companies that are sharing their preclinical methods and data for tests of nephrotoxicity, hepatotoxicity, vascular injury, and carcinogenicity. In this consortium, methods developed by one company that appear to best predict drug toxicity are veriﬁed by experiments performed by a second company. Over 160 scientists are participating, including 25 scientists from the FDA and its European counterpart, the EMEA, who participate in the meetings and discussions as advisors. The methods that are cross-validated by the companies are expected to eventually provide the scientiﬁc basis for regulatory guidance to be issued by the FDA and the EMEA. A goal of C-Path projects is to integrate new and advanced technologies into medical product development, especially those that accelerate pathways for innovative diagnostic tests and therapies. For example, C-Path’s project with the FDA and the University of Utah examines genetic tests for improved war-

farin dosage selection. The goal is to provide the scientiﬁcally based pathway for simultaneous development of drugs and genetic tests to improve a drug’s safety or effectiveness. Another of C-Path’s projects, the Molecular Assays and Targeted Therapeutics (MATT) project, is being conducted by a collaboration among the FDA, the NCI, and the CMS. MATT’s goal is to deﬁne a more rapid and efﬁcient process for integrated development of drugs, diagnostics, imaging, and other technologies that work together to help patients with cancer. The project is exploring a regulatory path by examining the utility of diagnostics and drugs that could enable targeted therapy of non-small-cell lung cancers that overexpress the epidermal growth factor receptor. C-Path’s Disease Model Registry (DMR) for orphan drugs (i.e., drugs intended for diseases affecting <200,000 persons in the United States) addresses the rising failure rate during the later phases of drug development. The overall goal of this project is to evaluate www.annualreviews.org • The FDA Critical Path Initiative

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methods and technologies that could improve understanding of the natural history of diseases and thereby identify clinical trial designs and methods that are more informative and have a greater chance for success. C-Path is creating the functional infrastructure for DMR and bringing together patients, the FDA’s Ofﬁce of Orphan Product Development, care providers, researchers, foundations, and commercial entities to help generate the data standards and scientiﬁc evidence needed to support the efﬁcient testing of new treatments for patients with orphan diseases. This work will also have relevance to personalized medicine because, similar to the situation with orphan diseases, the smaller marker size and the high cost of product development expected for personalized medicine have deterred investment in this new approach. The DMR will serve as a virtual control group, enabling developers to identify the optimal endpoints for clinical trials and thereby increase the likelihood of success in clinical trials. The DMR technology will also make possible online tracking of clinical outcomes, an essential element of newer innovations such as adaptive trial designs. The novel aspects of these C-Path projects are the core neutral funding and the scientiﬁcally qualiﬁed team leaders of the consortia. C-Path brings together scientists from highly competitive companies and then maintains a productive environment through modern project management techniques. Continued participation by the consortium members depends on the rewards they receive for the investment of time and effort. These rewards are expected to be science-based regulatory standards enabled by the work of the consortium, which deﬁne a development process that has the greatest possible efﬁciency and safety. The future of the Critical Path Initiative is increasingly secure because the many stakeholders in medical product development have come to recognize the value of and need for process improvement. They also recognize the importance of having a safe haven such as MIT’s Center for Biomedical Innovation

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or a neutral third party such as C-Path where members of the pharmaceutical industry and the FDA can work as scientists and not be inhibited by their usual roles as regulators and regulated. Likewise, industry scientists are ﬁnding it very rewarding to share with their competitors their knowledge and experiences, especially their failures, in precompetitive areas of development. Therefore, it is likely that the work of the critical path will continue indeﬁnitely. What is not yet clear is where it will take place and how it will be coordinated. The NIH is increasingly involved in critical path projects. The NCI collaborates with the FDA through the Oncology Biomarker Qualiﬁcation Initiative (OBQI). The National Heart, Lung, and Blood Institute has been working with the FDA to coordinate studies of the genetic testing of warfarin. However, tremendous potential remains for the NIH to play an important role in providing FDA with the data and scientiﬁc information needed to improve medical product development. Examples include the NIH roadmap initiatives, the facilities of the National Center for Research Resources, and the growing network of Clinical Translational Research Awards. These are almost all devoted to translational science and have the potential to interface directly with some of the 76 projects on the 2006 Critical Path Opportunities List. An additional opportunity to maximize the efﬁciency and impact of critical path research is being explored through increasing collaborations between FDA and Agency for Healthcare Research and Quality (AHRQ) scientists. AHRQ’s Centers for Education and Research on Therapeutics are working closely with the FDA to deﬁne the optimal approach for active surveillance and postmarketing evaluation of safety and effectiveness of medical products. Ongoing application of new surveillance techniques that are enabled by more widespread use of electronic health records should complement the improved understanding of drugs obtained prior to marketing. It is hoped that electronic records–based surveillance will

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result in rapid detection and analysis of unanticipated outcomes after the products are widely used, so that knowledge can be gained efﬁciently throughout the entire product life cycle. As of this writing, the US Congress is considering the FDA Revitalization Act. This legislation would create the Reagan-Udall Institute, a foundation established to advance FDA’s mission, that would engage in critical path research. Legislation being introduced in the House of Representatives would authorize funding for the FDA to create multiple critical path public-private partnerships. The European Commission is in the ﬁnal stages of approving the Innovative Medicines Initiative, a

partnership among the pharmaceutical industry, the European Union, and academia that would conduct research relevant to drug development, with funding divided equally between government and industry. The need for critical path research is not likely to end. Scientiﬁc advances will continue to create methodological challenges in medicine that will require changes in the way we develop new products. Ideally, a systematic approach to process improvement will become part of the fabric of translational research. A major question is whether the Critical Path Initiative can maintain its momentum and substantively contribute to improved drug development.

SUMMARY POINTS 1. The productivity of the pharmaceutical industry has decreased and the costs of producing a novel medicine have been rising sharply. 2. Some members of the public are calling for medicines to be more extensively studied prior to approval. There is also concern in the United States about pharmaceutical prices. 3. FDA’s Critical Path Initiative is intended to improve drug development and reduce uncertainty by applying new scientiﬁc tools to the development process. 4. The applied research needed to develop these tools requires collaboration across multiple public and private entities and may be best accomplished by various consortia. 5. The C-Path Institute was set up speciﬁcally to facilitate and conduct such research, and it has a number of important projects under way.

DISCLOSURE STATEMENT Dr. Woodcock directs the Critical Path Initiative at FDA. Dr. Woosley is president of the Critical Path Institute.

LITERATURE CITED 1. CMR International. 2005. Pharmaceutical R&D Factbook. Epsom, Surrey, UK: Cent. Med. Res. Intl. 2. 2006. Research and Development in the Pharmaceutical Industry: A CBO Study. Washington, DC: Congr. Budget Off. 3. DiMasi JA, Hansen H, Grabowski HG. 2003. The price of innovation: new estimates of drug development costs. J. Health Econ. 22:151–85 4. Adams CP, Brantner VV. 2006. Estimating the cost of new drug development: Is it really 802 million? Health Aff. 25:420–28 www.annualreviews.org • The FDA Critical Path Initiative

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5. CMR International. 2006/2007. Pharmaceutical R&D Factbook, Ch. 5, p. 2. Epsom, Surrey, UK: Cent. Med. Res. Intl. 6. Wood AJJ. 2006. A proposal for radical changes in the drug approval process. N. Engl. J. Med. 355:618–23 7. US Food and Drug Administration. 2004. Innovation or Stagnation: Challenge and Opportunity on the Critical Path to New Medical Products. http://www.fda.gov/oc/initiatives/ criticalpath/whitepaper.html 8. Wehling M. 2006. Translational science in medicine: implications for the pharmaceutical industry. Int. J. Pharm. Med. 20(5):303–10 9. US Food and Drug Administration. 2006. Innovation or Stagnation: Critical Path Opportunities Report and List. http://www.fda.gov/oc/initiatives/criticalpath/reports/ opp report.pdf 10. US Food and Drug Administration. 2007. Critical Path Opportunities Initiated During 2006. http://www.fda.gov/oc/initiatives/criticalpath/opportunities06.html 11. Woodcock J. 2007. The prospects for “personalized medicine” in drug development and drug therapy. Clin. Pharm. Ther. 81:164–69 12. Caskey CT. 2007. The drug development crisis: efﬁciency and safety. Annu. Rev. Med. 58:1–16 13. Huang SM, Goodsaid F, Rahman A, et al. 2006. Application of pharmacogenomics in clinical pharmacology. Toxicol. Mech. Methods 16:89–99 14. Lesko LJ. 2007. Paving the critical path: How can clinical pharmacology help achieve the vision? Clin. Pharm. Ther. 81:170–77 15. Clinical Data Interchange Standards Consortium (CDISC). 2007. CDISC and Industry Collaborative Group Lead FDA Critical Path Initiative Opportunity for Data Collection Standards. http://www.cdisc.org/news/PR33cdisccdashprojectfinal.pdf 16. Califf RM, Harrington RA, Madre LK, et al. 2007. Curbing the cardiovascular disease epidemic: aligning industry, government, payers, and academics. Health Aff. (Millwood) 26:62–74 17. Clabell CH, Noto TC, Krukoff MW. 2005. Clinical utility of the Food and Drug Administration Electrocardiogram Warehouse: a paradigm for the critical pathway initiative. J. Electocardiol. 38(Suppl.):175–79 18. Stanski DR. 2004. Model-Based Drug Development: A Critical Path Opportunity. http://www.fda.gov/oc/initiatives/criticalpath/stanski/stanski.html 19. US Food and Drug Administration—Center for Drug Evaluation and Research 2004. Pharmaceutical cGMPs for the 21st Century: A Risk-Based Approach, Final Report 2004. http://www.fda.gov/cder/gmp/gmp2004/GMP finalreport2004.htm

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237

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Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye,1 Masahiko Hoshijima,2 and Kenneth R. Chien3 1

Heart Failure Research Group, Baker Heart Research Institute, Melbourne, Victoria 8008, Australia; email: [email protected]

2

Institute of Molecular Medicine, University of California, San Diego, La Jolla, California 92093-0346

3

Cardiovascular Research Center, Massachusetts General Hospital and Harvard Stem Cell Institute, Harvard Medical School, Richard B. Simches Research Centre, Boston, Massachusetts 02114; email: [email protected]

Annu. Rev. Med. 2008. 59:13–28

Key Words

The Annual Review of Medicine is online at http://med.annualreviews.org

sarcoplasmic reticulum ATPase, phospholamban, excitation contraction coupling, gene therapy

This article’s doi: 10.1146/annurev.med.59.052407.103237 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0013$20.00

Abstract Heart failure is a major cardiovascular disease, characterized by considerable morbidity and mortality. Despite major advances in the pharmacotherapy of heart failure, the options for patients with severe end-stage symptoms remain limited. However, recent developments in the identiﬁcation of the molecular basis for the progressive nature of heart failure have identiﬁed a number of potentially important new therapeutic targets. In particular, key components of the cardiomyocyte calcium-handling pathway show characteristic changes in heart failure. A body of research examining the effect of restoration of these defects in experimental models of heart failure, whether in genetically engineered mouse models or by myocardial gene transfer, very strongly supports the calcium-handling pathway as a target for clinical intervention.

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INTRODUCTION

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Heart failure (HF) is a complex, progressive pathophysiological condition that is triggered by an initial myocardial defect. Most commonly the initiating insult is myocardial infarction that causes loss of contractile elements of the heart. This loss increases the load on the remaining myocardium, ultimately leading to loss of contractile reserve and a clinical HF phenotype. It is increasingly recognized that a number of genetic defects are responsible for cardiomyopathy, either directly or in conjunction with other triggers for myocardial injury. Although several classiﬁcations for HF exist, based on clinical severity or physiology, the epidemiology of symptomatic HF is well quantiﬁed. It is generally estimated that symptomatic HF currently affects 0.4%–2% of the general population (1–3). It is well known that the incidence of symptomatic HF rises substantially with age, and for individuals over 65 years of age, the prevalence of HF has been reported to be 6%–10% (1, 4, 5). Beyond simple measures of the number of patients affected by HF, further data indicate the huge impact of HF on society, health care resources, and the economy. For example, HF is the major reason for 12–15 million medical practitioner contacts per year and for 6.5 million hospital days per year (6). In its more advanced stages HF is characterized by considerable disability, and mortality rates may exceed 50% per annum in the most advanced stages (7–9). Driven by these facts, considerable effort has been exerted during the past two decades to identify the mechanisms that contribute to HF and consequently represent viable targets for intervention. A vast body of clinical trial data has led to the generation of systematic guidelines for the management of HF (1).

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FUNCTIONAL AND MOLECULAR PATHOPHYSIOLOGY OF HEART FAILURE Fundamentally, the physiological processes leading to the clinical proﬁle of the patient with HF are often considered in regard to changes in the systolic and diastolic properties of the myocardium. These can be integrated with structural considerations including (a) the loss of myocytes, (b) alteration in the geometry of the heart (including cellular hypertrophy, the development of secondary mitral regurgitation, and a spherical shape), (c) the development of myocardial ﬁbrosis, and perhaps most importantly (d ) reduction in myocardial contractility. Each of these processes contributes to the process of ventricular remodeling, a hallmark of HF. The loss of functioning contractile elements (i.e., cardiomyocytes) in the failing heart can come about in several ways. As indicated above, a substantial proportion of the burden of HF results from acute or chronic myocardial damage due to ischemia and/or infarction resulting in necrosis. It is noteworthy that myocardial necrosis can also result from several other processes, including drug toxicity (e.g., anthracyclines) and myocarditis. Perhaps of equal importance in the context of the chronically failing heart is cardiomyocyte loss via apoptosis. It is well known that the frequency of myocyte apoptosis is elevated in HF (10–12), and the mechanisms behind this are gradually becoming apparent. They probably include the effects of mechanical stress per se and increased local concentrations of neurohormones such as norepinephrine (acting via β-adrenergic receptors) and angiotensin II via the AT1 receptor (13–15). Cardiomyocyte hypertrophy is a key feature of ventricular remodeling in the early

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phase of the transition to HF, and the extent of remodeling correlates well with clinical outcome (16, 17). State-of-the-art techniques including genomics, proteomics, and gene manipulation, using either transgenic models or somatic cell gene transfer, have been employed to investigate the complex, interconnected signaling pathways that drive hypertrophy (17a, 18). Key hypertrophic stimuli include angiotensin II and noradrenaline, so the utility of antagonists of the renin-angiotensin system and adrenergic nervous system can be well appreciated. Experimental studies using more speciﬁc smallmolecule inhibitors have demonstrated the capacity for modulation of the hypertrophic program, but these approaches have commonly been limited by a lack of cardioselectivity and consequent toxicity (18–23). Alterations in the structure of the cardiac extracellular matrix are another key feature of the remodeling heart. This is a dynamic process that results in the accumulation of extracellular matrix proteins, stimulated by cytokines and hormones including transforming growth factor β, aldosterone, and angiotensin II (24, 25). In conjunction with the deposition of matrix proteins, the degradation of the extracellular matrix by matrix metalloproteinases appears to be enhanced during cardiac remodeling (26), thereby favoring chamber dilatation. Together, the complex balance of increased collagen deposition and matrix turnover favors the development of interstitial ﬁbrosis, further increasing wall stress and left ventricular diastolic pressure. It has been a key target of current and proposed therapies for HF (27–33).

Excitation-Contraction Coupling and the Failing Heart Excitation-contraction (EC) coupling (34, 35) is the sequence of cellular and molecular events that link the cellular action potential with contraction (Figure 1). In the myocardium, membrane depolarization causes the opening of voltage-dependent L-type

Ca2+ channels, resulting in the entry of a modest amount of Ca2+ . This process critically activates calcium release channels (ryanodine receptors, RyR) located immediately subjacent to the cell membrane, in the sarcoplasmic reticulum (SR), causing them to open and release a large amount of Ca2+ . The resulting large rise in the intracellular concentration of Ca2+ initiates contraction. Relaxation, an energy-requiring process, occurs by reaccumulation of Ca2+ into the SR via a speciﬁc pump, the SR Ca2+ -ATPase (SERCA). In the myocardium, the principal SERCA protein is the SERCA2a isoform (whilst SERCA1 is principally expressed in skeletal muscle). A closely associated protein, phospholamban (PLN), regulates SERCA2a activity in a manner that is dependent on its phosphorylation status (36, 37). In the dephosphorylated state, PLN exerts an inhibitory action on SERCA2a, slowing its enzymatic rate. When PLN is phosphorylated, classically at a serine residue located at position 16 by protein kinase A, the inhibitory action is lost, resulting in a net increase in SERCA activity. Physiologically, this situation typically occurs in the context of adrenergic activation with a resultant hastening of relaxation due to heightened SERCA activity. As indicated above, one of the major characteristics of the failing myocardium is depressed contractile reserve. Extensive studies using molecular biology, cell imaging, and gene manipulation have dissected in detail many of the mechanisms responsible for altered contractility. In particular, defects in the EC coupling process are a typical ﬁnding in both human HF and a variety of experimental models. Early studies (38–40) demonstrated a series of fundamental changes in intracellular Ca2+ handling, which reduced peak systolic Ca2+ concentration, elevated diastolic Ca2+ concentration, and prolonged the duration of the Ca2+ transient. Given considerable evidence (34) linking the amount of Ca2+ within the SR with contractility, particular emphasis has been placed on the role of SERCA in HF. Numerous studies have demonstrated

www.annualreviews.org • Reversing Ca 2+ Defects in Heart Failure

EC: excitationcontraction SR: sarcoplasmic reticulum SERCA: SR Ca2+ -ATPase PLN: phospholamban

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Figure 1 Key components of the cardiac excitation-contraction system and potential therapeutic targets. Ca2+ enters the cardiomyocyte via L-type Ca2+ channels, triggering release of Ca2+ from the sarcoplasmic reticulum (SR) via the ryanodine release channel (RyR), which is further regulated by the FK binding protein (FKBP). Removal of Ca2+ from the cardiomyocyte is principally mediated via uptake into the SR via the SR Ca2+ -ATPase (SERCA), which is regulated by phospholamban (PLN) depending on its state of phosphorylation. Extrusion of calcium via the Na+ -Ca2+ exchanger (NCX) also occurs. Regulation of several steps via protein kinase A (PKA) and CaM kinase are shown. βAR, beta adrenoceptor.

reductions in the mRNA and protein levels of SERCA2 are evident in the failing heart (41–44), and these ﬁndings are supported by biochemical studies conﬁrming the presence of reduced SERCA enzymatic activity (45). In conjunction with these studies, changes in the pattern of PLN expression have also been suggested to occur in HF. Observations in this regard include a decrease in the ratio of SERCA2 to PLN and hypophosphorylation of PLN due to desensitization of the βadrenergic signaling pathway (42, 46). Both of 16

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these changes in PLN would be expected to further impair SERCA2 function and hence contractility. In addition to changes in SERCA2 and PLN expression, changes in other related proteins may also contribute to altered EC coupling in HF. Some evidence points to a reduction in the expression of the L-type Ca2+ channel (47, 48), although its precise pathophysiological role in HF is controversial. More consistent observations implicate the SR Ca2+ release channel, RyR. Although

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levels of RyR protein per se do not appear to be altered in HF (42), RyR appears to be hyperphosphorylated in the failing heart, as does an associated regulatory protein, FKBP12.6 or calstabin. Both of these proteins increase the release of Ca2+ and may contribute to the elevated diastolic Ca2+ that occurs in HF (49). Beyond changes in critical elements of the EC pathway, other changes in contractile and regulatory proteins themselves are well known in HF. These include alterations in the isoform type of actin, myosin, troponin T, and myoﬁbrillar ATPase, among others (50–52).

ADRENERGIC NERVOUS SYSTEM, PROGRESSIVE HEART FAILURE, AND BETA BLOCKERS Heightened activity of the sympathetic nervous system is a hallmark pathophysiological feature in HF, as measured by a number of techniques (53–55). Although it is generally accepted that adrenergic stimulation of the failing heart is initially adaptive, the longer-term deleterious effects of prolonged adrenergic stimulation of the failing heart are equally evident (56, 57). In addition to the “toxic” effects of catecholamines on the myocardium, the adrenergic signaling pathway within cardiomyocytes is affected. One of the key changes in the failing heart is the downregulation of β-adrenoceptors, particularly the β1 -adrenoceptor (58), probably as a result of exposure to sustained high levels of noradrenaline. In contrast, the β2 adrenoceptor, located at both sympathetic presynaptic and postsynaptic neuroeffector junctions, is less prone to downregulation and may therefore assume a more prominent role in regulating myocardial function in the failing heart (59). The downregulation of β-adrenoceptors results from a combination of processes that include a reduction in β1 -adrenoceptor mRNA levels and an increase in the expression and activity of βadrenoceptor kinase (βARK), which desensitizes β-adrenoceptors and promotes their degradation. Transgenic animal studies have

conﬁrmed the key role of the sympathetic nervous system in the progression of HF. For example, β-adrenoceptor overexpression models (60) clearly show that sustained overactivation of the sympathetic nervous system contributes to the pathophysiology of progressive HF. Ultimately, of course, clinical trials conducted in HF patients across a spectrum of clinical severity demonstrated the beneﬁcial effects of adrenergic receptor antagonism using several different agents (61–63). The observations that adrenergic receptor antagonists improve the clinical outcome of HF, promote reverse remodeling, and improve contractility have prompted exploration of the molecular mechanisms that are responsible. Studies in both animal models and humans suggest that administration of beta blockers in the setting of chronic HF increases SERCA activity and improves myocardial function (64, 65). In a similar manner, it has also been observed that long-term mechanical support of the failing left ventricle may allow recovery of contractile function. This may in part be due to recovery of Ca2+ handling proteins, including SERCA2a (66, 67).

CALCIUM HANDLING: A TARGET FOR THERAPY IN HEART FAILURE? The recognition that abnormal Ca2+ handling is a pivotal component in the cellular biology of HF has attracted attention to this area as a potential therapeutic target (68). Interest is especially intense because there have been relatively few recent developments in the pharmacotherapy for HF (69). Moreover, for patients with end-stage HF, who have typically failed the routine armamentarium of pharmacotherapy and devices (biventricular pacemaker-deﬁbrillators), the only therapeutic options are heart transplantation and possibly long-term mechanical circulatory support. For most patients, however, both of these interventions are inappropriate

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for many reasons, including age and comorbid diseases. Research into the possible effect of manipulating the myocardial Ca2+ handling mechanisms in the setting of HF has utilized

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C

l tro n o

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Figure 2 Effect of restoration of SERCA levels on myocardial function in rats with heart failure induced by aortic banding. Top: End-systolic pressure. Bottom: End-diastolic pressure. ∗ p < 0.05 versus control, # p < 0.05 versus heart failure. Adapted with permission from Reference 74. 18

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various tools including transgenic and knockout animal models and gene transfer methodology. One experimental strategy has been to “restore” the expression of SERCA2a in the setting of HF. In a transgenic animal model system, overexpression of the SERCA2a gene within the myocardium protected the heart against the adverse effects of pressure overload (70). Speciﬁcally, in this study aortic constriction caused a substantial reduction in dP/dt in wild-type animals, while the transgenic overexpression of SERCA signiﬁcantly preserved left ventricular dP/dt under basal conditions, and this was accompanied by a hastening of the rate of relaxation. Several studies on the transfer of the SERCA2 gene have been conducted using virally mediated techniques after the onset of HF. Early studies showed that adenoviral transfer of SERCA2 to cardiomyocytes augmented contractility while also favorably affecting intracellular Ca2+ concentration (71). Subsequently, it was shown that adenovirally mediated transfer of SERCA to isolated cardiomyocytes from failing human heart samples signiﬁcantly improved indices of contractile function (72). Since then, a number of in vivo gene transfer studies have conﬁrmed the potential for augmenting SERCA2 expression to improve myocardial performance and also to restore the pattern of cardiac gene expression toward a more normal pattern. In a series of studies from Hajjar’s laboratory, gene transfer of SERCA2a attenuated ventricular remodeling in the aortic banding model of HF, preserved left ventricular contractility as assessed by pressure volume loop analysis (Figure 2), and improved survival (73, 74). The strong foundation of data regarding SERCA as a potential gene therapy target in human HF has recently provided the basis for a clinical trial that will explore the value of SERCA gene delivery in patients with HF (75). Considerable attention has also been directed toward the potential therapeutic value of manipulating PLN expression. Speciﬁcally, the intent is to inhibit the function

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delivery of S16E PLN was shown to increase cardiomyocyte contractility. Subsequently, in cardiomyopathic hamsters, we demonstrated that sustained overexpression of S16E PLN by adeno-associated virus (AAV) gene transfer blunted the progressive decline in left ventricular function (Figure 3). This study also showed that S16E PLN delivery abrogated

of PLN or to reduce its expression. Using a competitive approach, we engineered a PLN mutant, S16E PLN, in which the Ser16 amino acid residue was substituted for a glutamate. This substitution results in a conformational change that mimics the effect of phosphorylation of PLN at position 16 (76). Under basal conditions, the adenoviral

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Figure 3 Effect of inhibition of phospholamban (PLN) by a pseudophosphorylated PLN mutant (S16E) delivered via adeno-associated virus (AAV) gene therapy. Top: Effect of S16E PLN on LV ejection fraction and LV diastolic dimension in cardiomyopathic hamsters (from Reference 76 with permission). Bottom: Effect of S16E PLN on LV ejection fraction and LV diastolic dimension in sheep with pacing-induced cardiomyopathy (from Reference 78 with permission). # = p < 0.05, Λ = p < 0.01. www.annualreviews.org • Reversing Ca 2+ Defects in Heart Failure

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AAV: adeno-associated virus S16E PLN: an engineered PLN mutant in which the Ser16 amino acid residue was substituted for a glutamate, mimicking phosphorylation of PLN at position 16

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HF progression by reducing the extent of myocardial cell damage. A separate study examined the effect of AAV S16E PLN on HF progression after experimental myocardial infarction (77). In this series of experiments, S16E PLN gene delivery attenuated ventricular remodeling as determined by echocardiography, and this was accompanied by favorable effects on left ventricular contractility and diastolic function. Morphometric studies showed a beneﬁcial effect of S16E PLN delivery on the extent of cardiac ﬁbrosis. These ﬁndings have recently been further developed into a preclinical model of HF in large animals (78). By locally delivering the mutant PLN to the myocardium, it was also possible to show improved cardiac performance as well as evidence of reverse remodeling (Figure 3). Other approaches to the inhibition of PLN have also been employed and generally also demonstrate positive effects on cardiomyocyte and myocardial function. These include antisense strategies (79, 80), dominantnegative strategies (81), antibody methods (82), and RNA interference approaches (83, 84). In some studies, however, the effects of altered PLN expression or activity have not been consistent, possibly owing to the extent of PLN reduction. For example, in a study by Song and colleagues, PLN ablation (by knockout) in a transgenic model of HF augmented cardiomyocyte contractility and calcium handling, but this did not translate into a global improvement in cardiac performance (85).

APPROACHES TO CLINICAL TRANSLATION In attempting to translate laboratory observations regarding abnormalities of the EC coupling process in HF into clinical practice, it is important to take into account the selectivity of the approach for the myocardium and the potential for adverse effects. Although positive inotropic agents such as dobutamine or milrinone may improve myocardial con-

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tractility in the short term, their longer-term use is associated with increased incidence of ventricular and atrial arrhythmias (86). These agents act on aspects of the adrenergic signaling pathway. More speciﬁc agents acting on aspects of the EC pathway have been developed and are considered below, followed by a discussion of gene therapy.

Pharmacotherapy Although it does not speciﬁcally affect Ca2+ handling per se, levosimendan is an agent that dose-dependently improves contractility by stabilizing the molecular conformation of troponin C when it binds calcium (87). Levosimendan is also a vasodilator, which works by activating ATP-sensitive potassium channels (88). Very few other smallmolecule agents have been developed to modulate myocardial Ca2+ handling. JTV519, a 1,4-benzothiazepine derivative, stabilizes the ryanodine receptor (RyR), thereby improving contractility and inhibiting diastolic calcium leakage (89).

Gene Therapy: Choice of Delivery Vector Cellular and experimental in vivo studies have utilized an extensive range of gene delivery approaches, including nonviral and viral-based techniques. Many of the critical issues (outlined below) that will face clinicians and regulatory bodies as gene therapy moves forward into clinical trials are now coming to the fore. For example, in the context of HF therapy, high long-term expression of a transgene might be desired, given the expected low biohazard proﬁle. Early clinical studies particularly relied on gene transfer mediated by naked plasmid DNA because of its ease of production and safety proﬁle (90, 91). However, it is an inefﬁcient means of gene transfer, particularly when delivered intravascularly. The inclusion of various physicochemical agents to

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enhance the efﬁcacy of gene transfer may provide some clinical utility, but this has not been extensively explored. In the clinical setting, it is likely that unmodiﬁed DNA will be delivered only in very select and localized situations, if at all. The transfer of genes to cardiomyocytes and the myocardium in general is more difﬁcult than gene transfer for many other cell types. Recognition of this difﬁculty has led to the widespread use of virally mediated transfer techniques for preclinical research and in clinical trials. Recombinant adenoviral vectors are widely used experimentally and in a variety of clinical trials. These vectors can be produced relatively easily, in large quantity, and have been approved for use in a wide range of clinical trials (http://www. clinicaltrials.gov). However, for applications such as HF the relatively short duration of expression, typically less than one month, limits their potential value. Furthermore, issues have been raised in regard to the potential for adenovirus to initiate an inﬂammatory response, although it is possible that restriction of the delivery of adenovirus to selected sites may prevent these adverse effects by limiting systemic exposure (78). The production of newer recombinant adenoviral vectors that elicit less marked immune responses may lead to renewed interest in their use, and the reduction in immunogenicity may also afford higher and more sustained levels of gene expression. More recently, AAVs have been developed for use as gene transfer vectors. In particular, the AAV2 serotype has already been employed in clinical trials (93). These vectors offer the potential for longer-term expression with less immunogenicity. However, several technical issues limit the clinical utility of AAVs for gene therapy. These difﬁculties include the size of the potential gene insert that can be carried by AAVs, difﬁculty with manufacture, and the presence of preformed antibodies. At present, intense interest is also focused on the characterization of a number

of serotypes, with particular emphasis on their relative tissue and cell-type selectivity (94). In experimental studies, lentiviral vectors show promise as vehicles for highly efﬁcient gene transfer (95) with prolonged gene expression due to genomic integration.

Gene Therapy: Choice of Delivery Method The lessons learned from experimental studies using gene transfer in animal models of HF have proved invaluable in identifying potential therapeutic targets for clinical application. However, most of this work has been conducted in small animals. Therefore, the issue of scalability is important in determining the feasibility of gene delivery per se, as well as dosage. In broad terms, delivery of genes or possibly cells to the myocardium might be considered either as a localized therapy or more globally for a widespread myocardial process such as cardiomyopathy. For localized gene delivery, as might be exempliﬁed by a region of myocardial ischemia or an electrical focus for arrhythmogenesis, injection via a needle-tip catheter under ﬂuoroscopic control is well described. For example, this technique has been utilized in the delivery of proangiogenic factors in patients with refractory myocardial ischemia (96). Localized delivery via small coronary arterial branches may also possible in select cases. As mentioned above, in the context of a more widespread myocardial process, a technique that affords general delivery would be desirable. Gene therapy studies in large animals with experimental HF have been conducted using simple intracoronary delivery. For example, the intracoronary delivery of an adenovirus encoding adenylate cyclase was shown to result in increased myocardial expression of the enzyme and favorable hemodynamic effects (97, 98). However, in this study as in many others, the coinfusion of additional agents such as sodium nitroprusside was required to achieve signiﬁcant viral

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transfer. Other limitations of intracoronary delivery are that the single passage of the viral payload through the coronary circulation provides only a short time for gene transfer and exposes the systemic circulation to the possible adverse effects of gene expression or viral material in other organs. A variety of approaches have been proposed to improve uptake. Delivery of agents retrogradely via the coronary veins has also been tried with some success, although an epicardial predominance of delivery is typically noted (99). The high-pressure delivery of intracoronary perfusate with proximal balloon occlusion has been tested, but although it did augment gene

Coronary arterial catheter

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delivery, the procedure was complicated by signiﬁcant tissue barotrauma (100). Another approach has been the simultaneous use of balloon occlusion of both the coronary artery and relevant coronary vein to localize and augment gene delivery to a particular vascular territory (101). Other potentially translatable approaches to myocardial gene delivery have been somewhat more complex. Cardiac gene transfer using surgical cardiopulmonary bypass techniques has been described in neonatal pigs (102), with evidence of efﬁcient myocardial uptake and expression of the delivered transgene. In a somewhat analogous manner, we developed a percutaneous closed-loop system for myocardial gene delivery that could be applied to the failing heart in large animals and ultimately in man. We hypothesized that a closed-loop recirculation system for myocardial gene delivery might achieve several desirable features. First, by avoiding peripheral systemic delivery or even single-pass intracoronary infusion, this approach raises the concentration of vector reaching the myocardium. Furthermore, the required dose of viral vector may be reduced because dilution of loss is limited. This system has been safely deployed in large animals with severe HF (78, 103), as depicted in Figure 4.

CONCLUSION Coronary sinus recovery

Coronary artery infusion

Heart

Agent Pump

Oxygenator

Figure 4 Potential clinical means for gene delivery to the failing human heart. Upper panel shows the placement of catheters within the coronary arterial and coronary venous system, with gene recirculation mediated via the circuit depicted in the lower panel. 22

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Despite remarkable pharmacological advances in the treatment of patients with HF, the rate of development of new therapies, particularly for patients with moderate to severe HF, appears to have slowed. This is somewhat surprising given the wealth of experimental data that have identiﬁed a number of very promising targets, particularly involving the Ca2+ cycling pathway, for selective manipulation using gene transfer approaches in the failing heart. There is now cautious optimism about a potential role for gene therapy in patients with HF. The results of several phase I studies soon to commence are eagerly awaited.

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DISCLOSURE STATEMENT Dr. Kaye is a cofounder of V-Kardia Inc., which is responsible for developing the closed-loop cardiac gene therapy platform.

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56. Bristow MR, Ginsburg R, Minobe W, et al. 1982. Decreased catecholamine sensitivity and beta-adrenergic-receptor density in failing human hearts. N. Engl. J. Med. 307:205–11 57. Kaye DM, Lefkovits J, Jennings GL, et al. 1995. Adverse consequences of high sympathetic nervous activity in the failing human heart. J. Am. Coll. Cardiol. 26:1257–63 58. Bristow MR, Ginsberg R, Umans V, et al. 1986. β1- and β2-Adrenergic receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective β1 receptor down-regulation in heart failure. Circ. Res. 59:297–309 59. Xiao RP, Cheng H, Zhou YY, et al. 1999. Recent advances in cardiac β2 -adrenergic signal transduction. Circ. Res. 85:1092–100 60. Du XJ, Autelitano DJ, Dilley RJ, et al. 2000. β2 -Adrenergic receptor overexpression exacerbates development of heart failure after aortic stenosis. Circulation 101:71–77 61. Packer M, Bristow MR, Cohn JN, et al. 1996. The effect of carvedilol on morbidity and mortality in patients with chronic heart failure. U.S. Carvedilol Heart Failure Study Group. N. Engl. J. Med. 334:1349–55 62. MERIT-HF Trial. 1999. Effect of metoprolol CR/XL in chronic heart failure: Metoprolol CR/XL Randomised Intervention Trial in Congestive Heart Failure (MERIT-HF). Lancet 353:2001–7 63. CIBIS-II Trial. 1999. The Cardiac Insufﬁciency Bisoprolol Study II (CIBIS-II): a randomised trial. Lancet 353:9–13 64. Lowes BD, Gilbert EM, Abraham WT, et al. 2002. Myocardial gene expression in dilated cardiomyopathy treated with beta-blocking agents. N. Engl. J. Med. 346:1357–65 65. Sun YL, Hu SJ, Wang LH, et al. 2005. Effect of beta-blockers on cardiac function and calcium handling protein in postinfarction heart failure rats. Chest 128:1812–21 66. Kogler H, Schott P, Toischer K, et al. 2006. Relevance of brain natriuretic peptide in preload-dependent regulation of cardiac sarcoplasmic reticulum Ca2+ -ATPase expression. Circulation 113:2724–32 67. Rodrigue-Way A, Burkhoff D, Geesaman BJ, et al. 2005. Sarcomeric genes involved in reverse remodeling of the heart during left ventricular assist device support. J. Heart Lung Transplant. 24:73–80 68. Chien KR, Ross J Jr., Hoshijima H. 2003. Calcium and heart failure: the cycle game. Nat. Med. 9:508–9 69. Kaye DM, Krum H. 2007. Drug discovery for heart failure: a new era or the end of the pipeline? Nat. Rev. Drug Discov. 6:127–39 70. Muller OJ, Lange M, Rattunde H, et al. 2003. Transgenic rat hearts overexpressing SERCA2a show improved contractility under baseline conditions and pressure overload. Cardiovasc. Res. 59:380–89 71. Hajjar RJ, Schmidt U, Kang JX, et al. 1997. Adenoviral gene transfer of phospholamban in isolated rat cardiomyocytes. Rescue effects by concomitant gene transfer of sarcoplasmic reticulum Ca2+ -ATPase. Circ. Res. 81:145–53 72. del Monte F, Harding SE, Schmidt U, et al. 1999. Restoration of contractile function in isolated cardiomyocytes from failing human hearts by gene transfer of SERCA2a. Circulation 100:2308–11 73. del Monte F, Williams E, Lebeche D, et al. 2001. Improvement in survival and cardiac metabolism after gene transfer of sarcoplasmic reticulum Ca2+ ATPase in a rat model of heart failure. Circulation 104:1424–29 74. Sakata S, Lebeche D, Sakata N, et al. 2007. Restoration of mechanical and energetic function in failing aortic-banded rat hearts by gene transfer of calcium cycling proteins. J. Mol. Cell Cardiol. 42:852–61

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75. Ly H, Kawase Y, Yoneyama R, Hajjar RJ. 2007. Gene therapy in the treatment of heart failure. Physiology (Bethesda) 22:81–96 76. Hoshijima M, Ikeda Y, Iwanaga Y, et al. 2002. Chronic suppression of heart-failure progression by a pseudophosphorylated mutant of phospholamban via in vivo cardiac rAAV gene delivery. Nat. Med. 8:864–71 77. Iwanaga Y, Hoshijima M, Gu Y, et al. 2004. Chronic phospholamban inhibition prevents progressive cardiac dysfunction and pathological remodeling after infarction in rats. J. Clin. Invest. 113:727–36 78. Kaye D, Preovolos A, Marshall T, et al. 2007. Percutaneous cardiac recirculation mediated gene transfer of an inhibitory phospholamban peptide reverses advanced heart failure in large animals. J. Am. Coll. Cardiol. 50:253–60 79. Eizema K, Fechner H, Bezstarosti K, et al. 2000. Adenovirus-based phospholamban antisense expression as a novel approach to improve cardiac contractile dysfunction: comparison of a constitutive viral vs an endothelin-1-responsive cardiac promoter. Circulation 101:2193–99 80. del Monte F, Harding SE, Dec GW, et al. 2002. Targeting phospholamban by gene transfer in human heart failure. Circulation 105:904–7 81. Ziolo M, Martin J, Bossuyt J, et al. 2005. Adenoviral gene transfer of mutant phospholamban rescues contractile dysfunction in failing rabbit myocytes with relatively preserved SERCA function. Circ. Res. 96:815–17 82. Meyer M, Belke DD, Trost SU, et al. 2004. A recombinant antibody increases cardiac contractility by mimicking phospholamban phosphorylation. FASEB J. 18:1312–14 83. Watanabe A, Arai M, Yamazaki M, et al. 2004. Phospholamban ablation by RNA interference increases Ca2+ uptake into rat cardiac myocyte sarcoplasmic reticulum. J. Mol. Cell Cardiol. 37:691–98 84. Fechner H, Suckau L, Kurreck J, et al. 2007. Highly efﬁcient and speciﬁc modulation of cardiac calcium homeostasis by adenovector-derived short hairpin RNA targeting phospholamban. Gene Ther. 14:211–18 85. Song Q, Schmidt AG, Hahn HS, et al. 2003. Rescue of cardiomyocyte dysfunction by phospholamban ablation does not prevent ventricular failure in genetic hypertrophy. J. Clin. Invest. 111:859–67 86. Packer M, Carver JR, Rodeheffer RJ, et al. 1991. Effect of oral milrinone on mortality in severe chronic heart failure. The PROMISE Study Research Group. N. Engl. J. Med. 325:1468–75 87. Sorsa T, Heikkinen S, Abbott MB, et al. 2001. Binding of levosimendan, a calcium sensitizer, to cardiac troponin C. J. Biol. Chem. 276:9337–43 88. Yokoshiki H, Katsube Y, Sunagawa M, Sperelakis N. 1997. The novel calcium sensitizer levosimendan activates the ATP-sensitive K+ channel in rat ventricular cells. J. Pharmacol. Exp. Ther. 283:375–83 89. Wehrens XH, Lehnart SE, Marks AR. 2005. Ryanodine receptor–targeted antiarrhythmic therapy. Ann. NY Acad. Sci. 1047:366–75 90. Nabel EG, 1995. Gene therapy for cardiovascular disease. Circulation 91:541–48 91. Nabel GJ. 2004. Genetic, cellular and immune approaches to disease therapy: past and future. Nat. Med. 10:135–41 92. Deleted in proof 93. Manno CS, Pierce GF, Arruda VR, et al. 2006. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat. Med. 12:342–47 www.annualreviews.org • Reversing Ca 2+ Defects in Heart Failure

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94. Palomeque J, Chemaly ER, Colosi P, et al. 2007. Efﬁciency of eight different AAV serotypes in transducing rat myocardium in vivo. Gene Ther. 14:989–97. 95. Bonci D, Cittadini A, Latronico MV, et al. 2003. ‘Advanced’ generation lentiviruses as efﬁcient vectors for cardiomyocyte gene transduction in vitro and in vivo. Gene Ther. 10:630–36 96. Losordo DW, Vale PR, Hendel RC, et al. 2002. Phase 1/2 placebo-controlled, doubleblind, dose-escalating trial of myocardial vascular endothelial growth factor 2 gene transfer by catheter delivery in patients with chronic myocardial ischemia. Circulation 105:2012–18 97. Lai NC, Roth DM, Gao MH, et al. 2000. Intracoronary delivery of adenovirus encoding adenylyl cyclase VI increases left ventricular function and cAMP-generating capacity. Circulation 102:2396–401 98. Roth DM, Lai NC, Gao MH, et al. 2004. Nitroprusside increases gene transfer associated with intracoronary delivery of adenovirus. Hum. Gene Ther. 15:989–94 99. Boekstegers P, von Degenfeld G, Giehrl W, et al. 2000. Myocardial gene transfer by selective pressure-regulated retroinfusion of coronary veins. Gene Ther. 7:232–40 100. Emani SM, Shah AS, Bowman MK, et al. 2003. Catheter-based intracoronary myocardial adenoviral gene delivery: importance of intraluminal seal and infusion ﬂow rate. Mol. Ther. 8:306–13 101. Hayase M, Del Monte F, Kawase Y, et al. 2005. Catheter-based antegrade intracoronary viral gene delivery with coronary venous blockade. Am. J. Physiol. Heart Circ. Physiol. 288:H2995–3000 102. Davidson MJ, Jones JM, Emani SM, et al. 2001. Cardiac gene delivery with cardiopulmonary bypass. Circulation 104:131–33 103. Preovolos AC, Mennen MT, Bilney A, et al. 2006. Development of a novel perfusion technique to allow targeted delivery of gene therapy—the V-Focus system. J. Extra Corpor. Technol. 38:51–52

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237

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Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner1,2 and Mirella Ezban2 1

Department of Medicine, University of Lund, Sweden; email: [email protected]

2

Biopharmaceutical Research Unit, Novo Nordisk A/S, Maaloev, Denmark

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Key Words

First published online as a Review in Advance on September 10, 2007

hemophilia, recombinant FVIIa, TF-FVII pathway inhibitors, TFPI, inactivated rFVIIa

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.061606.095605 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0029$20.00

Abstract For hemophilia patients with inhibitors against FVIII or FIX, the development of recombinant factor VIIa (rFVIIa) raises the possibility of a therapeutic alternative whose availability and convenience of treatment are comparable to those of FVIII or FIX. In support of this new concept for the treatment of bleeding episodes, pharmacological doses of FVIIa have been shown to induce hemostasis. Pharmacological doses of rFVIIa enhance thrombin generation on thrombin-activated platelets, thereby facilitating the formation of strong, well-structured ﬁbrin plugs resistant to premature proteolysis. Modiﬁed rFVIIa molecules with a stronger hemostatic potential have been produced. Inhibition of the FVII-TF-dependent pathway (TFPI and rFVIIai) has been tried in attempts to prevent thrombosis, with promising results in animal models so far not conﬁrmed in clinical trials.

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INTRODUCTION rFVIIa: activated recombinant coagulation factor VII TF: tissue factor

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FV–FXIII: coagulation factor V–coagulation factor XIII; an “a” at the end indicates the active form of the protein TFPI: tissue factor pathway inhibitor AT: antithrombin OCS: open canicular system of platelets

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The development of recombinant factor VIIa (rFVIIa) stimulated research into the tissue factor (TF)- and FVII-dependent pathway of hemostasis. As a result, investigators have begun to explore ways of interfering in this system to enhance hemostasis (as in the treatment of bleeding episodes) or to limit it (as in the prevention of thromboembolism). The ﬁnding that pharmacological doses of rFVIIa provided hemostasis in patients with severe hemophilia in the absence of FVIII or FIX was a breakthrough in the understanding of the importance of FVII and TF for hemostasis. In the 1960s, the hemostatic process was modeled as a waterfall or cascade (1) in which a number of clotting factors existed as coenzymes activating each other in the circulating blood. In the cascade model, FVII and TF were recognized as the extrinsic pathway. However, they received little attention until it was demonstrated in the 1970s that the complex between FVII and TF activated not only FX but also FIX, which was part of the so-called intrinsic system (2). The cascade model did not include cells and platelets and their role for the localization of the hemostatic process. Neither was it able to explain why patients with a FXII deﬁciency do not bleed excessively. A potential role of platelets in the hemostatic process was identiﬁed already in the late 1800s, and subsequently, it was demonstrated that the rate of clotting and generation of thrombin increased as a function of platelet number. It was later shown that the exposure of phospholipids, especially phosphatidylserine (PS), on the outer surface of thrombin-activated platelets was important. Phosphatidylserine is also needed to maintain the anticoagulant activity of endothelium, as it enhances thrombomodulin activity. Furthermore, it was shown that platelets have complex coagulant activities that are not completely mimicked by phospholipids (1). Also important for regulating the hemostatic process is the presence of potent inhibitors in the

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circulation, such as the tissue factor pathway inhibitor (TFPI) and antithrombin (AT) (1, 3). It thus was recognized that the hemostatic process is strongly regulated by localization on cell surfaces and by plasma inhibitors. The so-called extrinsic and intrinsic pathways are much more complex than realized previously, and the idea of the two coagulation pathways—extrinsic and intrinsic—should be abandoned.

HEMOSTASIS According to the current concept, hemostasis occurs on cell surfaces, mainly involving the TF-bearing cells and the thrombinactivated platelets. A cell-based model system was used to study the hemostatic process, which allowed study of the TF-dependent and -independent processes together and apart (1). TF-expressing cells are present in the deeper layers of the vessel wall (4). When the vessel wall is injured, TF is exposed to circulating blood and forms complexes with FVIIa present in the circulation. Recently it was suggested that extravascular TF close to the vessels has bound FVII or FVIIa in the absence of an injury (5). TF may also originate from the circulating blood in the form of encrypted TF carried by cell elements such as white blood cells or microparticles (6). Furthermore, it was recently reported that washed platelets incubated with TF were able to take up TF in a process involving trafﬁc of vesicles through channels of the open canicular system (OCS). TF was identiﬁed in the OCS and occasionally in the alpha-granulae of the platelets (7). Because TF is a receptor protein anchored to cells by a transmembrane domain, the TFFVII/FVIIa complex is localized by cells to the site of injury. Recently the activity of this complex was found to be markedly inﬂuenced by lipid bilayer composition. Using a novel experimental model to regulate the lipid bilayers, and allowing the incorporation of membrane-spanning proteins, investigators

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demonstrated that full TF-FVIIa proteolytic activity required extremely high local concentrations of anionic phospholipids. The TFFVII/FVIIa complex activates FX into FXa as well as FIX into FIXa on the TF-bearing cells. Membrane microdomains with high phosphatidylserine (PS) content were shown to enhance the FX-activating capacity of the TFFVIIa complex (8). The importance of the membrane composition was also stressed by the ﬁnding that depletion of cholesterol from cell membranes impaired functional TF expression in ﬁbroblasts. The FXa formed converts limited amounts of prothrombin into thrombin sufﬁcient to activate FVIII and FV. This thrombin also activates FXI into FXIa, as well as platelets. The FXa activity is restricted to the TF-bearing cell surface. Any FXa that diffuses off the cell is immediately inhibited by the tissue factor pathway inhibitor (TFPI) and AT. As soon as FXa is formed, a complex including TF-FVIIa and FXa is formed, inhibited by TFPI, and internalized. TFPI-alpha contains an acidic N-terminal region followed by three tandem, Kunitz-type protease inhibitor domains and a basic C-terminal region (1). TFPI-beta is an alternatively spliced form of TFPI lacking the Kunitz domain 3 and the C-terminal part of TFPI-alpha. The C terminus of TFPI-alpha is presumably important for localizing the molecule to the cell surface through interactions with anionic phospholipids, glycosaminoglycans, or surface proteins. The majority of circulating TFPI is bound to plasma lipoproteins (9). Heparin releases the endothelial-cell-bound TFPI (3). Another pool of TFPI not released by heparin is bound to glycosylphosphatidylinositol (GPI), either directly (TFPI-beta) or indirectly (TFPI-alpha). Both forms of TFPI seem to be involved in the localization of TFPI to cell surfaces (9). TFPI has been found to enhance the TF-speciﬁc internalization and degradation of FVIIa, which requires the C-terminal domain of TFPI and FXa. Most of the internalized FVIIa is degraded, but a small fraction recycles back to the cell surface as an intact protein. In the

absence of TFPI, FVIIa bound to TF is internalized and degraded (10). The platelets activated by the initially formed thrombin will undergo the “ﬂip-ﬂop” reaction described by Bevers and coworkers (cited in Reference 1), which exposes negatively charged phospholipids on the surface (3). On this surface, the full thrombin burst necessary for effective hemostasis is generated (1). The binding of coagulation proteins on the platelet surface is facilitated by the combined stimulation of the platelet collagen receptor (GPVI) and thrombin receptor owing to the development of a subpopulation of platelets with an increased binding capacity (11; see also 12). Individual variations in such subpopulations may add to the variability of platelet procoagulant response. The binding of clotting factors to the platelet membrane depends on the PS content of the membrane (1). However, the binding of FIXa to the activated platelet surface is mediated not only by the membrane PS but also by speciﬁc sites promoting the formation of active FIXaFVIIIa complexes (“tenase complex”) (13). The tenase complex activates FX from the circulation into FXa on the platelet surface. FXa then associates with FVa on the surface and generates a burst of thrombin required to form a ﬁrm, well-structured ﬁbrin hemostatic plug, which is resistant to premature proteolysis and capable of maintaining hemostasis until the wound healing process is established. Full thrombin generation is necessary for the formation of a tight ﬁbrin structure and for the activation of FXIII, as well as the thrombinactivatable ﬁbrinolytic inhibitor (TAFI). Also the ﬁbrinogen concentration plays a key role in the formation of a dense and tight ﬁbrin network (6). FXIa activated by thrombin also associates with the platelet surface, where it activates more FIX into FIXa, thereby enhancing thrombin generation. Hemostasis in hemophilia is characterized by the formation of loose, fragile hemostatic ﬁbrin plugs, which may stop a bleeding initially but are easily dissolved by proteolytic enzymes bound to ﬁbrin. As a result, the

www.annualreviews.org • TF-FVII Pathway and Therapeutic Opportunities

PS: phosphatidylserine GPI: glycosylphosphatidylinositol GPVI: platelet collagen receptor TAFI: thrombin activatable ﬁbrinolytic inhibitor

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hemophilia patient rebleeds repeatedly and ﬁnally may succumb to extensive, profuse bleedings. In hemophilia the initial thrombin dependent on the TF-FVIIa complex is formed, but since FVIII or FIX is lacking, no tenase complex is formed on the platelet surface. Therefore, no full thrombin burst occurs in hemophilia patients, so the hemostatic plugs that form are loose and unstructured (6). In contrast, patients with FXI deﬁciency never have a bleeding disorder of the severity seen in hemophilia, since they do form basic tenase complexes on the activated platelet surface. However, because they miss the enhancement of thrombin generation on the platelet surface, their ﬁbrin plugs may be less strong and less resistant to proteolysis. Furthermore, the thrombin generated may not be sufﬁcient for full activation of the TAFI. As a result, patients with FXI deﬁciency often develop bleedings in association with surgery or major trauma, especially in areas of rich ﬁbrinolytic activity (e.g., mouth, nose, gastrointestinal tract), although they do not develop spontaneous bleedings as seen in hemophilia (14).

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FVIIa AS A HEMOSTATIC AGENT Approximately 20% of hemophilia patients have developed inhibitors against FVIII or FIX. These patients do not beneﬁt from concentrates of FVIII or FIX unless special measures are undertaken to overcome or remove the inhibitors. These procedures are not easy to perform and are not used to treat mild to moderate bleeding episodes. Prevention of joint bleedings diminishes the development of chronic disabling athropathy in hemophilia patients. In patients with inhibitors, “FVIII bypassing agents” (activated prothrombin complexes, APCCs) are used instead of concentrates of FVIII or FIX. The hemostatic effect of an APCC was found to vary between 50% and 65% in the only two controlled clinical studies performed (6). The APCCs, introduced in the 1970s, contain both zymogens and activated coagulation proteins and have been associated with thromboembolic events. 32

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A dog model was used in an attempt to identify potential thrombogenic factors in the APCCs. Coinjection of antithrombin and heparin prevented the signs of a systemic activation of the coagulation system as evidenced by preventing decreased ﬁbrinogen and thrombocytopenia. The conclusion drawn from these experiments was that FVIIa, which is not effectively inhibited by AT and heparin, should not be responsible for the thrombogenic effect of the APCCs. Furthermore, puriﬁed FVIIa did not induce any signs of a general activation of the coagulation system in the same dog model. However, FVIIa might well be a major contributor to the hemostatic effectiveness of APCCs. FVIIa occurs in the normal circulation in a concentration corresponding to ∼1% of the total FVII protein mass (6). In the 1970s, very little was known about the roles of FVII/FVIIa and TF in the hemostatic process, and FVIIa as a hemostatic agent was a new concept. FVIIa was puriﬁed from human plasma and infused into two hemophilia patients who had FVIII inhibitors. Hemostasis was achieved. No signs of a general activation of the coagulation system were observed (15). Development of rFVIIa required a design utilizing mammalian cells and recombinant technique. The need for a better treatment for hemophilia patients with inhibitors was obvious, and the observed hemostatic effect of plasma-derived puriﬁed FVIIa in severe hemophilia was encouraging in terms of the future usefulness of pharmacological doses of FVIIa. However, in the early 1980s, the risk of transmitting a pathogenic virus with blood products became evident. In addition, rFVIIa was tedious to prepare from plasma. Therefore, recombinant technology was chosen for preparing therapeutic rFVIIa with minimal risk of transmission of infectious agents (11). Between 1985 and 1988, rFVIIa was developed for treatment of hemophilia complicated by inhibitors against FVIII/FIX at Novo Nordisk A/S, Denmark. The vision at the time was to develop a therapy for hemophilia

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patients with inhibitors that would be as easily available and convenient as existing treatments for hemophilia patients without inhibitors. This meant developing an effective hemostatic agent that worked in the absence of FVIII/FIX, which would not only make major surgery possible but also decrease the number of bleedings. If the new agent were successful, there would be no further need for complicated, inconvenient, and expensive therapies such as induced immune tolerance treatment. Recombinant FVII is produced in a baby hamster kidney (BHK) cell line, which is similar to human plasma-derived FVII in its amino acid sequence, gamma-carboxylation, and glycosylation. During the puriﬁcation procedure, rFVII is autoactivated into rFVIIa. Preclinical development included an efﬁcacy study in dogs with hemophilia A and B, which showed that rFVIIa shortened (normalized) the canine cuticle bleeding time without adverse reactions (6). In the past, elective surgery has been more or less contraindicated in hemophilia patients with inhibitors because of the risk of uncontrollable bleeding. However, rFVIIa was demonstrated to have an efﬁcacy rate of 90%– 100% in major surgery, including major orthopedic surgery (6). Starting in 1988, rFVIIa was given to hemophilia patients with serious bleedings in the central nervous system, intraperitoneal and retroperitoneal bleedings, and severe intramuscular bleedings. The dosing schedule was essentially the same as that recommended in surgery, and the efﬁcacy rate was 83%–95%. As part of the vision of providing a treatment for hemophilia patients with inhibitors that would make them similar to patients without inhibitors, the effect of rFVIIa in a home-treatment setting was explored. An efﬁcacy rate of 92% was achieved; however, the average number of doses to achieve hemostasis was 2.3, which indicates that the dose used might not be optimal (14). The addition of pharmacological doses of rFVIIa, reaching plasma concentrations

of 25 nM or higher, induces hemostasis in the absence of FVIII or FIX, most probably by enhancing thrombin generation on the thrombin-activated platelet surface, which enables the formation of a tight, stable ﬁbrin hemostatic plug. In the cell-based model, rFVIIa binds to the thrombin-activated platelet surface with a low afﬁnity, requiring higher concentrations of rFVIIa than those found normally in circulating blood. The bound rFVIIa activates FX on the activated platelet surface independent of the presence of FVIII or FIX. The thrombin generation in the absence of FIX substantially improves following addition of rFVIIa in concentrations of 50 nM and up to 100 nM. Also, the clot lysis time in vitro in hemophilia plasma was found to be prolonged after addition of rFVIIa. Increased TAFI activation as a result of enhanced thrombin generation was suggested to contribute to the increased resistance to lysis. Furthermore, normalization of the ﬁbrin permeability was achieved by the addition of rFVIIa to hemophilia plasma containing platelets, an effect that was reﬂected in a tighter ﬁbrin structure. The hemostatic effect of exogenous rFVIIa in pharmacological doses thus seems to be mediated by an enhanced rate of thrombin generation on thrombin-activated platelet surfaces, resulting in an increased further activation of platelets at the site of injury, and increased platelet adhesion that may involve an enhanced platelet-platelet interaction initiated by thrombin binding to platelet glycoprotein Ib (GPIb) as well as other mechanisms. The enhanced thrombin generation ensures the formation of a tight ﬁbrin structure of the hemostatic plug, as well as full activation of TAFI and FXIII necessary for maintaining hemostasis (6).

Dose Adjustment The substitution therapy with FVIII in hemophilia A and FIX in hemophilia B is monitored by following the plasma concentrations of FVIII or FIX and adjusted

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by aiming at levels of >50% for hemostasis. The use of rFVIIa for treatment of bleeding in hemophilia is a new concept—a pharmacological instead of a substitution therapy. By increasing the physiological level of FVIIa, the nonspeciﬁc binding of rFVIIa to activated platelets is exploited. However, the exact relationship between the plasma concentration of FVII:C and the thrombin generation at the site of injury is not known. Furthermore, a perfect method for determining this localized effect does not exist. A number of assays for the measurement of thrombin generation have been described, but most of them measure thrombin formation in circulating blood rather than the localized thrombin. The recommended dose (90–120 μg/kg bolus i.v.) was based on test tube assays and dog experiments (16). Following the ﬁrst observation that rFVIIa normalized the activated partial thromboplastin time in plasma from hemophilia patients with inhibitors if a concentration of 3.8 μg/ml was used, similar doses of human rFVIIa were found to normalize the cuticle bleeding time in dogs with hemophilia A and B (6). Taking into account a recovery (FVII:C at 10 min after injection) of 40%–50% (17; see also 14), this would approximately correspond to a concentration of 40 nM of rFVIIa in plasma. The concentration of rFVIIa required to start thrombin generation on preactivated platelets in the cellbased in vitro hemostasis model was 50 nM (6). Despite these results, the doses initially used in hemophilia patients were lower. Clearance rate, recovery at 10 min after injection, and the capacity to generate thrombin on the platelet surface vary widely among individuals (14), so the optimal dose might show great variation in a wider population. The clearance rate in children below 15 years of age may be as much as three times the normal rate for adults, which suggests that they may require higher doses of rFVIIa in order to ensure formation of the ﬁrm, tight initial hemostatic plug that is necessary for maintaining hemostasis (14). Recently, a dose of 270 μg/kg was approved in Europe on the basis of a study comparing

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90 μg/kg three times per bleed with one single bolus of 270 μg/kg (11).

Effect of rFVIIa as Prophylaxis Several patients with severe hemophilia complicated by inhibitors against FVIII or FIX have been successfully treated with repeated daily doses of rFVIIa (18). These patients had all developed “target joints,” i.e., joints in which frequent bleeds had resulted in chronic swelling, inﬂammatory synovitis, and a tendency toward further bleeding. Bleedings in such a joint require a stable hemostatic plug for full hemostasis. Results in these patients supported the idea that rFVIIa could be used for prevention of chronic hemophilic arthropathy in hemophilia patients with inhibitors. In a recently published randomized prospective clinical trial (18a), rFVIIa was administered once daily in doses of 90 μg/kg or 270 μg/kg for three months. With oncedaily dosing of rFVIIa, the number of bleeds decreased, not only during the three-month treatment period but also during the observation period that followed (three months of no regular treatment). This outcome may mark another step toward the goal of making the treatment of hemophilia patients with inhibitors similar to that of noninhibitor patients. The decrease in bleeding during the treatment period was probably due to prevention of the repeated bleedings in target joints, resulting in amelioration of the inﬂammatory synovitis. However, it is not clear how this effect was achieved by once-daily administration of an agent with a plasma T/2 of 2–3 h. Another question related to this short halflife is why rFVIIa prophylaxis reduces the number of hemorrhagic events in the posttreatment period. Although this phenomenon may be due simply to a decrease in the inﬂammatory response, evidence related to the extravascular distribution of FVIIa suggests that extravascular coagulation may also play an important role in the prolonged reduction of bleeding episodes in hemophilia patients with

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inhibitors treated repeatedly on a long-term basis with rFVIIa. Already in 1974 it was shown that ﬁbrinopeptides are continuously cleaved from ﬁbrinogen at low levels in normal individuals (3). Furthermore, activation peptides from FIX, FX, and prothrombin have been identiﬁed in the blood of normal individuals. There is also abundant evidence for the presence of many coagulation proteins in the extravascular space (18). Functional FVIIaTF complexes were also demonstrated in an umbilical vein model system (18) as well as around dermal vessels in mice (5). A hypothesis to account for a prophylactic effect of rFVIIa would be that some of the injected rFVIIa reaches the extravascular space and increases the local concentration in this compartment, which may then facilitate the formation of rFVIIa-TF complexes. In vitro studies have shown that FVIIa-TF complexes form on cell surfaces and that the bound FVIIa is internalized and partially degraded in the cell. However, some of it will reappear on the cell surface and bind to TF recruited from the Golgi to the cell surface. The amount of FVIIa internalized is directly proportional to the amount of FVIIa bound to cell surface TF, and the process may continue for a long time if there is plenty of FVIIa present in the extravascular compartment (18). Assuming that a similar process occurs in vivo, continuous formation of rFVIIa-TF complexes on extravascular cell surfaces may facilitate thrombin generation on platelets that plug the leak in small blood vessels. Another possibility is that rFVIIa administered in pharmacological doses may bind to some other protein or compound on the vessel walls and serve as a reservoir for complex formation at any exposure of TF, thereby facilitating the formation of a hemostatic plug by increasing thrombin generation on activated platelets.

Clinical Experience with rFVIIa Other Than in Hemophilia Patients The ability of rFVIIa to enhance thrombin generation on the surface of activated platelets

makes it a potential hemostatic agent in any situation that requires the formation of a tight hemostatic plug. In the cell-based model, rFVIIa was shown to cause a dose-dependent shortening of the lag phase of platelet activation in the presence of platelet counts down to at least 10,000 μl−1 . Also a tighter ﬁbrin structure was observed in the presence of rFVIIa and low platelet counts. Furthermore, the addition of rFVIIa to whole blood made thrombocytopenic (<6000 μl−1 ) in a ﬂow-chamber model increased the ﬁbrin deposition. If the events observed in vitro also occur in vivo, these may contribute to the hemostatic effect of rFVIIa in many other conditions associated with uncontrolled hemorrhage (6). Anecdotal reports of a hemostatic effect in thrombocytopenic patients have been published, and following extensive reports on successful use of rFVIIa in patients with thrombasthenia, the use of rFVIIa (EU) in patients with Glanzmann’s thrombasthenia has been approved by the European Medicines Agency (EMEA). The dosage recommended is similar to the hemophilia dosage, 70–120 μg/kg every other hour in serious bleeding and surgery. The enhancement of thrombin generation on platelet surfaces by rFVIIa leads to a further activation of platelets (increased exposure of phospholipids) at the site of injury. It also increases platelet adhesion, suggested to involve an enhanced platelet-platelet interaction initiated by thrombin binding to GPIb as well as other mechanisms. Also, platelets from patients with Glanzmann’s thrombasthenia, deﬁcient of GPIIb/IIIa, aggregated in the presence of rFVIIa under ﬂow conditions using a collagen-coated surface; such aggregation may contribute to the effect of rFVIIa in Glanzmann’s thrombasthenia. A normalization of the ﬁbrin structure following the addition of rFVIIa was also observed in plasma from a patient with Glanzmann’s thrombasthenia (6). Thrombin generation may be impaired in patients subjected to multiple transfusions, including plasma substitutes and other types of ﬂuid that are used in patients sustaining

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major trauma, or extensive surgery with vast tissue and cell damage. In these patients a combination of dilution coagulopathy and local release of proteolytic enzymes that degrade coagulation proteins occurs. Owing to the dilution coagulopathy, loose, porous ﬁbrin deposits may form, which will be easy targets for extensive degradation by released enzymes. The result is profuse, diffuse bleeding from surfaces of damaged tissue, a process that may be mainly localized without signs of generally increased ﬁbrinolytic activity in the circulation. Successful use of rFVIIa in severely traumatized patients has been reported. rFVIIa has also been found effective in patients with uncontrollable hemorrhage unresponsive to conventional therapy (6, 14). Anecdotal reports of successful use of rFVIIa in patients with increased risk of bleeding due to treatment with anticoagulants have been published (19–22). Successful use of rFVIIa has also been reported in patients who did not have a preformed coagulation disorder but were subjected to a procedure expected to release an abundance of ﬁbrinolytic enzymes (6). The rFVIIa may have helped to generate extra thrombin, resulting in the formation of tight ﬁbrin plugs resistant to the fulminant ﬁbrinolysis occurring locally. A single dose of rFVIIa was also reported to limit the growth of an intracerebral hematoma in patients with intracerebral hemorrhage. In this situation, the formation of a stable ﬁbrin plug resistant to the ﬁbrinolytic activity surrounding the primary hematoma may contribute to the effect.

Safety Injection of rFVIIa in normal individuals increased plasma concentrations of F1+2 and D-dimer, but these did not exceed the normal range. No side effects have been observed in healthy volunteers, and the incidence of thrombotic events has been extremely low. A recent article reported thrombotic events in 1.5% of 11,000 patients who had received rFVIIa. Almost all of these occurred 36

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in nonhemophilia patients with an underlying condition predisposing them to thrombosis. Furthermore, not all thromboses could be attributed to rFVIIa, and all-cause mortality was 0.3%. The localized effect of rFVIIa through the binding to TF-expressing cells and activated platelets most probably makes the drug safe (11). No indication of the formation of antibodies against rFVIIa was seen in patients with hemophilia or in nonhemophilia patients treated with rFVIIa. However, FVII-deﬁcient patients are at risk for development of antibodies against FVII (23).

Modified rFVIIa The hemostatic effect of pharmacological doses of rFVIIa seems to be mediated by enhancement of thrombin generation on thrombin-activated platelets independent of TF. Several rFVIIa variants have been prepared in the effort to optimize this reaction by mimicking the allosteric effect of TF on FVIIa activity and stabilization. The approach of “rational mutagenesis” was used to construct variants of FVIIa with increased intrinsic activity (24). Several variants of rFVIIa with different levels of increased thrombin generation on preactivated platelets (TF-independent activity) were constructed. The most dramatic effect on the activity of FVIIa was obtained by substituting the residues occupying positions 158, 296 and 298 in thrombin/FIXa for those of FVIIa. The catalytic effect of FVIIaDVQ(V158D/E296V/M298Q) on FX activation was enhanced about 40-fold, and the amidolytic activity was increased about eightfold (24). By combining these mutations (DVQ) with others, a further increased rate of FX activation catalyzed up to 100-fold more than wild-type FVIIa (approximately one third of wild-type FVIIa-TF). These superactive mutants activate FX independently of TF at a greater rate than either free or bound wildtype FVIIa (24, 25). Subsequent work has shown that the explanation for this might be a facilitated insertion of the N-terminus tail

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of the protease domain into the FVII activation pocket and the formation of a stabilizing salt bridge (26) that is crucial for activity. The rFVIIa analogue with mutations DVQ (NN1731) has been further characterized in the cell-based reconstituted model system of coagulation (27). In this model, it was found that although rFVIIa and NN1731 resulted in similar rates of FX activation on TFbearing cells, NN1731 showed signiﬁcantly greater activity on the platelet surface. However, NN1731 did not directly activate the platelets, whereas the increased generation of FXa, which caused an increase in thrombin generation (four- to tenfold higher than rFVIIa), resulted in a higher rate of platelet activation, shorter time to clot formation, and greater clot stability. Under conditions of hemophilia, NN1731 shortened the time to onset of clot formation even more than rFVIIa, and both NN1731 and rFVIIa increased the rate of clot formation. NN1731 may therefore induce more rapid clot formation than rFVIIa. As the generation of FXa is similar to that of rFVIIa on TF-bearing cells, the increase in thrombin generation rate by NN1731 is likely to depend on increased FX activation on the surface of activated platelets. Furthermore, a clot lysis assay (11) demonstrated that rFVIIa has not only a procoagulant effect but also an antiﬁbrinolytic effect in plasma from hemophilia patients, which is at least partly due to enhanced TAFI activation. In these assays, the FVIIa analogues also had an enhanced effect. Overall, the data so far indicate that the modiﬁed rFVIIa, NN1731, employs a similar, albeit ampliﬁed, mechanism to promote clot formation and clot stabilization as rFVIIa. Because both procoagulant and antiﬁbrinolytic potentials are signiﬁcantly enhanced compared with rFVIIa, NN1731 may provide more prompt cessation of bleeding episodes and may reduce the risk of rebleeding. The FVIIa analogues, such as NN1731, show an increased rate of inhibition by AT (24). More extensive studies of the inhibition showed that free NN1731 was more readily

inhibited by TFPI and AT-heparin than was rFVIIa in the absence of TF. As for inhibition, no difference between rFVIIa and NN1731 was found in the presence of TF. AT inhibition of both rFVIIa and NN1731 is dependent on the presence of heparin, which probably means glucoseaminoglycans in vivo. Furthermore, only trace amounts of FXa were generated on the surface of unperturbed endothelium by rFVIIa, whereas signiﬁcantly higher amounts of FXa were generated by NN1731. The reaction was not TF-dependent and was inhibited by Annexin 5, indicating that anionic phospholipids were involved. Although higher amounts of activated FXa were generated by NN1731, no difference was observed between rFVIIa and NN1731 in thrombin generation on unperturbed endothelial cells in the presence of plasma. These results indicate that native endothelium does not support thrombin generation actively and that, although NN1731 may generate FXa on native endothelium, this does not enhance thrombin generation (28). Some of the modiﬁed rFVIIa molecules have been shown to reduce tail bleeding time and blood loss in a mouse hemophilia model (29).

NN1731: mutated FVIIa (V158D/E296V/ M298Q8)

INHIBITION OF FVII-TF-DEPENDENT HEMOSTASIS As knowledge of the TF-FVII-dependent pathway increased, the importance of regulating the initiation phase of coagulation in hemophilia was recognized (3). Inhibiting TFPI by adding a TFPI antibody was found to normalize the prolonged clotting time in plasma from hemophilia A and B patients, using a dilute prothrombin time assay (30). When TFPI is prevented from inhibiting the TF-FVIIa-FXa complex, thrombin generation in the absence of the prothrombinase complex is facilitated. This was further veriﬁed in an in vivo model (31) in which the cuticle bleeding time of rabbits made hemophilic was signiﬁcantly reduced after

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Summary of studies using FVIIai (32)

Model system

Results

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Immediate antithrombotic effect of rFVIIai Surgical carotid endarterectomy or femoral angioplasty in a baboon. Dosage: 1 mg/kg i.v. bolus

Decreased platelet and ﬁbrin deposition. Decrease in intimal thickening 30 days after surgery. Modest effect on bleeding

Femoral angioplasty in atherosclerotic rabbits. Dosage: 1 mg/kg i.v. bolus+ of 50 μg/kg/h for 3 days

Reduced angiographic restenosis and neointima hyperplasia 21 days after angioplasty

Recurrent arterial thrombosis in rabbits (Folts model). Dosage: i.v. 0.1 mg/kg/min for 10 min

Abolished cyclic ﬂow variations for up to 20 h

An ex vivo ﬂow system using a severely damaged porcine vessel in an extracorporeal shunt. Dosage: i.v. 0.5 mg and 1.0

Decreased platelet and ﬁbrin deposition

Micro-arterial thrombosis induced by deep vessel denudation in rabbits. Dosage: topical 0.5 mg

Increased patency rates after 30 min and 120 min

Venous thrombosis model (rabbit) induced by chemical destruction and restricted blood ﬂow. Dosage topical: application on the injured vessel

Increased patency and reduced thrombus weight

Effect of rFVIIai on postischemic reperfusion injury Temporary occlusion of the circumﬂex coronary artery (rabbit). Dosage: 1 mg/kg prior to reperfusion

Infarct size and no-ﬂow area decreased. Reduction in platelets and ﬁbrinogen

Isogenic rat aorta transplantation model. Dosage: i.v. bolus 1 mg/kg injected at surgery of the transplant

Reduction of chronic vascular changes such as intimal hyperplasia

Antiinflammatory effects of rFVIIai E. coli induced DIC and inﬂammation (baboon). Dosage: 280 μg/kg at 10 min, + 2, 4, 6, and 8 h after E. coli

Attenuation of inﬂammatory response, increased survival, and prevention of ﬁbrinogen consumption

ARDS-induced lung injury by E. coli (baboon). Dosage: i.v. bolus 1 mg/kg rFVIIai pulmonary

Reduction of pulmonary ﬁbrin and development of ARDS (38)

Abbreviations: DIC, disseminated intravascular coagulation; ARDS, adult respiratory distress syndrome.

the administration of an anti-TFPI antibody. Thus, enhancing the TF pathway either by adding pharmacological concentrations of rFVIIa or by blocking TFPI seems to induce hemostasis independent of the presence of FVIII or FIX. However, inhibition of TF-FVII-induced hemostasis may have a beneﬁcial effect on pathological situations characterized by ﬁbrin deposition in various organs. Recombinant TFPI (rTFPI) was produced in order to test this hypothesis. In several models, rTFPI was shown to inhibit or mitigate the development of disseminated intravascular coagulation. It reduced mortality in a baboon model of E. coli sepsis, prevented thrombosis in vascular trauma, and prevented arterial reocclusion after thrombolysis (32). However, a randomized controlled study including 1754 patients 38

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with severe sepsis and high international normal ratio (INR) found no effect on all-cause mortality (33).

TF Inhibition Apart from its role in the initiation of coagulation, TF-FVIIa also induces cell signaling that plays a role in several other biological processes, such as the inﬂammatory response and the pathophysiology of septicemia and cancer (34). Antibodies against TF were found to attenuate the coagulopathy induced by E. coli sepsis in baboons when given as a pretreatment (35; see also 36). A chimeric monoclonal antibody to TF blocking the binding of FX to the TF-FVIIa complex was tested in an open-label dose-escalation study including 26 patients with stable coronary

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artery disease who were taking aspirin. Spontaneous dose-dependent minor bleeding occurred in 14 patients, but no serious adverse events were seen (37). Despite dedicated work, no small-molecule inhibitors of TF are currently known. Such a discovery seems, in fact, unlikely given the high-energy contacts between TF and FVIIa. Active-site-inhibited rFVIIa (rFVIIai) would be a third possibility to achieve inhibition of the TF-FVII-dependent pathway. rFVIIai is prepared from rFVIIa by reaction with FFR (D-Phe-L-Phe-L-Argchloromethylketone). The tripeptide is covalently incorporated into the active site of rFVIIa, thereby blocking the reactive center. rFVIIai competes efﬁciently with endogenous FVIIa for binding to TF and thereby limits the formation of functional TF-FVIIa complexes (38).

The effects of rFVIIai have been studied in animal models of thrombus formation induced by mechanical injury, induced ischemia, and endotoxin infection. These studies are summarized in Table 1 (32). Recombinant nematode anticoagulant protein c2 (rNAPc2) is an 85-amino-acid serine protease inhibitor that directly inhibits the catalytic complex of TF-FVIIa. A potential anticoagulant effect was investigated in an open-label sequential dose-ranging study that included 293 patients undergoing total knee replacement. This study reported an overall rate of deep vein thrombosis of 12.2% and a proximal deep vein thrombosis rate of 1%–3%. Major bleedings occurred in 2%– 3% of patients. The efﬁcacy and safety proﬁle demonstrated for rNAPc2 was comparable to results reported with low-molecular-weight heparin prophylaxis (39).

DISCLOSURE STATEMENT Both authors are employees of Novo Nordisk A/S, the manufacturer of rFVIIa (NovoSeven).

LITERATURE CITED 1. Monroe DM, Hoffman M, Roberts HR. 2002. Platelets and thrombin generation. Art. Thromb. Vasc. Biol. 22:1381–89 2. Østerud B, Rapaport SI. 1977. Activation of factor IX by the reaction product of tissue factor and factor VII: additional pathway for initiating blood coagulation. Proc. Natl. Acad. Sci. USA 74:5260–64 3. Roberts HR, Monroe DM, Hoffman M. 2001. Molecular biology and biochemistry of the coagulation factors and pathways of hemostasis. In Williams Hematology, ed. E Beutler, MA Lichtman, BS Coller, TJ Kipps, U Seligsohn, pp. 1409–34. McGraw-Hill. 6th ed. 4. Rapaport SI, Rao LVM. 1995. The tissue factor pathway: how it has become a “prima ballerina.” Thromb. Haemost. 74:7–17 5. Hoffman M, Colina CM, McDonald AG, et al. 2007. Tissue factor around dermal vessels has bound factor VII in the absence of injury. J. Thromb. Haemost. 5:1403–8 6. Hedner U. 2006. Mechanism of action, development and clinical experience of recombinant FVIIa. J. Biotechnol. 124:747–57 7. Lopez-Vilchez I, Escolar G, Diaz-Ricart M, et al. 2007. Tissue factor enriched vesicles are taken up by platelets and induce platelet aggregation in the presence of factor VII. Thromb. Haemost. 97:202–11 8. Shaw AW, Pureza VS, Sligar SG, et al. 2007. The local phospholipid environment modulates the activation of blood clotting. J. Biol. Chem. 282:6556–63 9. Zhang J, Piro O, Lu L, Broze GJ Jr. 2003. Glycosyl phosphatidylinositol anchorage of tissue factor pathway inhibitor. Circulation 108:623–627 www.annualreviews.org • TF-FVII Pathway and Therapeutic Opportunities

1. Contains many references.

6. One of the most recent extensive reviews; includes many references.

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15. First demonstration of a hemostatic effect of pharmacological doses of FVIIa.

24. Describes the basis for modified FVII molecules.

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10. Ghosh S, Pendurthi UR, Steinoe A, et al. 2007. Endothelial cell protein C receptor acts as a cellular receptor for factor VII on endothelium. J. Biol. Chem. 282:11849–57 11. Hedner U. 2007. Recombinant factor VIIa: its background, development and clinical use. Curr. Opin. Hemat. 14:225–29 12. Kjalke M, Kjellev S, Røjkjær R. 2007. Preferential localization of recombinant factor VIIa to platelets activated with a combination of thrombin and a glycoprotein VI receptor agonist. J. Thromb. Haemost. 5:774–80 13. Melton LG, Li T, Stafford DW, et al. 2001. Location of the platelet binding site in zymogen coagulation factor IX. Blood Coag. Fibrinolysis 12:237–43 14. Hedner U, Erhardtsen E. 2003. Potential role of recombinant factor VIIa as a hemostatic agent. Clin. Adv. Hematol. Oncol. 1:112–19 15. Hedner U, Kisiel W. 2003. The use of human factor VIIa in the treatment of two hemophilia patients with high-titer inhibitors. J. Clin. Invest. 71:1836–41 16. Hedner U. 2004. Dosing with recombinant factor VIIa based on current evidence. Semin. Hemat. 41(Suppl. 1):35–39 17. Fridberg MJ, Hedner U, Roberts HR, et al. 2005. A study of the pharmacokinetics and safety of recombinant activated factor VII in healthy Caucasian and Japanese. Blood Coag. Fibrinolysis 16:259–66 18. Hedner U. 2006. Potential role of recombinant factor FVIIa in prophylaxis in severe hemophilia patients with inhibitors. J. Thromb. Haemost. 4:2498–500 18a. Konkle BA, Ebbesen LS, Erhardtsen E, et al. 2007. Randomized prospective clinical trial of rFVIIa for secondary prophylaxis in hemophilia patients with inhibitors. J. Thromb. Haemost. In press. doi: 12.1111/j.1538-7836.2007.02663.x 19. Bijsterveld NR, Moons AH, Boekholdt SM, et al. 2002. Ability of recombinant factor VIIa to reverse the anticoagulant effect of the pentasaccharide fondaparinux in healthy volunteers. Circulation 106:2550–54 20. Deveras RAE, Kessler CM. 2002. Reversal of warfarin-induced excessive anticoagulation with recombinant human factor VIIa concentrate. Ann. Int. Med. 137:884–88 21. Firozvi K, Acs P, Baidas S, et al. 2004. Efﬁcacious and safe use of recombinant activated factor VII (rFVIIa) in patients with enoxaparin (ENOX)-induced bleeding in patients with pre-existing hypercoagulable states. Blood 104(11, Pt 1):300A 22. Carnazzo SA, Saitta R, Laurentini GM, et al. 2005. Use of recombinant activated factor VII in an elderly female undergoing acenocoumarol thromboprophylaxis requiring an emergency laparotomy. J. Thromb. Thrombolysis 19:213–14 23. Nicolaisen EM. 1998. Antigenicity of activated recombinant factor VII followed through nine years of clinical experience. Blood Coag. Fibrinolysis 9(Suppl. 1):S119–23 24. Persson E, Kjalke M, Olsen OH. 2001. Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity. Proc. Natl. Acad. Sci. USA 98:13583–88 25. Persson E, Bak H, Ostergaard A, et al. 2004. Augmented intrinsic activity of factor VIIa by replacement of residues 305, 314, 337 and 374; evidence of two unique mutational mechanisms of activity enhancement. Biochem. J. 379:497–503 26. Rand KD, Jorgensen TJ, Olsen OH, et al. 2006. The allosteric activation of coagulation factor VIIa visualized by hydrogen exchange. J. Biol. Chem. 281:23018–24 27. Allen AG, Persson E, Campbell RA, et al. 2007. A variant of recombinant factor VIIa with enhanced procoagulant and antiﬁbrinolytic activities in an in vitro model of hemophilia. Arterioscler. Thromb. Vasc. Biol. 27:683–89 28. Ghosh S, Ezban M, Persson E, et al. 2007. Activity and regulation of factor VIIa analogs with increased potency at the endothelial cell surface. J. Thromb. Haemost. 5:336–46 Hedner

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29. Tranholm M, Kristensen K, Kristensen AT, et al. 2003. Improved hemostasis with superactive analogs of factor VIIa in a mouse model of hemophilia A. Blood 102:3615–20 30. Nordfang O, Valentin S, Beck T, et al. 1991. Inhibition of extrinsic pathway inhibitor shortens the coagulation time of normal plasma and of hemophilia plasma. Thromb. Hemost. 66:464–67 31. Erhardtsen E, Ezban M, Madsen MT, et al. 1995. Blocking of tissue factor pathway inhibitor (TFPI) shortens the bleeding time in rabbits with antibody induced haemophilia A. Blood Coag. Fibrinol. 6:388–94 32. Hedner U, Erhardtsen E. 2000. Future possibilities in the regulation of the extrinsic pathway: rFVIIa and TFPI. Ann. Med. 32(Suppl. 1):68–72 33. Abraham E, Reinhart K, Opal S, et al. 2003. Efﬁcacy and safety of tifacogin (recombinant tissue factor pathway inhibitor) in severe sepsis. JAMA 290:238–47 34. Rao LVM, Pendurthi UR. 2005. Tissue factor–factor VIIa signaling. Arterioscler. Thromb. Vasc. Biol. 25(1):47–56 35. Huang H, Norledge BV, Liu C, et al. 2003. Selective attenuation of the extrinsic limb of the tissue factor-driven coagulation protease cascade by occupancy of a novel peptidyl docking site on tissue factor. Biochemistry 42:10619–26 36. Taylor FB Jr, Chang ACK, Peer G, et al. 1998. Active site inhibited factor VIIa (DEGR VIIa) attenuates the coagulant and interleukin-6 and -8, but not tumor necrosis factor, responses of the baboon to LD100 Escherichia coli. Blood 91:1609–15 37. Morrow DA, Murphy SA, McCabe CH, et al. 2005. Potent inhibition of thrombin with a monoclonal antibody against tissue factor (Sunol-cH36): results of the PROXIMATETIMI 27 trial. Eur. Heart J. 26:682–88 38. Rao LVM, Ezban M. 2000. Active site-inactivated factor VIIa as an effective antithrombotic agent: mechanism of action (review). Blood Coag. Fibrinol. 11(Suppl. 1):135–43 ¨ 39. Lee A, Agnelli G, Buller H, et al. 2001. Dose-response study of recombinant factor VIIa/tissue factor inhibitor recombinant nematode anticoagulant protein c2 in prevention of postoperative venous thromboembolism in patients undergoing total knee replacement. Circulation 104:74–78

www.annualreviews.org • TF-FVII Pathway and Therapeutic Opportunities

30. First experiments suggesting a hemostatic effect of decreased or absent TFPI.

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237

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Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Therapy of Marfan Syndrome Annu. Rev. Med. 2008.59:43-59. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Daniel P. Judge1 and Harry C. Dietz2 1

Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine; 2 McKusick-Nathans Institute of Genetic Medicine and Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; email: [email protected]

Annu. Rev. Med. 2008. 59:43–59

Key Words

First published online as a Review in Advance on September 10, 2007

aortic aneurysm, ﬁbrillin-1, Loeys-Dietz syndrome, transforming growth factor beta

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.103106.103801 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0043$20.00

Abstract Marfan syndrome is a common inherited disorder of connective tissue caused by deﬁciency of the matrix protein ﬁbrillin-1. Effective surgical therapy for the most life-threatening manifestation, aortic root aneurysm, has led to a nearly normal lifespan for affected individuals who are appropriately recognized and treated. Traditional medical therapies, such as beta-adrenergic receptor blockade, are used to slow pathologic aortic growth and decrease the risk of aortic dissection by decreasing hemodynamic stress. New insights regarding the pathogenesis of Marfan syndrome have developed from investigation of murine models of this disorder. Fibrillin-1 deﬁciency is associated with excess signaling by transforming growth factor beta (TGFβ). TGFβ antagonists have shown great success in improving or preventing several manifestations of Marfan syndrome in these mice, including aortic aneurysm. These results highlight the potential for development of targeted therapies based on discovery of disease genes and interrogation of pathogenesis in murine models.

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INTRODUCTION Dolichocephaly: disproportionately long, narrow head Dolichostenomelia: long bone overgrowth

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Ectopia lentis: ocular lens dislocation MASS phenotype: mitral valve prolapse, myopia, borderline and nonprogressive aortic root enlargement, nonspeciﬁc skin and skeletal ﬁndings LDS: Loeys-Dietz syndrome TGFβ: transforming growth factor beta

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Marfan syndrome (MFS) is a systemic disorder of connective tissue with cardinal manifestations involving the ocular, skeletal, and cardiovascular systems. Conventional therapies have relied largely on hypothetical approaches to prevent life-threatening features such as aortic aneurysm and dissection. As our knowledge of the pathophysiology grows, emerging therapeutic strategies are predicted to surpass the traditional treatments for this condition. Before describing both traditional and emerging therapies, this review outlines the clinical features of MFS, as well as our current knowledge of the mechanisms of pathogenesis.

CLINICAL DIAGNOSIS In 1896, French pediatrician AntoineBernard Marfan ﬁrst described the skeletal features of Gabrielle P., a ﬁve-year-old girl with slender digits, dolichocephaly, and tall stature (1). Over the subsequent four decades, he compiled reports on >150 similar cases, recognizing Mendelian inheritance with cosegregation of dolichostenomelia, ectopia lentis, and mitral valve disease (2). The cardiac manifestations were described by Victor A. McKusick in 1955, who reported dilatation and dissection of the aorta and aortic valve regurgitation in MFS, which he classiﬁed among the heritable disorders of connective tissue (3). Despite these reports, the diagnosis of MFS was somewhat variable until the mid1980s. At the 1986 International Congress of Human Genetics in Berlin, consensus opinions were compiled regarding the clinical diagnosis (4). This conference led to a series of diagnostic criteria that attempted to draw distinctions within a broad spectrum of connective tissue disorders that showed overlap with the phenotypes seen in MFS. Accurate phenotypic assignment was important in subsequent linkage analyses and the eventual recognition that MFS is caused by mutations Judge

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Dietz

in FBN1, the gene encoding the extracellular matrix protein ﬁbrillin-1 (5, 6). However, the Berlin Nosology for the diagnosis of MFS was later recognized to be inappropriately inclusive of individuals with mild features within families segregating MFS (7). In 1996, revised diagnostic criteria for MFS were proposed, with continued reliance on a series of major and minor clinical manifestations in different organ systems (8). These criteria were more stringent (Table 1); they required at least four of eight skeletal features for assignment of the skeletal system as a major criterion, potentially included molecular testing, and increased requirements for family members of an unequivocally affected individual. Although it is currently estimated that >90% of people with classic MFS will have a deﬁnable FBN1 mutation, the large size of this gene and the extreme allelic heterogeneity characteristic of MFS have frustrated efﬁcient molecular diagnosis for this disorder (9). More importantly, many other conditions, such as mitral valve prolapse syndrome, MASS phenotype, familial ectopia lentis, Weill-Marchesani syndrome, and Shprintzen-Goldberg syndrome, have also been associated with mutations in FBN1 (10–14). It is often difﬁcult or impossible to predict the phenotype from the character or location of a mutation. Mutation analysis is best used to determine if a presymptomatic individual has inherited the predisposition for a deﬁned phenotype seen in the extended family. Although mutation testing can serve as an adjunct, diagnosis of MFS in a proband is currently made on clinical grounds. An important consideration in the differential diagnosis of MFS is a recently described condition known as Loeys-Dietz syndrome (LDS) (15). LDS is caused by heterozygous mutations in the genes encoding the transforming growth factor (TGFβ) receptors 1 and 2 (TGFBR1 or TGFBR2) (15). These mutations result in increased TGFβ signaling in both cells and involved tissues from affected patients (15). This disorder shares

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Table 1

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Diagnostic criteria for Marfan syndrome

Index case: • If the family/genetic history is not contributory, major criteria in ≥2 different organ systems and involvement of a third organ system. • If a mutation known to cause Marfan syndrome in others is detected, 1 major criterion in an organ system and involvement of a second organ system. Relative of an index case: • Presence of a major criterion in the family history, 1 major criterion in an organ system, and involvement of a second organ system.

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Organ systems: Skeletal: Major criteria: The presence of at least 4 of the following constitutes a major criterion in the skeletal system. • Pectus carinatum (protrusion of sternum) • Pectus excavatum (depression of sternum) requiring surgery • Reduced upper- to lower-segment ratio or arm span–to–height ratio >1.05 • Wrist and thumb signs • Scoliosis of >20◦ or spondylolisthesis (displacement of vertebra, usually in lumbar spine) • Reduced extension at the elbows (<170◦ ) • Medial displacement of the medial malleolus causing pes planus (ﬂat feet) • Protrusio acetabulae (protrusion of the femoral head into the pelvis) of any degree (ascertained on radiographs) Minor criteria: • Pectus excavatum of moderate severity • Joint hypermobility • Highly arched palate with crowding of teeth • Facial appearance [dolichocephaly, malar hypoplasia, enophthalmos (deeply set eyes), retrognathia (receding chin), down-slanting palpebral ﬁssures) For involvement of the skeletal system, at least 2 features contributing to major criteria, or 1 feature from the list contributing to the major criterion and 2 of the minor criteria must be present. Ocular system: Major criterion: • Ectopia lentis Minor criteria: • Abnormally ﬂat cornea • Increased axial length of globe • Hypoplastic iris or hypoplastic ciliary muscle causing decreased miosis For involvement of the ocular system, at least 2 of the minor criteria must be present. Cardiovascular system: Major criteria: • Dilation of the ascending aorta, with or without aortic regurgitation, and involving at least the sinuses of Valsalva • Dissection of the ascending aorta Minor criteria: • Mitral valve prolapse with or without mitral valve regurgitation • Dilatation of the main pulmonary artery, in the absence of valvular or peripheral pulmonic stenosis or any other obvious cause, younger than age 40 • Calciﬁcation of the mitral annulus younger than age 40 • Dilatation or dissection of the descending thoracic or abdominal aorta younger than age 50 For involvement of the cardiovascular system, only 1 of the minor criteria must be present. (Continued )

www.annualreviews.org • Therapy of Marfan Syndrome

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Table 1

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(Continued )

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Pulmonary system: Major criteria: none Minor criteria: • Spontaneous pneumothorax • Apical blebs For involvement of the pulmonary system, only 1 of the minor criteria must be present Skin and integument: Major criteria: none Minor criteria: • Striae atrophicae (stretch marks) without marked weight gain, pregnancy, or repetitive stress • Recurrent or incisional herniae For involvement of the skin and integument, only 1 of the minor criteria must be present Dura: Major criterion: • Lumbosacral dural ectasia (widening of the dural sac) Minor criteria: none Family/genetic history: Major criteria: • Having a parent, child, or sibling who meets these diagnostic criteria independently • Presence of a mutation in FBN1 that is known to cause MFS • Presence of a haplotype around FBN1, inherited by descent, known to be associated with unequivocally diagnosed MFS in the family Minor criteria: none

selected features with MFS (pectus deformity, scoliosis, and aortic root aneurysm) but can be distinguished on the basis of characteristic features, including hypertelorism, cleft palate, biﬁd or broad-based uvula, and the absence of ectopia lentis. Additional features that help to differentiate LDS from MFS are listed in Table 2. The arterial disease in LDS is notable for arterial tortuosity in large and medium-sized vessels (prominently of neck vessels) and the high risk of aneurysms and dissection throughout the arterial tree (16). Furthermore, aortic root aneurysms tend to dissect at younger ages and smaller dimensions than those seen in MFS. One series of 52 families reported an average age at death of 26 years among affected individuals (16). Because of the increased risk of aortic dissection at smaller aortic sizes in LDS, we advise affected adults to undergo prophylactic surgery when the aortic root diameter approaches 4.0 cm. Earlier intervention is con-

Hypertelorism: widely spaced eyes

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sidered depending on family history or an individual’s personal assessment of risk versus beneﬁt. Surgery in severely affected children is considered once the aortic annulus exceeds 1.8 cm, allowing placement of a graft of sufﬁcient size to accommodate growth. This approach differs substantially from our approach to MFS.

PATHOGENESIS MFS was traditionally considered to reﬂect a simple structural deﬁciency in connective tissues. Early hypotheses suggested that this condition might be due to a primary problem of the elastic ﬁber (3). Studies of both skin and aorta from affected individuals have shown decreased elastin with elastic ﬁber fragmentation (17, 18). Linkage analyses identiﬁed an associated locus on chromosome 15, excluding the elastin gene as the site of mutations causing this condition (5, 19). Upon recognition of FBN1 mutations resulting in MFS,

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Table 2

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Features differentiating Marfan and Loeys-Dietz syndromesa

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Marfan syndrome

Loeys-Dietz syndrome

Gene:

FBN1

TGFBR1 or TGFBR2

Ocular features: ectopia lentis myopia

typical typical

not associated observed

Skeletal features: arachnodactyly cervical spine instability club foot dolichostenomelia joint laxity pectus deformity scoliosis

typical not associated not associated typical typical typical typical

common common common observed (but subtle) typical and severe common common

Cardiovascular features: aortic root aneurysm arterial tortuosity childhood death/dissection congenital heart defectsb other aneurysms

typical not associated exceedingly rare not associated exceedingly rare

typical typical common common common

Craniofacial features: broad-based or biﬁd uvula cleft palate craniosynostosis hypertelorism

not associated not associated not associated not associated

typical common common typical

Neurodevelopmental features: Chiari I malformation developmental delay

not associated not associated

common observed

Skin/integument features: easy bruising dystrophic scars soft skin texture striae atrophicae (stretch marks) translucent skin

not associated not associated not associated typical not associated

typical common typical typical typical

a This table compares frequencies of clinical manifestations of Marfan syndrome and Loeys-Dietz syndrome, organized by organ systems. “Typical” indicates a majority of affected individuals, “common” indicates 20%–50% of affected individuals, “observed” indicates 1%–20% of affected individuals, and “not associated” indicates that affected individuals have a risk of this manifestation equal to the risk in an unaffected population. “Exceedingly rare” indicates that this feature occurs in <1% of affected individuals, but at a rate higher than in an unaffected population. b Includes patent ductus arteriosus, atrial septal defect, and bicuspid aortic valve.

this structural impairment was attributed to loss of ﬁbrillin-1 in the extracellular matrix. Consequently, in response to stress, such as hemodynamic forces in the proximal aorta, affected organ systems were thought to manifest this structural insufﬁciency with accelerated degeneration. In this traditional view, the

opportunity for effective treatments for MFS was greatly limited, as the bulk of elastic ﬁber formation occurs during embryogenesis. A dominant negative effect of mutant ﬁbrillin-1 was inferred from the dominant pattern of inheritance, the requirement for multimerization of ﬁbrillin-1 monomers to www.annualreviews.org • Therapy of Marfan Syndrome

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LTBP: latent transforming growth factor beta binding protein

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form microﬁbrils, and the dramatic paucity of ﬁbrillin-1 in immunostained samples derived from affected individuals (20, 21). This deﬁciency of ﬁbrillin-1 was thought to lead to a primary derangement of elastic ﬁber deposition, with ultimate failure of the connective tissues inevitable in affected tissues (22). Supporting this hypothesis is the observation that some (though not all) people with a premature termination codon in FBN1 have mild clinical manifestations of MFS (23, 24). This may be attributable to rapid and efﬁcient destruction of these transcripts by the nonsense-mediated mRNA decay pathway, and insufﬁcient mutant protein present to exert a dominant negative effect (25). Accordingly, one putative strategy for MFS therapy was to target the mutant allele with hammerhead ribozyme antisense technology (26). The role of haploinsufﬁciency in the pathogenesis of MFS was emphasized by investigation of murine models of this disorder (27). If dominant negative interference by the mutant allele is solely responsible for pathogenesis, then overexpression of a mutant FBN1 transgene on a wild-type background should result in similar manifestations. However, several strains of mice with robust expression of a transgene containing a cysteinesubstitution FBN1 mutation known to cause MFS, in addition to the normal two murine Fbn1 alleles, have no discernible aortic disease (27). In contrast, strains of mice with comparable targeted mutations in one endogenous murine Fbn1 allele have classic features of MFS, including progressive aortic root dilatation (27, 28). Finally, characterization of mice harboring both an endogenous Fbn1 mutation and a mutant FBN1 transgene revealed improvement in features of MFS (27). These data suggest that a threshold reduction in ﬁbrillin-1 function is needed for disease expression, that mutant ﬁbrillin-1 retains some of its function, and that dominant negative interference alone is insufﬁcient to trigger the pathogenetic sequence that culminates in MFS. From these experiments, it appears that strategies for therapy that promote producJudge

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tion of ﬁbrillin-1 (mutant and/or wild-type) may mitigate this disorder. These conclusions are strengthened by observations within families segregating hypomorphic FBN1 alleles and MFS. The level of expression of the normal allele in some of these families appears to correlate inversely with the severity of disease (29). Recent research has identiﬁed a regulatory role for ﬁbrillin-1 that appears to have important implications for the therapy of MFS. This extracellular matrix protein shares signiﬁcant homology with the latent transforming growth factor beta binding proteins (LTBPs), and binding studies have demonstrated their interaction (30, 31). TGFβ is secreted from the cell in the context of a large latent complex (LLC) that includes the mature cytokine, a dimer of its processed amino-terminal propetide called latency associated peptide (LAP), and one of three LTBP molecules. The possibility that failed matrix sequestration of the LLC by ﬁbrillin-1 leads to altered TGFβ signaling was ﬁrst analyzed in mice with reduced expression of ﬁbrillin-1 (32). These mice have defective alveolar septation, which causes enlarged airspaces. Immunostaining with an antibody speciﬁc for the free and active form of TGFβ identiﬁed increased amounts of this cytokine; reduced staining was seen for LAP suggesting increased activation (rather than production) of TGFβ in the ﬁbrillin-1 deﬁcient state (32). A transgenic strain was developed to produce green ﬂuorescent protein (GFP) in response to TGFβ stimulation. Production of GFP was progressively increased in developing lungs of mice with one or two mutant Fbn1 alleles. Further supporting the hypothesis that ﬁbrillin-1 deﬁciency leads to localized increase in TGFβ activity, treatment of these mice with a neutralizing antibody to this cytokine led to normalization of alveolar septation (32). Many manifestations of MFS are not logically explained by a structural deﬁciency resulting from FBN1 mutations. For instance,

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myxomatous atrioventricular valve leaﬂets are difﬁcult to reconcile with a pathogenetic model that singularly invokes tissue weakness. Mitral valves in mice homozygous for a Fbn1 mutation are longer and thicker than those in heterozygous mice, and both are longer and thicker than those in wild-type mice (33). These ﬁndings correlate with increased TGFβ activation and signaling, as well as augmented proliferation of cells within these valves, diminished apoptosis, and increased production of various targets of TGFβ activation (33). Once again, treatment of these mice with a neutralizing antibody to TGFβ improved atrioventricular valve length and thickness. The major life-threatening manifestation of MFS is aortic aneurysm with eventual dissection. Traditionally, aortic aneurysm has been attributed to a putative structural deﬁciency resulting from ﬁbrillin-1 mutation. Recently, a regulatory role for ﬁbrillin-1 has also been recognized as relevant in the aorta (34). Increased staining for markers of TGFβ signaling is present in heterozygous mutant mice with aortic aneurysm, as well as aortic wall from individuals with MFS (15). Neutralizing antibodies to TGFβ reduce aortic size and improve aortic wall architecture (34). However, therapeutic delivery of TGFβ neutralizing antibody is not currently available for people with MFS. Extensive evidence links angiotensin II signaling to TGFβ activation and signaling. Upon stimulation of the TGFβ receptors, a family of transcription factors called Smads is activated by sequential phosphorylation (35). Direct activation of the Smad pathway by angiotensin II, independent of TGFβ ligands, was reported in vascular smooth muscle cells (36). In addition, reports on therapy with angiotensin-converting enzyme inhibitors (ACE inhibitors) and angiotensin II receptor blockers (ARBs) in renal disorders mediated by excessive TGFβ further support the likelihood that either of these medications would ameliorate other disorders associated with excessive TGFβ (37, 38).

The two distinct receptors for angiotensin II, type 1 (AT1) and type 2 (AT2), appear to have opposing effects. For instance, Daugherty and colleagues reported that infusion of angiotensin II in apoE−/− mice results in abdominal aortic aneurysm (39). Subsequently, they assessed the relative contribution of AT1 and AT2 stimulation with use of selective antagonists (40). Aortic aneurysm was prevented in these mice with simultaneous infusion of angiotensin II and losartan, an AT1 antagonist, but worsened in both frequency and severity in mice that received angiotensin II and PD123319, a selective AT2 antagonist (40). Because losartan is speciﬁc for blocking the AT1 receptor, its use would not prevent stimulation of the AT2 receptor by angiotensin II, which appears to be beneﬁcial in aortic aneurysms. On the other hand, treatment with an ACE inhibitor should decrease angiotensin II stimulation of both the culprit AT1 and the potentially protective AT2 receptors. We tested the ability of losartan to delay or prevent ascending aortic aneurysm in a murine model of MFS (34). Treatment was initiated after moderate proximal aortic enlargement and compared with propranolol, a beta-adrenergic receptor antagonist (beta blocker), in doses that had similar hemodynamic effects. Mice treated with propranolol showed a relative reduction in aortic root growth rate compared to those receiving no treatment, but pathologic aortic root growth was completely prevented in these mice by treatment with losartan (34). Furthermore, losartan normalized aortic wall thickness and architecture, ﬁndings not observed in the propranolol-treated group. At the end of six months of treatment, the aortic wall of losartan-treated ﬁbrillin-1-deﬁcient mice could not be distinguished from that of wildtype mice, even though the mice already had established aortic enlargement when therapy was initiated (34) (Figure 1). These data suggest the potential for productive aortic wall remodeling after TGFβ inhibition. Remarkably, improvements in pulmonary and www.annualreviews.org • Therapy of Marfan Syndrome

ACE: angiotensinconverting enzyme ARB: angiotensin II receptor blocker AT1: angiotensin II, type 1 receptor AT2: angiotensin II, type 2 receptor

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Figure 1 Losartan prevents aortic aneurysm in mice. Representative mouse ascending aortas (arrowheads) are shown after latex injection. (A) Wild-type mouse. (B–F ) Mice heterozygous for Fbn1 mutation (C1039G), treated with placebo (B ), propranolol (C ), or losartan (D–F ). Scale bars: 4 mm. From Science (34), with permission.

skeletal muscle pathology also occurred with losartan treatment, further supporting the conclusion that this treatment works by decreasing TGFβ signaling rather than by reducing hemodynamic stress on structurally predisposed tissues (34, 41).

tions, (b) current pharmacologic approaches, (c) surgeries, (d ) endocarditis prophylaxis, and (e) emerging or experimental strategies. Most therapies that are currently available or under investigation seek to diminish aortic complications, such as aneurysm and dissection.

CURRENT AND FUTURE THERAPIES

Activity Restrictions

For the purpose of this review, therapies are categorized as follows: (a) activity restric50

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Because of the tall stature associated with dolichostenomelia (long bone overgrowth) in MFS, many affected individuals are drawn to

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athletic activities such as volleyball and basketball. However, strenuous physical exertion, competitive athletics, and particularly isometric activities are associated with increased risk of aortic dissection, ocular problems, and skeletal complications for those with MFS. Consensus guidelines have been developed for activity limitations (42). It is our practice to advise against isometric exercise such as weight lifting, and to recommend moderation in aerobic exertion. A more detailed review on exercise and MFS is available (43).

Current Pharmacologic Approaches Despite the limitations of the data supporting their use, beta blockers are widely considered the standard of care to slow the rate of enlargement of aortic aneurysm in people with MFS. This treatment was ﬁrst proposed in 1971 (44). Its rationale relies on the putative role of hemodynamic stress in the progressive enlargement of the proximal aorta. Beta blockers are probably beneﬁcial through both negative inotropic and chronotropic effects. Several small or retrospective studies have demonstrated the beneﬁt of beta blockers in slowing aortic root growth in MFS (45–48). Only one prospectively randomized trial has been reported to date assessing the efﬁcacy of beta blockers in MFS (47). Subjects included 70 individuals with a clinical diagnosis of MFS based on the Berlin criteria, which was the standard in 1994 when the study was published (4). In an open-label fashion, enrolled individuals were prospectively randomized to receive either no pharmacologic treatment (control) or propranolol (47). Importantly, the dose of propranolol was individualized for each treated case by targeting for a heart rate below 100 beats per minute during exercise or resulting in a 30% increase in the systolic time interval (corrected for the heart rate). This led to an average propranolol dose of 212 ± 68 mg per day. Fewer people who were treated with propranolol reached the primary clinical endpoints of aortic regurgita-

tion, aortic dissection, cardiovascular surgery, congestive heart failure, and death (ﬁve in treatment group, nine in control group). The rate of aortic enlargement was lower in the propanolol group than in the control group (47). Variation in the method of prescribing beta blockers for MFS is associated with disparate results. A recent meta-analysis reviewed 17 clinical trials and excluded 11 of these from the analysis because of incomplete or redundant information or absence of a control group (49). Of the remaining six trials, heart rate response to exercise was tested in only two, and only one was prospective (45, 47). The authors conclude that there is insufﬁcient evidence from these trials to support routine use of beta blockers in MFS. New studies are needed to prospectively address the utility of beta blockers (49). Side effects of beta blockers are common and are typically more evident at higher doses (50, 51). Use of cardiac-speciﬁc beta blockers may improve their tolerability, but some individuals do not tolerate any medications with this class of action. For such patients, limited clinical evidence supports use of verapamil, a calcium channel blocker, in an attempt to reduce cardiac inotropy and chronotropy and thereby reduce proximal aortic shear stress. One report describes 26 individuals with MFS who received pharmacologic treatment for aortic aneurysm (46). Of these, ﬁve were using verapamil. The rate of aortic growth was greater among 18 people who did not receive pharmacologic treatment than in the 26 who did (46). The subgroup receiving verapamil was too small to deﬁnitively establish that this therapy provided beneﬁt, and no prospectively randomized assessment of this strategy has been reported. Another strategy to delay pathologic aortic root growth involves use of ACE inhibitors. One trial compared 58 individuals with MFS who received either an ACE inhibitor (enalapril) or a beta blocker (propranolol or atenolol), reporting that those treated with enalapril had improved aortic www.annualreviews.org • Therapy of Marfan Syndrome

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distensibility, reduced aortic stiffness, and smaller aortic root diameter (52). It remains unknown whether increased aortic stiffness contributes to the pathogenesis of aneurysm and dissection. An important limitation of this nonrandomized trial was that beta blocker dosage was not titrated to physiologic response but rather was capped at 2 mg/kg once daily of atenolol or 1 mg/kg twice daily of propranolol (52). Moreover, the vast majority of patients who had previously been diagnosed with Marfan syndrome and were on beta blockers remained on beta blockers, whereas the bulk of “new patients,” presumably with a less well-deﬁned natural history of disease, chose to receive enalapril (52). Forty percent of new patients who originally chose to receive beta blockers subsequently switched to the enalapril group owing to subjective measures of intolerance (52). This practice might impose bias, as patients with a greater perception of risk may have greater motivation to continue standard therapy. At this point, the use of ACE inhibitors to slow pathologic aortic growth in MFS shares many of the uncertainties of other more established therapies. As discussed above, experimental evidence regarding other forms of aortic aneurysm suggests that signaling through the AT2 receptor is protective whereas signaling through the AT1 receptor contributes to pathogenesis. If relevant to MFS, these data suggest that reducing the production of angiotensin II, as achieved with ACE inhibitors, would not be optimal because the protective (AT2) pathway would also be blocked. Furthermore, ACE inhibitors would not address alternative tissue pathways for the production of angiotensin II, such as through the activity of chymase (53). Selective AT1 blockade would seem to be a better choice. (This concept is developed more completely in the subsection on emerging therapies for MFS.) Finally, it should be noted that many patients with MFS have been treated with ACE inhibitors for valve dysfunction. This usage has not been systematically assessed, but no pronounced effect on aortic root growth has been apparent.

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Surgery for Aortic Aneurysm Successful surgical repair of aortic root aneurysms may be the single most important cause of the improvement in life expectancy for individuals with MFS. Although the prognosis is highly variable, a 1972 publication stated that life expectancy for affected individuals was about two thirds that of unaffected people, with life-table mortality curves deviating in infancy (54). In that analysis, most of the deaths had a cardiovascular cause, including aortic dissection, heart failure, or cardiac valve disease. More recently, the average life expectancy for people with MFS was reported to be nearly normal, with a signiﬁcant increase in survival for those who underwent aortic root surgery after 1980 (55). In 1968, the surgical approach to aortic root aneurysm was revolutionized by publication of the ﬁrst description of replacement of the aortic root with a composite valve graft and direct coronary reanastomosis (56). Over the past 20 years, several modiﬁcations of this technique have been described. Some alternative methods have addressed different ways to construct the anastomosis of the coronary arteries (57, 58). A strategy to replace the aortic root with a pulmonary root autograft (Ross procedure) is not recommended owing to the possibility of progressive enlargement of the pulmonary root when it is exposed to systemic pressure, and eventual autograft failure, particularly in patients with a systemic connective tissue disorder (59, 60). Consistent with this concern, many patients with MFS show pulmonary artery dilation, although pulmonary pressures are lower than systemic pressures. It is now well established that aortic root surgery should be recommended for individuals with an aortic root diameter 5.0 cm or greater (61). Additional factors that may inﬂuence an earlier recommendation for aortic root surgery are a rapid rate of enlargement (>0.5–1.0 cm/y) or a family history of early aortic dissection. Gott and colleagues have demonstrated marked reduction in mortality

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for aortic root surgery if it is undertaken on an elective basis, rather than on an urgent or emergent basis (61). Preserving the native aortic valve at the time of aortic root repair is naturally very desirable because the need for lifelong anticoagulation with warfarin may be avoided (62, 63). The long-term durability of the native aortic valve in the setting of MFS remains somewhat uncertain. Several promising series have been reported (64–66). However, concerns include valvular insufﬁciency, late annular dilatation, and the possible need for reoperation to replace the valve (67). An ongoing registry by the National Marfan Foundation is currently enrolling individuals with MFS who receive aortic root surgery, comparing outcomes between those with “classic” aortic root and valve replacement to those with valve-sparing aortic root replacement. Until longer-term results of this operation are known, short- and medium-term results guide the clinical decision between composite graft and valve-sparing aortic root surgeries.

Endocarditis Prophylaxis Individuals with MFS often have multiple abnormal cardiac valves. Abnormalities include myxomatous thickening with prolapse and regurgitation of the mitral and tricuspid valves, as well as dilatation of the aortic and pulmonic roots, with insufﬁciency of these valve leaﬂets. Accordingly, these individuals are at greater risk of bacterial endocarditis than those without structural heart disease. Antibiotic prophylaxis was recommended to diminish the likelihood of infective endocarditis (IE) in this setting (68). Recently, the American Heart Association modiﬁed its recommendations for the use of prophylactic antibiotics during procedures that are associated with bacteremia, such as dental, gastrointestinal, or genitourinary interventions (69). In the absence of large randomized prospective placebo-controlled trials demonstrating efﬁcacy of antibiotic prophylaxis to prevent IE, these revised guidelines

recommend antibiotics only in patients considered to be at highest risk, such as those with prosthetic cardiac valves or with a history of IE, certain forms of congenital heart disease, or cardiac transplantation with subsequent cardiac valve disease (69). These conditions also pose the highest risk for adverse outcomes in the setting of IE. The pathogenesis of bacterial endocarditis is thought to rely on bloodstream infection, intracardiac turbulence, and abnormal endocardial surface of the affected valve (70). We consider individuals with MFS and myxomatous mitral or tricuspid valve disease with audible regurgitation not only to be at high risk of developing IE with dental, gastrointestinal, and genitourinary procedures, but also to be among those at highest risk for adverse outcome if they were to develop IE. In the absence of clinical trials performed speciﬁcally to address this question in this cohort of patients, we advise continued use of prophylactic antibiotics among individuals with MFS who have prosthetic cardiac valves, valve-sparing aortic root surgery, or myxomatous valve disease.

Emerging Therapies Several important insights have arisen from murine models of MFS, leading to therapies that are either currently being tested or are anticipated in the near future. Foremost among these is the strategy to modulate TGFβ activity with ARBs such as losartan that selectively block AT1 (34). ARBs have been shown to reduce the level of expression of TGFβ ligands and their receptors and to limit the production of activators of TGFβ such as thrombospondin-1 (TSP-1) (71). Increased TSP-1 expression has been identiﬁed in diseased tissues in mouse models of MFS, and this is attenuated or eliminated after treatment with losartan (41). Interestingly, TSP-1 appears to be a minor physiologic activator of TGFβ, as evidenced by the subtlety of phenotypes suggestive of TGFβ deﬁciency in Tsp1 knockout mice (72). In contrast, TSP-1 may www.annualreviews.org • Therapy of Marfan Syndrome

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be a major pathologic activator of TGFβ in MFS. If so, TSP-1 may prove to be an ideal therapeutic target because antagonists would have a high therapeutic index. Both TSP-1 antagonists and AT2 agonists are currently being tested in mouse models of MFS. Enthusiasm regarding the possibility of completely halting pathologic aortic root growth in affected individuals, as occurred in treated mice with a similar genetic predisposition to aortic aneurysm, is tempered by the lack of clinical trial data. In a clinical trial coordinated by the Pediatric Heart Network of the National Heart, Lung and Blood Institute of the National Institutes of Health, 19 sites within the United States, Canada, and Belgium are enrolling individuals with MFS, aged six months to 25 years, with aortic root Z-score >3.0. Enrolled subjects will receive either a beta blocker (atenolol) or ARB (losartan) for a period of three years. The primary outcome will be the rate of change in normalized aortic root size. Secondary outcomes will include size and rate of change in other segments of aorta; time to aortic dissection, surgery, or death; and measures of cardiac size and aortic stiffness. Several studies have shown the ability of antibodies that antagonize TGFβ to modulate manifestations of MFS in mice, including defective pulmonary alveolar septation, myxomatous atrioventricular valves, skeletal muscle myopathy, and aortic root aneurysms (32–34, 41). A humanized anti-TGFβ1 monoclonal antibody (CAT-192) has been produced and is currently under investigation for treatment of other disorders in which this cytokine has been determined to play a pathogenetic

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role (73). Direct inhibition of TGFβ with neutralizing antibodies remains a promising strategy to ameliorate many manifestations of MFS. Insights derived from the study of MFS may prove relevant to other conditions. The ﬁnding that other inherited arteriopathies (LDS, arterial tortuosity syndrome, and autosomal recessive cutis laxa) also relate to excessive TGFβ signaling (15, 74, 75) suggests that this may be a common ﬁnal pathway for aneurysm formation, and that ARBs may prove effective regardless of the underlying reason for aneurysm. There is already evidence derived from a mouse model of Duchenne muscular dystrophy that losartanmediated restoration of muscle regeneration, architecture, and function (as initially observed in MFS) may develop into a productive treatment strategy (41).

CONCLUSIONS Successful treatment of MFS relies on several important steps: ﬁrst, recognition of the disorder with anticipation of potential complications; second, early intervention with activity restrictions and pharmacologic therapies; and third, surgical intervention when appropriate. Investigation of the pathophysiologic basis for many aspects of MFS in murine models has led to remarkable insights. We anticipate that translation of these research discoveries to clinical trials will demonstrate effectiveness of a pharmacologic approach in which target selection is based on our improved understanding of the molecular basis for pathologic features of this condition.

SUMMARY POINTS 1. The diagnosis of Marfan syndrome is made through clinical criteria (Table 1). It is important to exclude the diagnosis of Loeys-Dietz syndrome (Table 2). 2. Many manifestations of Marfan syndrome, including aortic aneurysm, are caused by increased activation of and signaling by transforming growth factor beta (TGFβ).

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3. For individuals with Marfan syndrome, we advise moderation in aerobic activity and avoidance of isometric exercise. 4. Beta-adrenergic receptor blockers appear to slow pathologic aortic root growth, but the data supporting their use is limited.

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5. Elective repair of aortic root aneurysm is recommended with aortic root diameter of 5 cm or greater in adults with Marfan syndrome. Guidelines for surgery in children are less precise and require a more individualized approach. 6. Because of high lifetime risk of infective endocarditis, as well as high risk for adverse outcomes in the setting of endocarditis, we continue to advise patients with Marfan syndrome and other connective tissue disorders who have myxomatous valves or valvesparing aortic root surgery to use antibiotic prophylaxis for dental, gastrointestinal, and genitourinary procedures. 7. Angiotensin II type 1 (AT1) receptor blockers such as losartan prevent pathologic aortic root growth, normalize aortic wall architecture and thickness, and improve noncardiovascular manifestations such as pulmonary and skeletal muscle pathology in a murine model of Marfan syndrome. Clinical trials are under way to test the efﬁcacy of this treatment in people with Marfan syndrome.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS The authors acknowledge the William S. Smilow Center for Marfan Syndrome Research, the Dana and Albert “Cubby” Broccoli Center for Aortic Diseases, and the National Marfan Foundation.

LITERATURE CITED 1. Marfan A-B. 1896. Un cas de deformation congenitale des quarte membres plus prononc´ee aux extremit´es caracteris´ee par l’allongement des os avec un certain degr`e d’amincissement. Bull. Mem. Soc. Med. Hop. Paris 13:220–26 2. Marfan A-B. 1938. La dolichost´enom´elie [dolichom´elie arachnodactylie]. Ann. Med. 44:5– 29 3. McKusick VA. 1955. The cardiovascular aspects of Marfan’s syndrome. Circulation 11:321 4. Beighton P, de Paepe A, Danks D, et al. 1988. International Nosology of Heritable Disorders of Connective Tissue, Berlin, 1986. Am. J. Med. Genet. 29:581–94 5. Dietz HC, Pyeritz RE, Hall BD, et al. 1991. The Marfan syndrome locus: conﬁrmation of assignment to chromosome 15 and identiﬁcation of tightly linked markers at 15q15–q21.3. Genomics 9:355–61 6. Dietz HC, Cutting GR, Pyeritz RE, et al. 1991. Marfan syndrome caused by a recurrent de novo missense mutation in the ﬁbrillin gene. Nature 352:337–39 www.annualreviews.org • Therapy of Marfan Syndrome

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7. Pereira L, Levran O, Ramirez F, et al. 1994. A molecular approach to the stratiﬁcation of cardiovascular risk in families with Marfan’s syndrome. N. Engl. J. Med. 331:148–53 8. De Paepe A, Devereux RB, Dietz HC, et al. 1996. Revised diagnostic criteria for the Marfan syndrome. Am. J. Med. Genet. 62:417–26 9. Loeys B, De Backer J, Van Acker P, et al. 2004. Comprehensive molecular screening of the FBN1 gene favors locus homogeneity of classical Marfan syndrome. Hum. Mutat. 24:140–46 10. Glesby MJ, Pyeritz RE. 1989. Association of mitral valve prolapse and systemic abnormalities of connective tissue. A phenotypic continuum. JAMA 262:523–28 11. Montgomery RA, Geraghty MT, Bull E, et al. 1998. Multiple molecular mechanisms underlying subdiagnostic variants of Marfan syndrome. Am. J. Hum. Genet. 63:1703–11 12. Kainulainen K, Karttunen L, Puhakka L, et al. 1994. Mutations in the ﬁbrillin gene responsible for dominant ectopia lentis and neonatal Marfan syndrome. Nat. Genet. 6:64–69 13. Faivre L, Gorlin RJ, Wirtz MK, et al. 2003. In frame ﬁbrillin-1 gene deletion in autosomal dominant Weill-Marchesani syndrome. J. Med. Genet. 40:34–36 14. Sood S, Eldadah ZA, Krause WL, et al. 1996. Mutation in ﬁbrillin-1 and the Marfanoidcraniosynostosis (Shprintzen-Goldberg) syndrome. Nat. Genet. 12:209–11 15. Loeys BL, Chen J, Neptune ER, et al. 2005. A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2. Nat. Genet. 37:275–81 16. Loeys BL, Schwarze U, Holm T, et al. 2006. Aneurysm syndromes caused by mutations in the TGF-beta receptor. N. Engl. J. Med. 355:788–98 17. Halme T, Savunen T, Aho H, et al. 1985. Elastin and collagen in the aortic wall: changes in the Marfan syndrome and annuloaortic ectasia. Exp. Mol. Pathol. 43:1–12 18. Tsuji T. 1986. Marfan syndrome: demonstration of abnormal elastic ﬁbers in skin. J. Cutan. Pathol. 13:144–53 19. Kainulainen K, Pulkkinen L, Savolainen A, et al. 1990. Location on chromosome 15 of the gene defect causing Marfan syndrome. N. Engl. J. Med. 323:935–39 20. Sakai LY, Keene DR, Engvall E. 1986. Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microﬁbrils. J. Cell. Biol. 103:2499–509 21. Hollister DW, Godfrey M, Sakai LY, et al. 1990. Immunohistologic abnormalities of the microﬁbrillar-ﬁber system in the Marfan syndrome. N. Engl. J. Med. 323:152–59 22. Kielty CM, Shuttleworth CA. 1995. Fibrillin-containing microﬁbrils: structure and function in health and disease. Int. J. Biochem. Cell Biol. 27:747–60 23. Dietz HC, McIntosh I, Sakai LY, et al. 1993. Four novel FBN1 mutations: signiﬁcance for mutant transcript level and EGF-like domain calcium binding in the pathogenesis of Marfan syndrome. Genomics 17:468–75 24. Schrijver I, Liu W, Odom R, et al. 2002. Premature termination mutations in FBN1: distinct effects on differential allelic expression and on protein and clinical phenotypes. Am. J. Hum. Genet. 71:223–37 25. Frischmeyer PA, Dietz HC. 1999. Nonsense-mediated mRNA decay in health and disease. Hum. Mol. Genet. 8:1893–900 26. Montgomery RA, Dietz HC. 1997. Inhibition of ﬁbrillin 1 expression using U1 snRNA as a vehicle for the presentation of antisense targeting sequence. Hum. Mol. Genet. 6:519–25 27. Judge DP, Biery NJ, Keene DR, et al. 2004. Evidence for a critical contribution of haploinsufﬁciency in the complex pathogenesis of Marfan syndrome. J. Clin. Invest. 114:172–81 28. Pereira L, Andrikopoulos K, Tian J, et al. 1997. Targeting of the gene encoding ﬁbrillin-1 recapitulates the vascular aspect of Marfan syndrome. Nat. Genet. 17:218–22

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29. Hutchinson S, Furger A, Halliday D, et al. 2003. Allelic variation in normal human FBN1 expression in a family with Marfan syndrome: a potential modiﬁer of phenotype? Hum. Mol. Genet. 12:2269–76 30. Sinha S, Nevett C, Shuttleworth CA, et al. 1998. Cellular and extracellular biology of the latent transforming growth factor-beta binding proteins. Matrix Biol. 17:529–45 31. Isogai Z, Ono RN, Ushiro S, et al. 2003. Latent transforming growth factor beta-binding protein 1 interacts with ﬁbrillin and is a microﬁbril-associated protein. J. Biol. Chem. 278:2750–57 32. Neptune ER, Frischmeyer PA, Arking DE, et al. 2003. Dysregulation of TGF-beta activation contributes to pathogenesis in Marfan syndrome. Nat. Genet. 33:407–11 33. Ng CM, Cheng A, Myers LA, et al. 2004. TGF-beta-dependent pathogenesis of mitral valve prolapse in a mouse model of Marfan syndrome. J. Clin. Invest. 114:1586–92 34. Habashi JP, Judge DP, Holm TM, et al. 2006. Losartan, an AT1 antagonist, prevents aortic aneurysm in a mouse model of Marfan syndrome. Science 312:117–21 35. Massague J, Seoane J, Wotton D. 2005. Smad transcription factors. Genes. Dev. 19:2783– 810 36. Rodriguez-Vita J, Sanchez-Lopez E, Esteban V, et al. 2005. Angiotensin II activates the Smad pathway in vascular smooth muscle cells by a transforming growth factor-betaindependent mechanism. Circulation 111:2509–17 37. Lavoie P, Robitaille G, Agharazii M, et al. 2005. Neutralization of transforming growth factor-beta attenuates hypertension and prevents renal injury in uremic rats. J. Hypertens. 23:1895–903 38. Houlihan CA, Akdeniz A, Tsalamandris C, et al. 2002. Urinary transforming growth factor-beta excretion in patients with hypertension, type 2 diabetes, and elevated albumin excretion rate: effects of angiotensin receptor blockade and sodium restriction. Diab. Care 25:1072–77 39. Daugherty A, Manning MW, Cassis LA. 2000. Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deﬁcient mice. J. Clin. Invest. 105:1605–12 40. Daugherty A, Manning MW, Cassis LA. 2001. Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis. Br. J. Pharmacol. 134:865–70 41. Cohn RD, van Erp C, Habashi JP, et al. 2007. Angiotensin II type 1 receptor blockade attenuates TGF-beta-induced failure of muscle regeneration in multiple myopathic states. Nat. Med. 13:204–10 42. Maron BJ, Chaitman BR, Ackerman MJ, et al. 2004. Recommendations for physical activity and recreational sports participation for young patients with genetic cardiovascular diseases. Circulation 109:2807–16 43. Braverman AC. 1998. Exercise and the Marfan syndrome. Med. Sci. Sports Exerc. 30:S387– 95 44. Halpern BL, Char F, Murdoch JL, et al. 1971. A prospectus on the prevention of aortic rupture in the Marfan syndrome with data on survivorship without treatment. Johns Hopkins Med. J. 129:123–29 45. Salim MA, Alpert BS, Ward JC, et al. 1994. Effect of beta-adrenergic blockade on aortic root rate of dilation in the Marfan syndrome. Am. J. Cardiol. 74:629–33 46. Rossi-Foulkes R, Roman MJ, Rosen SE, et al. 1999. Phenotypic features and impact of beta blocker or calcium antagonist therapy on aortic lumen size in the Marfan syndrome. Am. J. Cardiol. 83:1364–68 www.annualreviews.org • Therapy of Marfan Syndrome

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47. Shores J, Berger KR, Murphy EA, et al. 1994. Progression of aortic dilatation and the beneﬁt of long-term beta-adrenergic blockade in Marfan’s syndrome. N. Engl. J. Med. 330:1335–41 48. Ladouceur M, Fermanian C, Lupoglazoff J-M, et al. 2007. Effect of beta-blockade on ascending aortic dilatation in children with the Marfan syndrome. Am. J. Cardiol. 99:406– 9 49. Gersony DR, McClaughlin MA, Jin Z, et al. 2007. The effect of beta-blocker therapy on clinical outcome in patients with Marfan’s syndrome: a meta-analysis. Int. J. Cardiol. 114:303–8 50. Dahlof C, Dimenas E, Kendall M, et al. 1991. Quality of life in cardiovascular diseases. Emphasis on beta-blocker treatment. Circulation 84:VI108–18 51. Gleiter CH, Deckert J. 1996. Adverse CNS-effects of beta-adrenoceptor blockers. Pharmacopsychiatry 29:201–11 52. Yetman AT, Bornemeier RA, McCrindle BW. 2005. Usefulness of enalapril versus propranolol or atenolol for prevention of aortic dilation in patients with the Marfan syndrome. Am. J. Cardiol. 95:1125–27 53. Takai S, Jin D, Sakaguchi M, et al. 1999. Chymase-dependent angiotensin II formation in human vascular tissue. Circulation 100:654–58 54. Murdoch JL, Walker BA, Halpern BL, et al. 1972. Life expectancy and causes of death in the Marfan syndrome. N. Engl. J. Med. 286:804–8 55. Silverman DI, Burton KJ, Gray J, et al. 1995. Life expectancy in the Marfan syndrome. Am. J. Cardiol. 75:157–60 56. Bentall H, De Bono A. 1968. A technique for complete replacement of the ascending aorta. Thorax 23:338–39 57. Cabrol C, Pavie A, Gandjbakhch I, et al. 1981. Complete replacement of the ascending aorta with reimplantation of the coronary arteries: new surgical approach. J. Thorac. Cardiovasc. Surg. 81:309–15 58. Svensson LG, Crawford ES, Hess KR, et al. 1992. Composite valve graft replacement of the proximal aorta: comparison of techniques in 348 patients. Ann. Thorac. Surg. 54:427–37; discussion 438–39 59. Ross DN. 1967. Replacement of aortic and mitral valves with a pulmonary autograft. Lancet 290:956–58 60. Luciani GB, Casali G, Favaro A, et al. 2003. Fate of the aortic root late after Ross operation. Circulation 108:II-61–67 61. Gott VL, Greene PS, Alejo DE, et al. 1999. Replacement of the aortic root in patients with Marfan’s syndrome. N. Engl. J. Med. 340:1307–13 62. Sarsam MA, Yacoub M. 1993. Remodeling of the aortic valve anulus. J. Thorac. Cardiovasc. Surg. 105:435–38 63. David TE, Feindel CM. 1992. An aortic valve-sparing operation for patients with aortic incompetence and aneurysm of the ascending aorta. J. Thorac. Cardiovasc. Surg. 103:617– 21; discussion 22 64. Miller DC. 2007. Valve-sparing aortic root replacement: current state of the art and where are we headed? Ann. Thorac. Surg. 83:S736–39; discussion S85–90 65. Patel ND, Williams JA, Barreiro CJ, et al. 2006. Valve-sparing aortic root replacement: early experience with the De Paulis Valsalva graft in 51 patients. Ann. Thorac. Surg. 82:548– 53 66. David TE, Feindel CM, Webb GD, et al. 2006. Long-term results of aortic valve-sparing operations for aortic root aneurysm. J. Thorac. Cardiovasc. Surg. 132:347–54

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67. Bethea BT, Fitton TP, Alejo DE, et al. 2004. Results of aortic valve-sparing operations: experience with remodeling and reimplantation procedures in 65 patients. Ann. Thorac. Surg. 78:767–72 68. Dajani AS, Taubert KA, Wilson W, et al. 1997. Prevention of bacterial endocarditis. Recommendations by the American Heart Association. JAMA 277:1794–801 69. Wilson W, Taubert KA, Gewitz M, et al. 2007. Prevention of infective endocarditis. Guidelines from the American Heart Association. Circulation. Epub ahead of print 70. Thiene G, Basso C. 2006. Pathology and pathogenesis of infective endocarditis in native heart valves. Cardiovasc. Pathol. 15:256–63 71. Naito T, Masaki T, Nikolic-Paterson DJ, et al. 2004. Angiotensin II induces thrombospondin-1 production in human mesangial cells via p38 MAPK and JNK: a mechanism for activation of latent TGF-beta1. Am. J. Physiol. Renal Physiol. 286:F278–87 72. Lawler J, Sunday M, Thibert V, et al. 1998. Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia. J. Clin. Invest. 101:982– 92 73. Denton CP, Merkel PA, Furst DE, et al. 2007. Recombinant human antitransforming growth factor beta1 antibody therapy in systemic sclerosis: a multicenter, randomized, placebo-controlled phase I/II trial of CAT-192. Arthritis Rheum. 56:323–33 74. Coucke PJ, Willaert A, Wessels MW, et al. 2006. Mutations in the facilitative glucose transporter GLUT10 alter angiogenesis and cause arterial tortuosity syndrome. Nat. Genet. 38:452–57 75. Hanada K, Vermeij M, Garinis GA, et al. 2007. Perturbations of vascular homeostasis and aortic valve abnormalities in ﬁbulin-4 deﬁcient mice. Circ. Res. 100:738–46

RELATED RESOURCES National Marfan Foundation, http://www.marfan.org, 22 Manhasset Avenue, Port Washington, NY 11050; 1-800-862-7326

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237

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Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

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Preeclampsia and Angiogenic Imbalance Sharon Maynard,1 Franklin H. Epstein,2 and S. Ananth Karumanchi2 1

Renal Division, Department of Medicine, George Washington University School of Medicine; 2 Departments of Medicine, Obstetrics and Gynecology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215; email: [email protected]

Annu. Rev. Med. 2008. 59:61–78

Key Words

First published online as a Review in Advance on October 15, 2007

hypertension in pregnancy, VEGF, sFlt1, endothelial dysfunction

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.110106.214058 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0061$20.00

Abstract Preeclampsia is a systemic syndrome of pregnancy that originates in the placenta and is characterized by widespread maternal endothelial dysfunction. Until recently, the molecular pathogenesis of preeclampsia was largely unknown, but recent work suggests a key role for altered expression of placental antiangiogenic factors. Soluble Flt1 and soluble endoglin, secreted by the placenta, are increased in the maternal circulation weeks before the onset of preeclampsia. These antiangiogenic factors produce systemic endothelial dysfunction, resulting in hypertension, proteinuria, and the other systemic manifestations of preeclampsia. The molecular basis for placental dysregulation of these pathogenic factors remains unknown, and the role of angiogenic proteins in early placental vascular development is just beginning to be explored. These discoveries have exciting clinical implications and are likely to transform the detection and treatment of preeclampsia in the future.

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INTRODUCTION

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Preeclampsia is a systemic syndrome that occurs in pregnant women. It is characterized by the new onset of hypertension and proteinuria usually after 20 weeks gestation. Preeclampsia affects ∼3%–5% of pregnancies worldwide (1). Despite many advances in our understanding of the pathophysiology of preeclampsia, delivery of the placenta remains the only deﬁnitive treatment. Preeclampsia is a leading cause of preterm birth and consequent neonatal morbidity and mortality in the developed world. In developing countries, where access to safe, emergent delivery is less readily available, preeclampsia claims the lives of >60,000 mothers every year (1). This article describes recent discoveries concerning the pathogenesis of preeclampsia, with emphasis on the emerging role of angiogenic factors as potential mediators of the clinical signs and symptoms of preeclampsia. This work clariﬁes the pathogenesis of this enigmatic disease and holds promise for the prediction, prevention, and treatment of preeclampsia.

centa is a product of both mother and father (5). Several large genome-wide scans seeking a speciﬁc linkage to preeclampsia have been fairly discordant and disappointing, with signiﬁcant LOD scores in isolated Finnish (2p25, 9p13) (6) and Icelandic (2p12) (7) populations. Speciﬁc genetic mutations consistent with these loci have not been identiﬁed. Several medical conditions are associated with increased preeclampsia risk, including chronic hypertension, diabetes mellitus, renal disease, obesity, and hypercoagulable states. Women with preeclampsia in a prior pregnancy have a high risk of preeclampsia in subsequent pregnancies. Conditions associated with increased placental mass, such as multifetal gestations and hyatidiform mole, also are associated with increased preeclampsia risk. Interestingly, smoking during pregnancy is thought to reduce the risk of preeclampsia (8). Although none of these risk factors is fully understood, they have provided insights into pathogenesis.

CLINICAL FEATURES EPIDEMIOLOGY AND RISK FACTORS Most cases of preeclampsia occur in healthy, nulliparous women, in whom the incidence of preeclampsia may be as high as 7.5%. Although it is classically a disorder of ﬁrst pregnancies, multiparous women who are pregnant with a new partner are said to have an elevated preeclampsia risk similar to that of nulliparous women (2). This effect may be due to increased interpregnancy interval rather than the change in paternity per se (3). Although most cases of preeclampsia occur in the absence of a family history, the presence of preeclampsia in a ﬁrst-degree relative increases a woman’s risk of severe preeclampsia two- to fourfold (4), suggesting a genetic contribution to the disease. A history of preeclampsia in the father’s mother also confers an increased risk, recalling that the pla62

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The new onset of hypertension and proteinuria are the cardinal features of preeclampsia. The severity of hypertension in preeclampsia can vary widely, from mild blood pressure elevations easily managed with bed rest, to severe hypertension resistant to multiple medications, often associated with headache and visual changes. The degree of proteinuria in preeclampsia also varies, from minimal to nephrotic range. Occasionally, gestational hypertension without proteinuria is associated with features of severe preeclampsia, such as alterations in liver function, hemolysis, or seizures (9). Although edema was historically part of the diagnostic triad for preeclampsia, it is also a common feature of normal pregnancy, diminishing its usefulness as a speciﬁc pathological sign. Still, the sudden onset of severe edema–especially edema of the hands and face–can be an important symptom in this

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otherwise insidious disease and is often the only change detectable by the patient. Serum uric acid is elevated in preeclampsia primarily as a result of enhanced tubular urate reabsorption. Hyperuricemia may contribute to the pathogenesis of preeclampsia by inducing endothelial dysfunction (10). Serum uric acid levels are correlated with the presence and severity of preeclampsia and with adverse pregnancy outcomes (11), even in gestational hypertension without proteinuria (12). Nevertheless, uric acid is of limited clinical utility in either distinguishing preeclampsia from other hypertensive disorders of pregnancy or predicting adverse outcomes (13). Uncommon but serious complications of preeclampsia include acute renal failure, seizures, pulmonary edema, acute liver injury, hemolysis, and/or thrombocytopenia. The latter three signs frequently occur together as part of the HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome. The HELLP syndrome is considered a severe variant of preeclampsia and is associated with a higher risk of maternal and neonatal adverse outcomes than preeclampsia alone. Seizures (eclampsia) complicate ∼2% of preeclampsia cases in the United States. Although eclampsia most often occurs in the setting of hypertension and proteinuria, it can occur without these warning signs. Up to one third of eclampsia cases occur postpartum, sometimes days to weeks after delivery (14). Complications affecting the developing fetus and the neonate include iatrogenic prematurity (and its associated sequelae), fetal growth restriction, oligohydramnios, and placental abruption. Although the exact pathogenesis of these complications is unknown, impaired uteroplacental blood ﬂow or placental infarction are likely to contribute.

Maternal and Neonatal Mortality Approximately 500,000 women die in childbirth each year worldwide, and preeclampsia/eclampsia is estimated to account for 10%–15% of these deaths. Maternal death is

most often due to eclampsia, cerebral hemorrhage, renal failure, hepatic failure, or the HELLP syndrome. Adverse maternal outcomes can often be avoided with timely delivery; hence, in the developed world, the burden of morbidity and mortality falls on the neonate.Worldwide, preeclampsia is associated with a perinatal and neonatal mortality rate of 10% (15). Neonatal death is most commonly due to iatrogenic premature delivery undertaken to preserve the health of the mother, but it can also result from placental abruption or intrauterine fetal death.

HELLP: hemolysis, elevated liver enzymes, and low platelets

Long-Term Cardiovascular Complications Traditionally, women with preeclampsia have been reassured that the syndrome remits completely after delivery, with no long-term consequences aside from increased preeclampsia risk in future pregnancies. Epidemiologic studies have tempered this claim. Approximately 20% of women with preeclampsia develop hypertension or microalbuminuria within seven years of a pregnancy complicated by preeclampisa, compared with only 2% among women with uncomplicated pregnancies (16). The long-term risk of cardiovascular and cerebrovascular disease is doubled in women with preeclampsia and gestational hypertension compared with age-matched controls (17, 18). This increase in subsequent cardiovascular disease is observed for both preeclampsia and gestational hypertension (17), suggesting either common risk factors or a common pathophysiology in these two syndromes. Severe preeclampsia, recurrent preeclampsia, preeclampsia with preterm birth, and preeclampsia with intrauterine growth restriction (IUGR) are most strongly associated with adverse cardiovascular outcomes. Preeclampsia, especially in association with low birthweight, also carries an increased risk of later maternal kidney disease requiring a kidney biopsy (19). Preeclampsia and cardiovascular disease share many common risk factors, such as

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chronic hypertension, diabetes, obesity, renal disease, and the metabolic syndrome. Still, the increase in long-term cardiovascular mortality holds even for women who develop preeclampsia in the absence of any overt vascular risk factors. Whether the increase in long-term cardiovascular events results from vascular damage or persistent endothelial dysfunction caused by preeclampsia, or instead reﬂects the common risk factors shared by preeclampsia and cardiovascular disease, remains speculative.

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HIF: hypoxia-inducible factor

PLACENTAL VASCULAR DEVELOPMENT The Role of the Placenta Observational evidence suggests the placenta has a central role in preeclampsia. Preeclampsia only occurs in the presence of a placenta— though not necessarily a fetus, as in the case of hyatidiform mole—and almost always remits after its delivery. In a case of preeclampsia with extrauterine pregnancy, removal of the fetus alone was not sufﬁcient; symptoms persisted until the placenta was delivered (20). Cases of postpartum eclampsia have been associated with retained placental fragments, with rapid improvement after uterine curettage (21). Severe preeclampsia is associated with pathologic evidence of placental hypoperfusion and ischemia. Findings include acute atherosis, a lesion of diffuse vascular obstruction that includes ﬁbrin deposition, intimal thickening, necrosis, atherosclerosis, and endothelial damage. Placental infarcts, probably due to occlusion of maternal spiral arteries, are also common. Although these ﬁndings are not universal, they appear to be correlated with severity of clinical disease (22). Abnormal uterine artery Doppler ultrasound, consistent with decreased uteroplacental perfusion, is observed before the clinical onset of preeclampsia. Unfortunately, this ﬁnding is nonspeciﬁc, so it is not diagnostically useful if used alone (23). In women residing at high altitude (24), there are alterations 64

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in placental hypoxia-inducible factor (HIF) and its targets similar to those seen in women with preeclampsia (25, 26) (see below). This observation suggests that hypoxia may be a contributing factor in preeclampsia pathogenesis. Hypertension and proteinuria can be induced by constriction of uterine blood ﬂow in pregnant primates and other mammals (27, 28). These observations suggest that placental ischemia may be an early event in preeclampsia, at least in some patients. However, evidence that placental ischemia causes preeclampsia remains circumstantial, and several observations call the hypothesis into question. For example, the animal models based on uterine hypoperfusion fail to induce several of the multiorgan features of preeclampsia, including seizures and the HELLP syndrome. In most cases of preeclampsia, there is no evidence of growth restriction or fetal intolerance of labor, expected consequences of placental ischemia. Conversely, cases of fetal growth restriction, where placental insufﬁciency is the rule, often occur without preeclampsia. Hence, overt placental ischemia may be neither universal nor speciﬁc for preeclampsia but instead may be an important secondary event observed in severe cases.

Placental Vascular Remodeling Early in normal placental development, extravillous cytotrophoblasts invade the uterine spiral arteries of the decidua and myometrium. These invasive fetal cells replace the endothelial layer of the uterine vessels, transforming them from small resistance vessels to ﬂaccid, high-caliber capacitance vessels (Figure 1). This vascular transformation allows the increase in uterine blood ﬂow needed to sustain the fetus through the pregnancy. In preeclampsia, this transformation is incomplete. Cytotrophoblast invasion of the arteries is limited to the superﬁcial decidua, and the myometrial segments remain narrow (29). Fisher et al. have shown that in normal placental development, invasive

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Figure 1

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Placentation abnormalities in preeclampsia (from Reference 97 with permission). In normal placental development (top), invasive cytotrophoblasts of fetal origin invade the maternal spiral arteries, transforming them into high-caliber capacitance vessels capable of providing adequate placental perfusion to sustain the growing fetus. During this process, the cytotrophoblasts take on an endothelial phenotype (pseudovasculogenesis). In preeclampsia (bottom), cytotrophoblasts fail to adopt an invasive endothelial phenotype. Instead, invasion of the spiral arteries is shallow and they remain small-caliber resistance vessels.

cytotrophoblasts downregulate the expression of adhesion molecules characteristic of their epithelial cell origin and adopt an endothelial cell-surface adhesion phenotype, a process dubbed pseudovasculogenesis (30) or vascular mimicry. In preeclampsia, cytotrophoblasts do not undergo this switching of cell-surface integrins and adhesion molecules, and they fail to adequately invade the myometrial spiral arteries (31).

The factors that regulate this process are just beginning to be elucidated. Angiogenic factors, including Flt1 (VEGFR-1), VEGFR-2, Tie-1, and Tie-2, are essential for normal placental vascular development; mice deﬁcient in these receptors have defective placental vasculogenesis and die in utero (32). HIF-1, which modulates expression of these angiogenic proteins, is increased in preeclampsia. Invasive cytotrophoblasts

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express several other angiogenic factors, also regulated by HIF, including vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and VEGFR-1 (Flt1); expression of these proteins by immunolocalization is altered in preeclampsia (33). Another HIF target, transforming growth factor beta3 (TGF-β3), may block cytotrophoblast invasion (34). A genetic study recently identiﬁed polymorphisms in STOX1, a paternally imprinted gene and member of the winged helix gene family, in a Dutch preeclampsia cohort (35). The authors hypothesized that loss-of-function mutations in this gene could result in defective polyploidization of extravillous trophoblast, leading to loss of cytotrophoblast invasion. More work is needed to uncover the molecular signals governing cytotrophoblast invasion early in placentation, defects in which may underlie the early stages of preeclampsia. The lack of a naturally occurring animal model for preeclampsia and lack of access to human placental samples from early gestation have greatly limited research in this area.

VEGF: vascular endothelial growth factor

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PlGF: placental growth factor

MATERNAL ENDOTHELIAL DYSFUNCTION Although preeclampsia appears to begin in the placenta, the target organ is the maternal endothelium. The clinical manifestations of preeclampsia reﬂect widespread endothelial dysfunction, resulting in vasoconstriction and end-organ ischemia. Incubation of endothelial cells with serum from women with preeclampsia results in endothelial dysfunction; hence, it has been hypothesized that circulating factors, probably originating in the placenta, are responsible for the manifestations of the disease (36). Dozens of serum markers of endothelial activation and endothelial dysfunction are deranged in women with preeclampsia, including von Willebrand antigen, cellular ﬁbronectin, soluble tissue factor, soluble Eselectin, platelet-derived growth factor, and endothelin. There is evidence of oxidative 66

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stress and platelet activation (37). Inﬂammation is often present; for example, neutrophil inﬁltration is observed in the vascular smooth muscle of subcutaneous fat, with increased vascular smooth muscle expression of IL-8 and intercellular adhesion molecule1 (ICAM-1) (38). Several of these aberrations occur well before the onset of symptoms, supporting the central role of endothelial dysfunction in the pathogenesis of preeclampsia.

Hemodynamic Changes The decreases in peripheral vascular resistance and arterial blood pressure that occur during normal pregnancy are absent or reversed in preeclampsia. Systemic vascular resistance is high and cardiac output is low owing to widespread vasoconstriction. Interestingly, some have suggested that patients destined to develop preeclampsia start out with a hyperdynamic circulation during the preclinical stage and cross over to the vasoconstricted stage during the hypertensive phase (39). There is exaggerated sensitivity to vasopressors such as angiotensin II and norepinephrine (36). Women who go on to develop preeclampsia have impaired endothelium-dependent vasorelaxation (40) and subtle increases in blood pressure and pulse pressure (41) prior to onset of overt hypertension and proteinuria, suggesting that changes in endothelial function are present early in the course of the disease.

Renal Pathology The most characteristic pathologic changes in preeclampsia are seen in the kidney. In 1959, Spargo et al. coined the term glomerular endotheliosis to describe ultrastructural changes in renal glomeruli, including generalized swelling and vacuolization of the endothelial cells and loss of the capillary space (Figure 2a–c). There are deposits of ﬁbrin within and under the endothelial cells, and electron microscopy shows loss of glomerular endothelial fenestrae. The primary injury appears to be to the endothelial cells; the

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a

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Figure 2 Glomerular endotheliosis (from Reference 98 with permission). All light micrographs taken at identical original magniﬁcation of 40×. (a) Normal human glomerulus, H & E. (b) Human preeclamptic glomerulus, H & E. Patient is a 33-year-old woman with twin gestation and severe preeclampsia at 26 weeks gestation with urine protein/creatinine ratio of 26 at the time of biopsy. (c) Electron microscopy of glomerulus of same patient. Note occlusion of capillary lumen with cytoplasm and expansion of subendothelial space with electron-dense material (asterisks). Podocyte cytoplasm shows protein resorption droplets (arrow) and relatively intact foot processes (arrow). Original magniﬁcation 1500×. (d ) Control rat glomerulus, H & E. Note normal cellularity and open capillary loops. (e) Glomerulus of rat treated with soluble fms-like tyrosine kinase-1 (sFlt-1), H & E. Note occlusion of capillary loops with minimal increase in cellularity. ( f ) Electron microscopy of sFlt-1 treated rat. Note occlusion of capillary loops by cytoplasm (asterisk) with relative preservation of podocyte foot processes (arrows). Original magniﬁcation 2500×.

podocyte foot processes are relatively intact early in disease, a ﬁnding atypical of other nephrotic diseases. Although glomerular endotheliosis was once considered pathognomonic for preeclampsia, recent studies have shown that mild glomerular endotheliosis also occurs in pregnancy without preeclampsia, especially in gestational hypertension (42). This suggests the endothelial dysfunction of preeclampsia may be an exaggeration of a process present near term in many normal pregnancies.

Cerebral Edema Cerebral edema and intracerebral parenchymal hemorrhage are common autopsy ﬁndings in women who died from eclampsia. The

presence of cerebral edema in eclampsia correlates with markers of endothelial damage but not the severity of hypertension (43), suggesting the edema is secondary to endothelial dysfunction rather than a direct result of blood pressure elevation. Findings on head CT and MRI are similar to those seen in hypertensive encephalopathy, with vasogenic cerebral edema and infarctions in the subcortical white matter and adjacent gray matter, predominantly in the parietal and occipital lobes (14). A syndrome that includes these characteristic MRI changes, along with headache, seizures, altered mental status, and hypertension, has been described in patients with acute hypertensive encephalopathy in the setting of renal disease, eclampsia, or immunosuppression (44). This syndrome, termed reversible

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posterior leukoencephalopathy, has subsequently been associated with the use of antiangiogenic agents for cancer therapy (45). This association supports the involvement of innate antiangiogenic factors in the pathophysiology of preeclampsia/eclampsia, as detailed in the next section.

sFlt1: soluble fms-like tyrosine kinase-1

ALTERATIONS IN ANGIOGENIC FACTORS

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Recent work suggests that changes in circulating angiogenic factors play a key role in the pathogenesis of preeclampsia. Increased expression of soluble fms-like tyrosine kinase-1 (sFlt1), associated with decreased PlGF and VEGF signaling, were the ﬁrst abnormalities described (46, 47). sFlt1 is a truncated splice variant of the membrane-bound VEGF receptor Flt1, also called VEGFR1. sFlt1 consists of the extracellular ligand-binding domain without the transmembrane and

intracellular signaling domains. Hence, sFlt1 is secreted rather than bound to the cell surface, and it has biological activity as an antagonist to both VEGF and PlGF, binding them in the circulation and preventing interaction with their endogenous receptors (48) (Figure 3). Placental expression of soluble Flt1 is increased in preeclampsia and is associated with a marked increase in maternal circulating sFlt1 (46). Several investigators have conﬁrmed that the increase in maternal circulating sFlt1 precedes the onset of clinical disease (49–52) and is correlated with disease severity (51, 53). In vitro effects of sFlt1 include vasoconstriction and endothelial dysfunction. Exogenous sFlt1 administered to pregnant rats produces a syndrome resembling preeclampsia, including hypertension, proteinuria, and glomerular endotheliosis (46) (Figure 2d–f ). Thus, sFlt1 may be a key part of the mechanism linking the placenta with maternal endothelial dysfunction.

Figure 3 sFlt1 and soluble endoglin (sEng) cause endothelial dysfunction by antagonizing VEGF and TGF-β1 signaling (from Reference 99 with permission). There is mounting evidence that VEGF and TGF-β1 are required to maintain endothelial health in several tissues, including the kidney and perhaps the placenta. During normal pregnancy, vascular homeostasis is maintained by physiological levels of VEGF and TGF-β1 signaling in the vasculature. In preeclampsia, excess placental secretion of sFlt1 and sEng (two endogenous circulating antiangiogenic proteins) inhibits VEGF and TGF-β1 signaling, respectively, in the vasculature. This results in endothelial cell dysfunction, including decreased prostacyclin and nitric oxide production, and release of procoagulant proteins. 68

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Derangements in other angiogenic molecules have also been observed. Maternal serum levels of endostatin, an inhibitor of angiogenesis, are elevated in preeclampsia (54). Soluble endoglin (sEng) is upregulated in preeclampsia in a pattern similar to that of sFlt1. sEng is a truncated form of endoglin (CD105), a cell surface receptor for TGF-β, which binds and antagonizes TGF-β. sEng ampliﬁes the vascular damage mediated by sFlt1 in pregnant rats, inducing a severe preeclampsia-like syndrome with features of the HELLP syndrome (55). This effect may be mediated by interference with nitric oxide–mediated vasodilation (Figure 3). As with sFlt1, circulating sEng levels are elevated weeks prior to preeclampsia onset (56). The precise role of sEng in preeclampsia and its relationship with sFlt1 is currently being explored.

(62). This suggests that VEGF deﬁciency— induced by excess sFlt1—can produce the characteristic renal manifestations of preeclampsia. It is also interesting to note that women with a history of preeclampsia have a decreased risk of malignancy (18, 63), suggesting that the antiangiogenic milieu of preeclampsia may extend beyond pregnancy. The physiologic role of PlGF is less well understood than that of VEGF, but PlGF appears to stimulate angiogenesis under conditions of ischemia, inﬂammation, and wound healing (64) and may contribute to atherosclerosis (65). Blockade of both VEGF and PlGF is required to produce preeclampsialike changes in pregnant rats (46), signifying that PlGF blockade may be important in the pathogenesis of sFlt1-induced endothelial dysfunction.

VEGF Signaling and Endothelial Cell Health

Angiogenic Signals in Placental Vascular Development

Circumstantial evidence supports the hypothesis that interference with VEGF/PlGF signaling could mediate endothelial dysfunction in preeclampsia. VEGF is important in the stabilization of endothelial cells in mature blood vessels. VEGF is particularly important in the health of the fenestrated and sinusoidal endothelium found in the renal glomerulus, brain, and liver (57)—organs severely affected in preeclampsia. VEGF is highly expressed by glomerular podocytes, and VEGF receptors are present on glomerular endothelial cells (58). Anti-VEGF therapies given to adult animals cause glomerular endothelial damage with proteinuria (59, 60). In a podocytespeciﬁc VEGF knockout mouse, heterozygosity for VEGF-A resulted in renal disease characterized by proteinuria and glomerular endotheliosis (61). The most striking experimental illustration of the effect of VEGF antagonism in humans comes from antiangiogenesis cancer trials, where anti-VEGF antibodies produce proteinuria, hypertension, and loss of glomerular endothelial fenestrae

Angiogenic factors are likely to be important in the regulation of placental vasculogenesis. VEGF ligands and receptors are highly expressed by placental tissue in the ﬁrst trimester. sFlt1 decreases cytotrophoblast invasiveness in vitro (33). Circulating sFlt1 levels are relatively low early in pregnancy and begin to rise in the third trimester. This may reﬂect a physiologic antiangiogenic shift in the placental milieu toward the end of pregnancy, corresponding to completion of the vasculogenic phase of placental growth. It is intuitive to hypothesize that placental vascular development might be regulated by a local balance between pro- and antiangiogenic factors and that alterations in these pathways in early gestation could contribute to inadequate cytotrophoblast invasion in preeclampsia. By the third trimester, excess placental sFlt1 is detectable in the maternal circulation, producing end-organ effects. In this case, placental ischemia may not be causative but rather reﬂective of this derangement of angiogenic balance.

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sEng: soluble endoglin

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Mechanistic Insights into Clinical Risk Factors

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The discovery of the importance of sFlt1 has provided insight into certain preeclampsia risk factors. Higher sFlt1 levels have been noted in ﬁrst versus second pregnancies (66), twin versus singleton pregnancies (67), and pregnancies with fetuses having trisomy 13 (68)— all established risk factors for preeclampsia. Conversely, decreased levels of sFlt1 in smokers (69) may explain the protective effect of smoking in preeclampsia. This effect may be mediated by carbon monoxide, a byproduct of cigarette smoking, which diminishes VEGFinduced sFlt1 and sEng release from cultured endothelial cells (70).

Preeclampsia and IUGR: Shared Clinical and Pathophysiologic Features It has long been recognized that preeclampsia and intrauterine growth restriction (IUGR) share many common clinical and pathologic features. IUGR is a common complication of preeclampsia, and abnormal uterine blood ﬂow by Doppler ultrasound in early pregnancy is associated with an increased risk for both disorders. Why some women with placental insufﬁciency manifest the systemic syndrome of preeclampsia, while others have small-for-gestational-age babies without these maternal symptoms, is unknown. With regard to angiogenic factors, there appears to be some overlap between the two syndromes. Some (71–73) but not all (49, 74) studies report that women with IUGR but without preeclampsia have increased circulating sFlt1 and sEng and decreased PlGF. Alterations in angiogenic factors in IUGR without preeclampsia, when detected, are less pronounced than in preeclampsia. Given the pathologic and clinical overlap between IUGR and preeclampsia, it is our belief that the two conditions probably share common pathophysiologic underpinnings, at least at the level of insufﬁcient placental vascular de70

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velopment. Variability in clinical phenotype is probably attributable to individual environmental and genetic differences that alter the maternal response to the placental disease.

OTHER PIECES OF THE PREECLAMPSIA PUZZLE The Renin-Angiotensin-Aldosterone System Women with preeclampsia have increased vascular responsiveness to angiotensin II and other vasoconstrictive agents. However, plasma renin levels are suppressed relative to normal pregnancy as a secondary response to systemic vasoconstriction and hypertension. Wallukat et al. (75) identiﬁed agonistic angiotensin II type 1 (AT1) receptor autoantibodies in women with preeclampsia. They hypothesized that these antibodies, which activate the AT1 receptor, may account for the increased angiotensin II sensitivity of preeclampsia. The same investigators later showed that these AT1 receptor autoantibodies, like angiotensin II itself, stimulate endothelial cells to produce tissue factor, an early marker of endothelial dysfunction. Xia et al. (76) found that AT1 receptor autoantibodies decreased invasiveness of immortalized human trophoblasts in an in vitro invasion assay, suggesting that these autoantibodies might contribute to defective placental pseudovasculogenesis as well. AT1 receptor autoantibodies are not limited to pregnancy; they also appear to be increased in malignant renovascular hypertension and vascular rejection in nonpregnancy (77). Work by Abdalla et al. (78) has suggested that heterodimerization of AT1 receptors with bradykinin 2 receptors may contribute to angiotensin II hypersensitivity in preeclampsia. This work remains to be validated in other studies.

Oxidative Stress and Inflammation Oxidative stress—i.e., the presence of reactive oxygen species in excess of antioxidant

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buffering capacity—is a prominent feature of preeclampsia. It has been hypothesized that in preeclampsia, placental oxidative stress is transferred to the systemic circulation, resulting in oxidative damage to the maternal vascular endothelium. However, the absence of any clinical beneﬁt of antioxidant supplementation for the prevention of preeclampsia suggests that oxidative stress is probably a secondary phenomenon in preeclampsia and not a promising therapeutic target (79). Circulating placental cytotrophoblast debris and the accompanying inﬂammation have also been proposed as a pathogenic mechanism to explain the maternal endothelial dysfunction, but causal evidence for this hypothesis is still lacking (80).

important role in immune tolerance required for normal placental development, have been recently noted to induce angiogenic factors and vascular remodeling (84). Moreover, genetic studies suggest that the susceptibility to preeclampsia may be inﬂuenced by polymorphic human leukocyte antigen C (HLA-C) ligands and the killer immunoglobulin receptors (KIRs) present on NK cells (85). These data suggest that decidual NK cells secrete cytokines and angiogenic factors that play an important role during normal placental vascular remodeling and differentiation, and that alterations in NK cell signaling may mediate abnormal placentation noted in preeclampsia.

CLINICAL IMPLICATIONS OF RECENT ADVANCES

Immunologic Intolerance Immune maladaption remains an intriguing but unproven theory of the pathogenesis of preeclampsia. Normal placentation requires the development of immune tolerance between the fetus and the mother. Preeclampsia occurs more often in ﬁrst pregnancies, after a change in paternity (81), or with long interpregnancy interval (3). Prolonged maternal exposure to paternal ejaculate appears to be associated with a decreased risk of preeclampsia. For example, women using contraceptive methods that reduce exposure to sperm have increased preeclampsia incidence (82). Women impregnated by intracytoplasmic sperm injection (ICSI) in which sperm were surgically obtained (i.e., the woman was never exposed to partner’s sperm in intercourse) had a threefold increased risk of preeclampsia compared to ICSI cases where sperm were obtained by ejaculation (i.e., the woman was likely to have prior exposure to paternal ejaculate via intercourse) (83). These observations suggest preeclampsia may involve an abnormal maternal immune response to novel paternally derived fetal antigens. Natural killer (NK) cells at the maternal/ fetal interface, which are thought to play an

Screening and Prediction Although there is not yet any deﬁnitive therapeutic or preventive strategy for preeclampsia, clinical experience suggests that early detection, monitoring, and supportive care are beneﬁcial to the patient and the fetus. Reliable prediction of preeclampsia would allow closer prenatal monitoring, early diagnosis, and timely intervention with steroids to enhance fetal lung maturity, magnesium for seizure prophylaxis, antihypertensive medications, bed rest, and—when indicated— expeditious delivery. Furthermore, a robust biomarker for preeclampsia would enable targeted studies of therapies and preventive strategies for preeclampsia, including existing (e.g., antiplatelet agents), controversial (calcium, antioxidants), and novel approaches. However, no screening test has yet proven accurate enough for widespread clinical use (86). Alterations in circulating levels of angiogenic factors occur weeks prior to the onset of preeclampsia and are promising biomarkers for screening and/or diagnosis. Signiﬁcant elevations in maternal sFlt1 and sEng are observed from mid-gestation onward

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(49, 87) and appear to rise 5–8 weeks prior to preeclampsia onset (51, 56). Maternal sFlt1 levels are particularly elevated in severe preeclampsia, early-onset preeclampsia, and preeclampsia complicated by a small-forgestational-age baby (51, 88). Serum levels of PlGF are lower in women who go on to develop preeclampsia from the ﬁrst (89) or early second (51, 90, 91) trimester. The ratio of sFlt1 to PlGF has been proposed as an index of antiangiogenic activity that reﬂects alterations in both biomarkers (92) and is a better predictor of preeclampsia than either measure alone (56). Because PlGF is small enough to pass into the urine, changes in urinary PlGF may be a potential marker for preeclampsia. Urinary levels of PlGF are signiﬁcantly lower in women who develop preeclampsia from the late second trimester (92) and may prove useful in screening and diagnosis of preeclampsia, especially in early-onset and severe disease (93). SEng is also elevated in the maternal circulation prior to preeclampsia onset, with gestational patterns similar to those of sFlt1. From the late second and early third trimester, elevations in both sEng and the sFlt1:PlGF ratio (but not either biomarker alone) are associated with very high risk (odds ratio >30) for the development of preterm preeclampsia (56). The timing, source (i.e., serum versus urinary), and combination of biomarkers and other tests that will prove most predictive of preeclampsia and its sequelae are now being explored in prospective studies. For example, a recent cohort study found that the combination of abnormal uterine artery Doppler and low serum PlGF in the second trimester was strongly associated with both early-onset and severe preeclampsia, with odds ratios of 35–45 (94). Other work suggests that rapid changes in angiogenic factor levels with advancing gestation may be more predictive of preeclampsia than levels at any single point in gestation (95, 96). It is likely that these discoveries will enhance our ability to identify women at high risk much earlier in gestation, which

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would improve clinical management and open up possibilities for early intervention.

Novel Treatment Strategies Currently, the only deﬁnitive treatment for preeclampsia is delivery of the fetus and placenta. In the late second and early third trimester, preeclampsia requiring delivery to preserve the health of the mother can result in signiﬁcant neonatal morbidity and mortality due to prematurity. The identiﬁcation of sFlt1 and sEng as a key pathogenic link between placental pathology and maternal endothelial damage provides hope that these biomarkers may also be effective therapeutic targets. Potential therapies may be directed at restoring normal angiogenic balance in the maternal circulation—that is, the relative biologic activity of proangiogenic factors such as VEGF and PlGF relative to antiangiogenic factors such as sFlt1 and sEng. For example, VEGF121 was recently shown to diminish hypertension and proteinuria in a rat model of sFlt1induced preeclampsia, without apparent harm to the fetus (96a). Such therapies may transform the way preeclampsia is treated; an intervention that allows clinicians to safely postpone delivery for even a few weeks could have a tremendous impact on neonatal morbidity and mortality in select cases. Although much more work is needed, we soon may see real improvements in the management of this ancient syndrome.

SUMMARY AND FUTURE DIRECTIONS The past ﬁve years have provided exciting advances in our understanding of the pathogenesis of preeclampsia. Although the initiating events in preeclampsia are still not known, recent work suggests that excess circulating antiangiogenic factors (sFlt1 and sEng) may be a pathophysiologic link between the placental disease and the systemic maternal manifestations. The implications for the management of preeclampsia may be profound. More

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work is needed to further deﬁne the regulation of placental vascular development and expression of these factors in normal pregnancy

and in preeclampsia, and the mechanisms responsible for variability in the maternal response.

DISCLOSURE STATEMENT S.E.M. and S.A.K are listed as coinventors on provisional patents ﬁled by the Beth Israel Deaconess Medical Center for the diagnosis and therapy of preeclampsia. S.A.K is a consultant to Beckman Coulter, Johnson & Johnson, and Abbott Diagnostics.

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ACKNOWLEDGMENTS S.A.K. is supported by NIH grants (DK065997 and HL079594). S.E.M. is supported by the Partnership for Cures, Charles E. Culpeper Scholarship in Medical Sciences.

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34. Caniggia I, Mostachﬁ H, Winter J, et al. 2000. Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGFbeta(3). J. Clin. Invest. 105:577–87 35. van Dijk M, Mulders J, Poutsma A, et al. 2005. Maternal segregation of the Dutch preeclampsia locus at 10q22 with a new member of the winged helix gene family. Nat. Genet. 37:514–19 36. Roberts JM, Taylor RN, Musci TJ, et al. 1989. Preeclampsia: an endothelial cell disorder. Am. J. Obstet. Gynecol. 161:1200–4 37. Roberts JM, Gammill HS. 2005. Preeclampsia: recent insights. Hypertension 46:1243–49 38. Leik CE, Walsh SW. 2004. Neutrophils inﬁltrate resistance-sized vessels of subcutaneous fat in women with preeclampsia. Hypertension 44:72–77 39. Easterling TR. 1992. The maternal hemodynamics of preeclampsia. Clin. Obstet. Gynecol. 35:375–86 40. Khan F, Belch JJ, MacLeod M, et al. 2005. Changes in endothelial function precede the clinical disease in women in whom preeclampsia develops. Hypertension 46:1123–28 41. Duckitt K, Harrington D. 2005. Risk factors for pre-eclampsia at antenatal booking: systematic review of controlled studies. BMJ 330:565 42. Strevens H, Wide-Swensson D, Hansen A, et al. 2003. Glomerular endotheliosis in normal pregnancy and pre-eclampsia. Br. J. Obstet. Gynaecol. 110:831–36 43. Schwartz RB, Feske SK, Polak JF, et al. 2000. Preeclampsia-eclampsia: clinical and neuroradiographic correlates and insights into the pathogenesis of hypertensive encephalopathy. Radiology 217:371–76 44. Hinchey J, Chaves C, Appignani B, et al. 1996. A reversible posterior leukoencephalopathy syndrome. N. Engl. J. Med. 334:494–500 45. Ozcan C, Wong SJ, Hari P. 2006. Reversible posterior leukoencephalopathy syndrome and bevacizumab. N. Engl. J. Med. 354:980–82 46. Maynard SE, Min JY, Merchan J, et al. 2003. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J. Clin. Invest. 111:649–58 47. Ahmad S, Ahmed A. 2004. Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeclampsia. Circ. Res. 95:884–91 48. Kendall RL, Thomas KA. 1993. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc. Natl. Acad. Sci. USA 90:10705– 9 49. Wathen KA, Tuutti E, Stenman UH, et al. 2006. Maternal serum-soluble vascular endothelial growth factor receptor-1 in early pregnancy ending in preeclampsia or intrauterine growth retardation. J. Clin. Endocrinol. Metab. 91:180–84 50. McKeeman GC, Ardill JE, Caldwell CM, et al. 2004. Soluble vascular endothelial growth factor receptor-1 (sFlt-1) is increased throughout gestation in patients who have preeclampsia develop. Am. J. Obstet. Gynecol. 191:1240–46 51. Levine RJ, Maynard SE, Qian C, et al. 2004. Circulating angiogenic factors and the risk of preeclampsia. N. Engl. J. Med. 350:672–83 52. Hertig A, Berkane N, Lefevre G, et al. 2004. Maternal serum sFlt1 concentration is an early and reliable predictive marker of preeclampsia. Clin. Chem. 50:1702–3 53. Chaiworapongsa T, Romero R, Espinoza J, et al. 2004. Evidence supporting a role for blockade of the vascular endothelial growth factor system in the pathophysiology of preeclampsia. Young Investigator Award. Am. J. Obstet. Gynecol. 190:1541–47 www.annualreviews.org • Preeclampsia and Angiogenic Imbalance

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54. Hirtenlehner K, Pollheimer J, Lichtenberger C, et al. 2003. Elevated serum concentrations of the angiogenesis inhibitor endostatin in preeclamptic women. J. Soc. Gynecol. Invest. 10:412–17 55. Venkatesha S, Toporsian M, Lam C, et al. 2006. Soluble endoglin contributes to the pathogenesis of preeclampsia. Nat. Med. 12:642–49 56. Levine RJ, Lam C, Qian C, et al. 2006. Soluble endoglin and other circulating antiangiogenic factors in preeclampsia. N. Engl. J. Med. 355:992–1005 57. Esser S, Wolburg K, Wolburg H, et al. 1998. Vascular endothelial growth factor induces endothelial fenestrations in vitro. J. Cell Biol. 140:947–59 58. Maharaj ASR, Saint-Geniez M, Maldonado AE, et al. 2006. Vascular endothelial growth factor localization in the adult. Am. J. Pathol. 168:639–48 59. Sugimoto H, Hamano Y, Charytan D, et al. 2003. Neutralization of circulating vascular endothelial growth factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces proteinuria. J. Biol. Chem. 278:12605–8 60. Kitamoto Y, Takeya M, Tokunaga H, et al. 2001. Glomerular endothelial cells are maintained by vascular endothelial growth factor in the adult kidney. Tohoku. J. Exp. Med. 195:43–54 61. Eremina V, Sood M, Haigh J, et al. 2003. Glomerular-speciﬁc alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J. Clin. Invest. 111:707– 16 62. Zhu X, Wu S, Dahut WL, et al. 2007. Risks of proteinuria and hypertension with bevacizumab, an antibody against vascular endothelial growth factor: systematic review and meta-analysis. Am. J. Kidney Dis. 49:186–93 63. Aagaard-Tillery KM, Stoddard GJ, Holmgren C, et al. 2006. Preeclampsia and subsequent risk of cancer in Utah. Am. J. Obstet. Gynecol. 195:691–99 64. Carmeliet P, Moons L, Luttun A, et al. 2001. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat. Med. 7:575–83 65. Luttun A, Tjwa M, Moons L, et al. 2002. Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat. Med. 8:831–40 66. Wolf M, Shah A, Lam C, et al. 2005. Circulating levels of the antiangiogenic marker sFLT-1 are increased in ﬁrst vs second pregnancies. Am. J. Obstet. Gynecol. 193:16–22 67. Maynard SE, Moore-Simas T, Solitro M, et al. 2007. Circulating angiogenic factors in singleton vs. multiple gestation pregnancies. Am. J. Obstet. Gyencol. 197:244.e1–8 68. Bdolah Y, Palomaki GE, Yaron Y, et al. 2006. Circulating angiogenic proteins in trisomy 13. Am. J. Obstet. Gynecol. 194:239–45 69. Belgore FM, Lip GY, Blann AD. 2000. Vascular endothelial growth factor and its receptor, Flt-1, in smokers and nonsmokers. Br. J. Biomed. Sci. 57:207–13 70. Cudmore M, Ahmad S, Al-Ani B, et al. 2007. Negative regulation of soluble Flt-1 and soluble endoglin release by heme oxygenase-1. Circulation 115:1789–97 71. Tsatsaris V, Gofﬁn F, Munaut C, et al. 2003. Overexpression of the soluble vascular endothelial growth factor receptor in preeclamptic patients: pathophysiological consequences. J. Clin. Endocrinol. Metab. 88:5555–63 72. Stepan H, Kramer T, Faber R. 2007. Maternal plasma concentrations of soluble endoglin in pregnancies with intrauterine growth restriction. J. Clin. Endocrinol. Metab. 92(7):2831– 34

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73. Crispi F, Dominguez C, Llurba E, et al. 2006. Placental angiogenic growth factors and uterine artery Doppler ﬁndings for characterization of different subsets in preeclampsia and in isolated intrauterine growth restriction. Am. J. Obstet. Gynecol. 195:201–7 74. Shibata E, Rajakumar A, Powers RW, et al. 2005. Soluble fms-like tyrosine kinase 1 is increased in preeclampsia but not in normotensive pregnancies with small-for-gestationalage neonates: relationship to circulating placental growth factor. J. Clin. Endocrinol. Metab. 90:4895–903 75. Wallukat G, Homuth V, Fischer T, et al. 1999. Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor. J. Clin. Invest. 103:945–52 76. Xia Y, Wen H, Bobst S, et al. 2003. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J. Soc. Gynecol. Invest. 10:82–93 77. Fu ML, Herlitz H, Schulze W, et al. 2000. Autoantibodies against the angiotensin receptor (AT1) in patients with hypertension. J. Hypertens. 18:945–53 78. Abdalla S, Lother H, el Massiery A, et al. 2001. Increased AT(1) receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness. Nat. Med. 7:1003–9 79. Poston L, Briley AL, Seed PT, et al. 2006. Vitamin C and vitamin E in pregnant women at risk for pre-eclampsia (VIP Trial): randomised placebo-controlled trial. Lancet 367:1145– 54 80. Redman CW, Sargent IL. 2005. Latest advances in understanding preeclampsia. Science 308:1592–94 81. Tubbergen P, Lachmeijer AMA, Althuisius SM, et al. 1999. Change in paternity: a risk factor for preeclampsia in multiparous women? J. Reprod. Immunol. 45:81–88 82. Klonoff-Cohen HS, Savitz DA, Cefalo RC, et al. 1989. An epidemiologic study of contraception and preeclampsia. JAMA 262:3143–47 83. Wang JX, Knottnerus A-M, Schuit G, et al. 2002. Surgically obtained sperm, and risk of gestational hypertension and pre-eclampsia. Lancet 359:673–74 84. Hanna J, Goldman-Wohl D, Hamani Y, et al. 2006. Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat. Med. 12:1065–74 85. Hiby SE, Walker JJ, O’Shaughnessy KM, et al. 2004. Combinations of maternal KIR and fetal HLA-C genes inﬂuence the risk of preeclampsia and reproductive success. J. Exp. Med. 200:957–65 86. Conde-Agudelo A, Villar J, Lindheimer M. 2004. World Health Organization systematic review of screening tests for preeclampsia. Obstet. Gynecol. 104:1367–91 87. Park CW, Park JS, Shim SS, et al. 2005. An elevated maternal plasma, but not amniotic ﬂuid, soluble fms-like tyrosine kinase-1 (sFlt-1) at the time of mid-trimester genetic amniocentesis is a risk factor for preeclampsia. Am. J. Obstet. Gynecol. 193:984–89 88. Powers RW, Roberts JM, Cooper KM, et al. 2005. Maternal serum soluble fms-like tyrosine kinase 1 concentrations are not increased in early pregnancy and decrease more slowly postpartum in women who develop preeclampsia. Am. J. Obstet. Gynecol. 193:185– 91 89. Thadhani R, Mutter WP, Wolf M, et al. 2004. First trimester placental growth factor and soluble fms-like tyrosine kinase 1 and risk for preeclampsia. J. Clin. Endocrinol. Metab. 89:770–5 90. Taylor RN, Grimwood J, Taylor RS, et al. 2003. Longitudinal serum concentrations of placental growth factor: evidence for abnormal placental angiogenesis in pathologic pregnancies. Am. J. Obstet. Gynecol. 188:177–82 91. Polliotti BM, Fry AG, Saller DN, et al. 2003. Second-trimester maternal serum placental growth factor and vascular endothelial growth factor for predicting severe, early-onset preeclampsia. Obstet. Gynecol. 101:1266–74 www.annualreviews.org • Preeclampsia and Angiogenic Imbalance

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92. Levine RJ, Thadhani R, Qian C, et al. 2005. Urinary placental growth factor and risk of preeclampsia. JAMA 293:77–85 93. Buhimschi CS, Norwitz ER, Funai E, et al. 2005. Urinary angiogenic factors cluster hypertensive disorders and identify women with severe preeclampsia. Am. J. Obstet. Gynecol. 192:734–41 94. Espinoza J, Romero R, Nien JK, et al. 2007. Identiﬁcation of patients at risk for early onset and/or severe preeclampsia with the use of uterine artery Doppler velocimetry and placental growth factor. Am. J. Obstet. Gynecol. 196:326 e1–13 95. Vatten LJ, Eskild A, Nilsen TI, et al. 2007. Changes in circulating level of angiogenic factors from the ﬁrst to second trimester as predictors of preeclampsia. Am. J. Obstet. Gynecol. 196:239 e1–6 96. Moore Simas T, Solitro M, Nadkarni S, et al. 2007. Angiogenic factors for the prediction of preeclampsia in high-risk women. Am. J. Obstet. Gynecol. 197:244.e1–244.e8 96a. Li Z, Zhang Y, Ying Ma J, et al. 2007. Recombinant vascular endothial growth factor 121 attenuates hypertension and improves kidney damage in a rat model of preeclampsia. Hypertension 50:686–92 97. Lam C, Lim KH, Karumanchi SA. 2005. Circulating angiogenic factors in the pathogenesis and prediction of preeclampsia. Hypertension 46:1077–85 98. Karumanchi SA, Maynard SE, Stillman IE, et al. 2005. Preeclampsia: a renal perspective. Kidney Int. 67:2101–13 99. Karumanchi SA, Epstein FH. 2007. Placental ischemia and soluble fms-like tyrosine kinase 1—cause or consequence of preeclampisa? Kidney Int. 71(10):959–61

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237

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Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

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Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg1 and Daniel J. Rader2 1

Division of Cardiovascular Medicine, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; email: [email protected]

2

Cardiovascular Institute; Institute for Diabetes, Obesity and Metabolism; and Institute for Translational Medicine and Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; email: [email protected]

Annu. Rev. Med. 2008. 59:79–94

Key Words

First published online as a Review in Advance on October 15, 2007

cholesterol, triglycerides, high-density lipoproteins, cardiovascular risk

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.121206.112237 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0079$20.00

Abstract Lipid-modifying therapy has been proven to signiﬁcantly reduce cardiovascular events and total mortality. Most of the data have come from statin trials. Statin therapy is generally well-tolerated and safe, and for patients who are at higher than average risk of cardiovascular disease, the beneﬁt of lipid-modifying therapy far exceeds the risk. Careful risk assessment is a critical component of effective lipidmodifying therapy. In the foreseeable future, low-density lipoprotein cholesterol (LDL-C) will remain the primary therapeutic target, and combination therapy is likely to become the norm. The major questions are how low to treat and how to achieve increasingly aggressive targets in lipid-lowering therapy. Many patients on LDL-lowering therapy continue to have abnormalities of the triglyceride–highdensity lipoprotein (TG-HDL) axis, so additional drug therapy is often considered for such patients. In this review, we brieﬂy discuss new developments in cardiovascular risk assessment, then discuss recent developments in treatment to reduce LDL, and ﬁnally discuss current concepts regarding therapy targeting the TG-HDL axis.

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INTRODUCTION CAD: coronary artery disease

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HDL-C: high-density lipoprotein cholesterol TG-HDL axis: balance of triglycerides and HDL-C. Even with successful LDL-lowering therapy, many patients have residual high triglyceride, low HDL-C, or both CHD: coronary heart disease FRS: Framingham Risk Score

Dyslipidemia is an important risk factor for coronary artery disease (CAD) and other atherosclerotic vascular disease. Elevated levels of atherogenic lipoproteins [low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL)] and reduced levels of antiatherogenic lipoproteins [high-density lipoproteins (HDL)] are targets for therapeutic intervention. It has been clearly demonstrated that lowering LDL cholesterol (LDLC) levels with diet and drugs signiﬁcantly reduces the risk of cardiovascular events and total mortality, and guidelines for treatment to reduce LDL-C have become increasingly aggressive, particularly in high-risk patients. Evidence supporting treatment to lower triglycerides or raise HDL-C levels is much less abundant, and formal guidelines are nonexistent. The decision to employ drug therapy targeted to the “TG-HDL axis” is left to the clinical judgment of the physician. Accurate risk assessment is imperative so that patients receive an appropriate level of lipid-modifying drug therapy. In this review, we focus ﬁrst on cardiovascular risk assessment as a critical determinant of the intensity of lipid-modifying therapy. We then discuss recent developments in treatment to reduce LDL and other atherogenic lipoproteins. Finally, we discuss current concepts regarding utilization of therapy that targets the TG-HDL axis.

SCREENING FOR ALL ADULTS The Adult Treatment Panel (ATP) III guidelines of the National Cholesterol Education Program (NCEP) of the National Heart Lung and Blood Institute (1) recommended that all adults over age 20 undergo a fasting full lipid panel to evaluate triglycerides, total cholesterol, HDL-C, and calculated LDL-C. [The LDL-C is calculated from the other lipid values using the following equation: LDL-C = total cholesterol – (triglycerides/5) – HDLC.] The guidelines also recommended the 80

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clinical use of the “non-HDL-C” level as a secondary target of therapy in patients with fasting triglyceride levels >200 mg/dl. The guidelines strongly emphasize risk assessment and stratiﬁcation as a guide to intensity of lipid-modifying drug therapy.

CARDIOVASCULAR RISK ASSESSMENT All decisions regarding lipid management are conditioned by the level of cardiovascular risk, including short-term, 10-year, and lifetime risk. Decisions such as whether to initiate lipid-modifying drug therapy, what target to set for the LDL-C level, and whether to employ combination therapy targeted to the TGHDL axis are all inﬂuenced by the clinician’s assessment of cardiovascular risk. The key categories of cardiovascular risk, as established by the NCEP ATPIII guidelines (1) and further modiﬁed by a subsequent update (2), are as follows: 1. Recent acute coronary syndrome 2. Pre-existing stable atherosclerotic cardiovascular disease 3. Diabetes mellitus, considered a “coronary heart disease (CHD) risk equivalent” condition 4. Absolute 10-year risk of a cardiovascular event >20% as determined by the Framingham Risk Score (FRS), also a CHD risk equivalent condition 5. Absolute FRS 10-year risk of 10%– 20%, considered “moderate risk” 6. Absolute FRS 10-year risk of <10%, considered “low risk” In addition, a very high LDL-C level, such as that seen in heterozygous familial hypercholesterolemia, confers a very high lifetime risk even though the FRS 10-year risk may be low, especially in a younger individual. Patients diagnosed with the metabolic syndrome are also at higher risk; therapeutic lifestyle changes and possibly more aggressive drug therapy are appropriate for them. The ATPIII guidelines also introduced diagnostic criteria for the metabolic syndrome,

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a common condition usually associated with obesity. Indeed, the ﬁve criteria for diagnosis of metabolic syndrome include increased waist circumference. Other criteria are elevated blood pressure, elevated triglycerides, low HDL-C, and impaired fasting glucose (1). Patients who have at least three of the ﬁve criteria can be formally diagnosed as having the metabolic syndrome. More than one third of the adult US population meets sufﬁcient criteria for diagnosis (3). Subsequent studies have established that the diagnosis of metabolic syndrome confers an increased cardiovascular risk above and beyond the individual traditional risk factors and the FRS (4, 5). Thus, clinicians should make an effort to formally diagnose the metabolic syndrome, and when treating dyslipidemia in patients who are so diagnosed, clinicians should bear in mind their elevated cardiovascular risk (see below). The FRS has limitations. It does not take family history into account, and thus a strong family history of premature CHD should be considered an important risk factor even if the FRS is low. Furthermore, a variety of other biomarkers have been proposed to add value beyond traditional risk factors in predicting future coronary events. Perhaps the most celebrated of these is C reactive protein (CRP) (6); others include apolipoprotein B (apoB), lipoprotein(a) [Lp(a)], lipoprotein particle size and number, and lipoproteinassociated phospholipase A2 (Lp-PLA2). Selective use of such tests may help to identify additional high-risk individuals who should be considered for drug therapy when their LDL-C is in a “gray zone.” Recognizing the need for an updated risk scoring system, Ridker and colleagues recently proposed the Reynolds Risk Score, which includes family history and CRP. Using this tool, they reclassiﬁed 40%–50% of women at intermediate risk into higher- or lower-risk categories (7). Whether this scoring system will be more widely adopted remains to be seen, but the concepts and data on which it is based are valid.

The use of noninvasive imaging of atherosclerotic disease is of conceptual interest (8, 9). Abundant data indicate that the carotid intimal medial thickness (IMT), measured by ultrasound, is predictive of coronary events. Accumulating data suggest that coronary artery calciﬁcation, measured by computed tomography, is also predictive, independent of traditional risk factors. The use of noninvasive imaging as a tool for cardiovascular risk assessment is likely to grow over the next several years.

REDUCING LDL-C The evidence supporting therapy to reduce LDL-C levels in the interest of improving cardiovascular outcomes and mortality is overwhelming. Lower is better—but how low is low, and how do we get there?

“Lower is Better” Statin trials have provided the major base of evidence for the beneﬁt of LDL-lowering therapy; prior to the introduction of statins (HMG-CoA reductase inhibitors), the evidence was encouraging but limited. Placebocontrolled statin trials have consistently shown a signiﬁcant reduction of cardiovascular events, and beneﬁt extends even to patients whose LDL-C levels are average or below average. This was most strikingly demonstrated by the Heart Protection Study (10), which enrolled 20,536 subjects aged 40 to 80 years old with established CHD, other atherosclerotic vascular disease, or diabetes; the only lipid-related entry criterion was a total cholesterol level >135 mg/dl. Treatment with simvastatin 40 mg was associated with a highly signiﬁcant 24% reduction in major coronary events, 25% reduction in stroke, and 13% reduction in total mortality. Beneﬁt of simvastatin therapy was evident regardless of baseline cholesterol, even among the group with LDL-C <100 mg/dl at baseline. Other highly positive placebo-controlled statin trials in high-risk groups with average

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LDL-C levels were the Anglo-Scandinavian Cardiac Outcomes Trial Lipid-Lowering Arm (ASCOT-LLA) with atorvastatin, which enrolled hypertensive patients with at least three other risk factors (11), and the Collaborative Atorvastatin Diabetes Study (CARDS), which enrolled type 2 diabetic patients (12). In a new generation of trials, a more intensively treated group was compared to a less intensively treated group. In the chronic CAD setting, the Treat to New Targets (TNT) trial (13) compared atorvastatin 10 mg or 80 mg daily in patients with stable CAD and LDL-C <130 mg/dl. Atorvastatin 80 mg (mean ontreatment LDL-C 77 mg/dl) was associated with a signiﬁcant 22% reduction in major cardiovascular events compared with atorvastatin 10 mg (mean LDL-C 101 mg/dl). In the Incremental Decrease in End Points Through Aggressive Lipid Lowering (IDEAL) trial, simvastatin 20 mg was compared to atorvastatin 80 mg in stable CAD patients. Although the 11% decrease in the primary composite endpoint was not statistically signiﬁcant ( p = 0.07), there was a highly signiﬁcant reduction in secondary coronary disease endpoints (14). In the Pravastatin or Atorvastatin Evaluation and Infection Therapy (PROVE-IT) study in patients presenting with acute coronary syndromes, more intensive lowering of LDL (mean on-treatment LDL-C 62 mg/dl) with atorvastatin 80 mg was superior to less intensive lowering with pravastatin 40 mg (mean on-treatment LDL-C 95 mg/dl) at reducing death, myocardial infarction, or unstable angina requiring hospitalization (15). The Aggrastat to Zocor (A to Z) trial, which randomized acute coronary syndrome patients to simvastatin 40 mg versus placebo for four months, followed by titration to simvastatin 80 mg versus simvastatin, failed to demonstrate a signiﬁcant difference between the two arms with regard to the primary endpoint, but a signiﬁcant reduction in cardiovascular events during the follow-up period starting at four months was demonstrated (16). In the aggregate, these studies (10–16) reinforced the concept that “lower is better”

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with regard to reducing LDL-C, especially in patients at higher risk. The most recent full ATPIII guidelines, in 2001, preceded these studies, but an update was issued in 2004 (2) (Table 1) after some of them had been reported. The updated report recommended a new optional LDL target of <70 mg/dl for patients with established atherosclerotic vascular disease or diabetes, and an optional LDL target of <100 mg/dl for patients with intermediate risk (two or more risk factors and a FRS of 10%–20%). These optional guidelines have been rapidly adopted by cardiologists and a majority of internists. Indeed, it is considered “state of the art” preventive care to attempt to reduce LDL-C to <70 mg/dl in patients with atherosclerotic vascular disease or diabetes, and to <100 mg/dl in the majority of patients with two or more risk factors and/or a FRS >10%. Current debate centers on (a) whether LDL-C should be reduced even further than 70 mg/dl in very high risk patients, (b) whether even moderaterisk patients should be reduced to LDL-C <70 mg/dl, (c) which patients with average cholesterol levels should be started on statin therapy, and (d ) how early to initiate statin therapy. The ﬁnding that genetically low LDL-C levels (due to loss-of-function mutations in the PCSK9 gene) confer an eightfold reduction in lifetime risk of CHD (17) provided strong genetic evidence to support early and substantial LDL-C lowering.

Strategies for Achieving Aggressive LDL-C Targets Despite the remarkable consensus that highrisk patients should be treated aggressively, a substantial number of patients do not achieve their LDL-C goals. Although this failure is partly due to poor compliance, it is also due to the difﬁculty of achieving aggressive LDLC goals, particularly <70 mg/dl. Therapeutic lifestyle changes are an important component in the reduction of LDLC levels. Dietary saturated fat and cholesterol should be restricted; plant stanol and sterol

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Table 1 ATPIII LDL-C goals and cutpoints for therapeutic lifestyle changes (TLC) and drug therapy in different risk categories and proposed modifications based on recent clinical trial evidence Risk category CHDa

LDL-C goal equivalentsb

Initiate TLC mg/dlh

Consider drug therapyi ≥100 mg/dlj (<100 mg/dl: consider drug options)i

<100 mg/dl (optional goal: <70 mg/dl)f

≥100

Moderately high risk: 2+ risk factorsc (10-year risk 10% to 20%)d

<130 mg/dlg

≥130 mg/dlh

≥130 mg/dl (100–129 mg/dl; consider drug options)k

Moderate risk: 2+ risk factorsc (10-year risk <10%)d

<130 mg/dl

≥130 mg/dl

≥160 mg/dl

Lower risk: 0–1 risk factore

<160 mg/dl

≥160 mg/dl

≥190 mg/dl (160–189 mg/dl: LDL-lowering drug optional)

High risk: or CHD risk (10-year risk >20%)

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a

CHD (coronary heart disease) includes history of myocardial infarction, unstable angina, stable angina, coronary artery procedures (angioplasty or bypass surgery), or evidence of clinically signiﬁcant myocardial ischemia. b CHD risk equivalents include clinical manifestations of noncoronary forms of atherosclerotic disease [peripheral arterial disease, abdominal aortic aneurysm, and carotid artery disease (transient ischemic attacks or stroke of carotid origin or >50% obstruction of a carotid artery)], diabetes, and 2+ risk factors with 10-year risk for hard CHD >20%. c Risk factors include cigarette smoking, hypertension (blood pressure ≥140/90 mm Hg or on antihypertensive medication), low HDL cholesterol (<40 mg/dl), family history of premature CHD (CHD in male ﬁrst-degree relative <55 years of age; CHD in female ﬁrst-degree relative <65 years of age), and age (men ≥45 years; women ≥55 years). d Electronic 10-year risk calculators are available at http://www.nhlbi.nih.gov/guidelines/cholesterol. e Almost all people with 0–1 risk factor have a 10-year risk <10%, and 10-year risk assessment in people at this risk level is thus not necessary. f Very high risk favors the optional LDL-C goal of <70 mg/dl, and in patients with high triglycerides, non-HDL-C <100 mg/dl. g Optional LDL-C goal <100 mg/dl. h Any person at high risk or moderately high risk who has lifestyle-related risk factors (e.g., obesity, physical inactivity, elevated triglyceride, low HDL-C, or metabolic syndrome) is a candidate for TLC to modify these risk factors regardless of LDL-C level. i When LDL-lowering drug therapy is employed, it is advised that intensity of therapy be sufﬁcient to achieve at least a 30%–40% reduction in LDL-C levels. j If baseline LDL-C is <100 mg/dl, institution of an LDL-lowering drug is a therapeutic option on the basis of available clinical trial results. If a high-risk person has high triglycerides or low HDL-C, combining a ﬁbrate or nicotinic acid with an LDL-lowering drug can be considered. k For moderately high-risk persons, when LDL-C level is 100–129 mg/dl at baseline or with TLC, initiation of an LDL-lowering drug to achieve an LDL-C level <100 mg/dl is a therapeutic option on the basis of available clinical trial results.

esters, psyllium, soy protein, and Chinese red yeast rice (which contains lovastatin) can all have modest cholesterol-lowering effects. Most other herbal approaches, such as ingestion of garlic and guggulipid, have not been shown to reduce LDL-C. No controlled studies have been performed in which several of these nonpharmacologic options have been combined to evaluate their additive or synergistic effects. Most patients who are more than 5%–10% above their LDL-C goal will require drug therapy to reach it. Statins are the mainstay of therapy and are the ﬁrst choice in patients who require LDL-C reduction. By inhibiting cholesterol synthesis in the liver, they bring about compensatory upregulation of the hep-

atic LDL receptor, thus promoting LDL removal by the liver and reduced plasma LDLC levels. The six statins currently available are lovastatin, pravastatin, simvastatin, ﬂuvastatin, atorvastatin, and rosuvastatin. A substantial number of patients in clinical practice do not achieve their LDL-C goal with the dose of statin they are started on, and doubling the dose produces a fairly predictable further reduction of only 6% (18). The clinician should be prepared to titrate statins appropriately and to use combination therapy in order to achieve LDL-C goals. There is a surprising lack of statin titration in clinical practice, and even when statins are titrated the results are often disappointing. Many patients require combination therapy to

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reach their LDL-C goal. Several options exist; the most common current approach relies on a new class of drugs known as cholesterol absorption inhibitors (CAIs). The small intestine mediates the absorption of cholesterol, which is derived from both the diet (about one third) and the liver via the bile (about two thirds). Inhibiting absorption results in reduced hepatic cholesterol stores and thus upregulation of hepatic LDL receptor expression, which in turn leads to reduction in LDLC levels. The ﬁrst CAI, ezetimibe, has been on the market since 2003 (19). Ezetimibe 10 mg was shown to inhibit cholesterol absorption in humans by almost 60% (20). On average, ezetemibe reduces LDL-C levels by ∼18% as monotherapy (21), and to a similar extent when combined with a statin (22). Ezetimibe is mostly used in combination with statins to further reduce LDL-C levels, and a ﬁxeddose combination of simvastatin and ezetimibe is available. No cardiovascular outcome data have yet been reported with ezetimibe, but a study called IMPROVE-IT comparing ezetimibe-simvastatin to simvastatin alone is currently under way. A substantial minority of patients do not achieve their LDL-C goal even on statin plus CAI, particularly high-risk patients with a high baseline LDL-C whose LDL-C target is <70 mg/dl. Bile acid sequestrants (cholestyramine, colestipol, colesevalam), which have been available for several decades, bind bile acids in the intestine, prevent their reabsorption, and accelerate their loss in the feces. The liver diverts cholesterol to bile acid synthesis, resulting in decreased hepatic cholesterol and upregulation of hepatic LDL receptor expression. Bile acid sequestrants reduce LDLC by up to ∼20% and are clearly additive to statin therapy with regard to LDL reduction (23). As monotherapy, bile acid sequestrants have been shown to reduce clinical cardiovascular events. Their tendency to increase plasma triglyceride levels has limited their use, as has the need to swallow a large number of pills or drink a slurry of the drug. However, for patients on high-dose statin plus ezetimGlassberg

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ibe who are not yet at their LDL target, the addition of a bile acid sequestrant should be considered. Statins are overall safe and generally welltolerated. Approximately 3%–5% of patients develop muscle fatigue or pain on statin therapy. There is a much smaller risk of severe myopathy, deﬁned as muscle pain plus substantial creatinine kinase (CK) elevation. An even rarer but potentially life-threatening effect is rhabdomyolysis, deﬁned as myopathy with renal involvement (24). The risk of myopathy and rhabdomyolysis increases in a dose-dependent fashion with statins. Risk factors for statin-associated myopathy include advanced age, female gender, low body weight, renal insufﬁciency, and occult hypothyroidism. An important risk factor is concomitant administration of drugs that interfere with the cytochrome P450 (particularly 3A4) metabolism of statins, such as erythromycin-type antibiotics, antifungal agents, immunosuppressive drugs, amiodarone, gemﬁbrozil, and HIV protease inhibitors. Routine measurement of serum CK is unnecessary. If a patient taking a statin complains of muscle pain, a normal CK level does not exclude the possibility that the symptoms are due to the drug. Conversely, an elevated CK in an asymptomatic patient on a statin does not necessarily mandate discontinuation. In clinical practice, the biggest issue regarding patients on statins is not myopathy but myalgias. The CK is usually normal, and the relationship to the statin is often uncertain. Nevertheless, myalgias drive many patients to decide they are unable to take statins. There is substantial art involved on the part of the clinician in working with patients who have had myalgias on a statin. This includes attempting to determine the character of the muscle pain and its temporal relationship to statin administration, as well as trying other statins at low doses and sometime creative regimens. In many cases, the myalgias do not recur on a different (or even the same) statin. However, a subset of patients have recurrent symptoms and are labeled statin-intolerant.

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It is particularly difﬁcult for such patients to reach their target LDL-C level. Of course, the generally well-tolerated CAI ezetimibe is useful in this setting, as are bile acid sequestrants. Niacin, discussed in the next section, could also be considered. Some patients cannot achieve their LDLC goal even with combination drug therapy at the maximal tolerated dose. A subset of them are statin-intolerant, and another subset simply cannot achieve the LDL-C goal, usually because of a genetic disorder. Partial ileal bypass was developed as a surgical procedure to decrease LDL-C levels in the 1960s. Similar to the mechanism of the bile acid sequestrants, partial ileal bypass interrupts the enterohepatic circulation of bile acids, resulting in upregulation of the hepatic LDL receptor. The clinical utility of partial ileal bypass at this time is limited to hypercholesterolemic patients with established CAD who are unable to tolerate standard lipid-lowering medications and who do not wish to have LDL apheresis. LDL apheresis is the preferred option for refractory or drug-resistant hypercholesterolemia. The patient’s plasma is passed over columns that selectively remove LDL; then the LDL-depleted plasma is returned to the patient (25). Most patients have it performed biweekly, although severely hypercholesterolemic patients may have it weekly. Clinical trials have indicated that LDL apheresis can retard progression or cause regression of CAD in patients with severe drugresistant hypercholesterolemia (26, 27), and have even suggested a decrease in clinical cardiovascular events (27, 28). LDL apheresis is approved by the US Food and Drug Administration (FDA) and is reimbursed by most insurance plans. Candidates for LDL apheresis are patients who, on maximal tolerated combination drug therapy, either have CHD and LDL-C >200 mg/dl or have no CHD and LDL-C >300 mg/dl. New LDL-lowering therapies are clearly needed for those patients unable to achieve aggressive LDL goals on existing therapies.

TARGETING THE TG-HDL AXIS WITH DRUG THERAPY This section addresses two questions. First, is the TG-HDL axis an appropriate target for drug therapy? And if so, how should therapy be approached?

Outcome Studies The most common dyslipidemia associated with increased CHD risk is elevated triglycerides accompanied by low HDL-C (5). Indeed, even after patients have been treated with LDL-lowering therapy, many have residual high triglyceride, low HDL-C, or both. Abnormalities of the TG-HDL axis in statintreated patients are predictive of recurrent cardiovascular events. For example, in the Scandinavian Simvastatin Survival Study, the subset of 458 patients with both high triglyceride (>159 mg/dl) and low HDL (<39 mg/dl) had the highest event rate: 36% over the course of the study (29). Thus, the issue of whether treatment targeted to the TGHDL axis, such as ﬁbrates, niacin, or omega-3 fatty acids, will further reduce the residual risk compared to statin therapy alone is of critical importance. Unfortunately, to date the only data available are from monotherapy studies that were not performed on the background of statin therapy, although several relevant studies are ongoing. Fibric acid derivatives, or ﬁbrates, are agonists of PPARα. They include cloﬁbrate, gemﬁbrozil, fenoﬁbrate, and bezaﬁbrate. Fibrates have the greatest effect on lowering triglycerides (up to 40%) and can also modestly raise HDL-C (5%–20% depending on the triglyceride levels), but they have relatively minimal effects on LDL-C. Randomized controlled clinical trials suggest that ﬁbrate monotherapy reduces cardiovascular events, particularly in patients with insulin resistance and abnormalities of the TG-HDL axis (30). The Helsinki Heart Study compared gemﬁbrozil 1200 mg to placebo in men with elevated non-HDL-C >200 mg/dl and demonstrated

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a signiﬁcant 34% reduction in combined fatal and nonfatal myocardial infarction (the primary endpoint) (31). The Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial (VA-HIT) tested the beneﬁt of gemﬁbrozil 1200 mg in men with CAD who had average LDL-C levels (mean 112 mg/dl) but low HDL-C levels (mean 32 mg/dl). VAHIT showed a 22% reduction in the primary endpoint (nonfatal myocardial infarction and coronary death) compared to placebo (32). The Bezaﬁbrate Infarction Prevention trial in patients with established CAD, LDL-C <180 mg/dl, and HDL-C <45 mg/dl randomized to either bezaﬁbrate 400 mg daily or placebo failed to demonstrate a signiﬁcant reduction in the primary endpoint of fatal or nonfatal myocardial infarction, but a post hoc analysis in the subgroup with high baseline triglycerides (>200 mg/dl) suggested a significant (39%) reduction in events (33). Most recently, the FIELD trial assessed the effect of fenoﬁbrate 200 mg in diabetic patients with or without known CAD and mean baseline lipid values of LDL-C 118 mg/dl, HDL-C 42 mg/dl, and triglycerides 153 mg/dl (34). There was a nonsignﬁcant 11% reduction in the primary outcome of ﬁrst myocardial infarction or CHD death, although the reduction in some secondary endpoints was significant. It has been postulated that the limited effect seen with fenoﬁbrate was possibly due to a greater “drop-in” of statin therapy in the placebo group. Interestingly, there has never been a ﬁbrate trial targeted speciﬁcally to individuals with elevated triglyceride levels, even though ﬁbrates are most effective in reducing triglycerides. A large placebo-controlled clinical outcome study called the Action to Control Cardiovascular Risk in Diabetes (ACCORD) is testing the effect of fenoﬁbrate on the background of statin therapy in patients with type 2 diabetes mellitus. This study is important because it more closely mirrors current clinical practice. Nicotinic acid, or niacin, is the most effective pharmacologic agent available for raising HDL-C (up to ∼30%), and it also reduces

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triglycerides and LDL-C (35). Niacin acts on a G protein–coupled receptor in adipocytes called GPR109A to reduce lipolysis and release of free fatty acids, which is thought to contribute to its lipid-modifying effects (36). The only large randomized controlled outcome trial to evaluate niacin has been the Coronary Drug Project, which demonstrated a modest beneﬁt. Immediate-release niacin decreased the risk of coronary events (CHD death or nonfatal myocardial infarction) by 15% and decreased the risk of nonfatal myocardial infarction by 26% compared with placebo (37). It was associated with reduced mortality at 15 years of follow-up (38). Several other clinical trials have found niacin to be effective alone or in combination with other drugs in slowing atherosclerotic disease progression. The ARBITER trial evaluated the effect of the addition of Niaspan 1000 mg daily versus placebo upon carotid intimal medial thickness (IMT) among patients with CAD who were at LDL goal on statin therapy (39). Niaspan raised HDL-C by 25% after 24 months of treatment and blunted the progression of IMT. In the HDL Atherosclerosis Treatment Study (HATS), 160 patients with CAD who had low HDL-C and normal LDL-C were randomized to simvastatin plus niacin versus double placebo, and the active treatment group was found to have reduced progression of angiographic disease (40). The composite clinical endpoint was reduced by 89% in patients treated with simvastatinniacin compared with double placebo ( p = 0.04), but the sample size was too small to warrant deﬁnitive statements regarding outcomes. Furthermore, the lack of a statinonly comparator group makes it difﬁcult to discern the contribution of the niacin to the angiographic and outcome beneﬁt. Two large clinical outcome studies are ongoing in which the effects of adding extendedrelease niacin to a statin are being compared with the statin alone: the Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides and Impact on Global Health Outcomes (AIM-HIGH)

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and the Heart Protection Study 2/THRIVE. These studies represent how niacin is used in clinical practice and address the incremental beneﬁt of adding niacin to statin therapy. Omega-3 fatty acids reduce triglyceride levels and appear to reduce cardiovascular risk (41, 42). Both epidemiologic and experimental studies have demonstrated the beneﬁt of both dietary ﬁsh and concentrated ﬁsh oils in the reduction of CHD events. Abundant data demonstrate a reduction in cardiovascular risk associated with dietary ﬁsh intake (43). A large randomized controlled trial demonstrated that daily intake of 1 g of omega3 fatty acids was associated with a signiﬁcant reduction in cardiovascular events (44). It is not clear that the cardioprotective effect of omega-3 fatty acids is related to their triglyceride-lowering effects, as 1 g has relatively little effect on triglyceride levels but was shown to be cardioprotective.

Current Approaches to Targeting the TG-HDL Axis Therapeutically The treatment guidelines developed by NCEP ATPIII remain focused on LDL-C as the primary target for therapy but acknowledge that elevated triglyceride and low HDL-C are risk factors, even when LDLC is “treated to goal.” However, as noted above, clinical trial data supporting the treatment of the TG-HDL axis are less compelling. The NCEP ATPIII guidelines introduced the concept of non-HDL-C as a secondary target of therapy in patients with triglyceride levels >200 mg/dl. Non-HDLC is calculated simply as the total cholesterol minus the HDL-C and is intended to represent all of the atherogenic apoB-containing lipoproteins. The goals for non-HDL-C are 30 mg/dl higher than the goals for LDL-C. Thus, many patients with abnormalities of the TG-HDL axis have elevated non-HDL-C levels and require intensiﬁcation of therapy to reduce non-HDL-C to recommended goals. The guidelines acknowledge that HDL-C can be considered a target of therapy at the dis-

cretion of the clinician but do not set speciﬁc HDL targets. Management of the patient with abnormalities of the TG-HDL axis should focus initially on therapeutic lifestyle changes. In general, patients should be advised to limit total fat intake to 30% of total calories, and limit the intake of simple carbohydrates, while increasing complex carbohydrates such as whole grains, fruits, and vegetables. Lowcarbohydrate diets have been shown to reduce triglyceride by up to 20% and in some instances also raise HDL-C by up to 20% (45, 46). However, longer-term data investigating broader cardiovascular surrogates and clinical outcomes are needed before these types of diets can be broadly recommended. Aerobic exercise independent of weight loss has been shown to modestly reduce triglyceride and increase HDL-C levels in a dose-dependent fashion. Finally, weight loss is important: Estimates from meta-analyses suggest that for every 4.5 kg (∼10 lb) of stable weight reduction, triglyceride levels are reduced by 6 mg/dl and HDL-C levels are increased by ∼2 mg/dl (5). Patients with persistent abnormalities in the TG-HDL axis despite attempts at therapeutic lifestyle changes are often at a level of cardiovascular risk that merits consideration of drug therapy. Of course, if the LDLC is also elevated or the patient has established atherosclerotic cardiovascular disease or type 2 diabetes, such that therapy for primary LDL reduction is indicated as discussed above, the decision to initiate statin therapy is straightforward. However, many patients with dyslipidemia related to the TGHDL axis do not have especially elevated LDL-C, so they do not qualify for LDLlowering therapy based on existing guidelines. In this setting, the decision of whether to initiate drug therapy, and if so, with which drug class, is based on clinical judgment and not addressed by formal guidelines. If the fasting triglycerides are especially elevated in a range predisposing to risk of acute pancreatitis (triglyceride >1000 mg/dl), then therapeutic lifestyle changes accompanied by primary

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triglyceride-lowering drug therapy, generally a ﬁbrate or omega-3 fatty acids, are clearly indicated. However, when the triglycerides are not in this range, and particularly when they are in the 200–500 mg/dl range, there are relatively few clinical trials to guide selection of drug therapy. No outcomes trials have been performed in which hypertriglyceridemia was a criterion for entry, and in fact most of the statin trials, and even some of the ﬁbrate trials, have excluded individuals with signiﬁcantly elevated triglyceride levels. However, the strongest data to suggest appropriate treatment of such patients come from the statin trials. For example, a subgroup analysis of the Scandinavian Simvastatin Survival Study found that subjects with both elevated triglyceride and low HDL beneﬁted from simvastatin therapy (29), although all subjects had elevated LDL-C at entry. More compellingly, the Heart Protection Study did not exclude subjects based on their triglyceride levels, and a subgroup analysis showed that all strata of baseline triglyceride and HDL levels beneﬁted from simvastatin (10). Thus, it is our opinion that a patient with dyslipidemia of the TG-HDL axis who has tried therapeutic lifestyle changes and is a high enough cardiovascular risk to merit consideration of drug therapy should be advised to start with statin therapy. The large body of data that now exists suggests that such patients will beneﬁt from statin therapy even if their baseline LDL-C is average or low. The one exception may be in patients with extremely elevated triglyceride levels of >500 mg/dL, in whom triglyceride-lowering therapy should be initiated ﬁrst. The more difﬁcult clinical conundrum, however, is how to approach the patient who has already been treated with a statin but has persistent abnormalities of the TG-HDL axis. Many patients with dyslipidemia require therapy with more than one lipid-modifying drug (47). This situation raises the question of adding a second drug to modify the TGHDL axis. As noted above, no completed outcome trials address this question directly, and

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no guidelines recommend a course of action in this situation. The NCEP ATPIII guidelines do recommend that if LDL-C is at goal but triglyceride is >200 mg/dl, the clinician should calculate the non-HDL-C as a secondary target of therapy and attempt to reduced it to goal (i.e., 30 mg/dl greater than the LDL-C goal in the same patient, as mentioned above). In some cases, non-HDL-C might be reduced by intensifying statin therapy or adding a CAI. However, it may be reasonable in many cases to add a drug that acts on the TG-HDL axis in order to reduce nonHDL-C as well as raise HDL-C and reduce cardiovascular risk. However, data demonstrating effects of combination lipid-lowering therapy on clinical endpoints are sparse. Thus, the potential beneﬁts of this approach in improving the lipid proﬁle and reducing cardiovascular events must be weighed against the tolerability and safety of adding a second drug. Adding a fibrate to statin therapy. A relatively high-risk patient on a statin in whom the major remaining lipid abnormality is an elevated triglyceride level might be considered for the addition of a ﬁbrate. The lipid effects of ﬁbrates complement those of statins, and several controlled trials have documented the efﬁcacy of adding a ﬁbrate to a statin. For example, the addition of fenoﬁbrate to simvastatin was compared with simvastatin alone in patients with combined hyperlipidemia (48). Combination therapy reduced median triglyceride levels by 43% compared with 20% for simvastatin monotherapy and increased HDL-C levels by 19% compared with 10% for simvastatin alone. However, although a statin-ﬁbrate combination is more effective in treating the lipid proﬁle in patients with mixed hyperlipidemia than either drug alone, there are no data indicating that addition of a ﬁbrate to a statin further reduces cardiovascular risk. The major issue with the clinical use of this combination is safety, particularly related to myopathy (24). Indeed, cerivastatin was withdrawn because it increased the incidence

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of myopathy and rhabdomyolysis, especially when combined with gemﬁbrozil (24). It appears that gemﬁbrozil affects the pharmacokinetics of most statins; fenoﬁbrate has a more modest effect (49). This may be secondary to the differential effect of these two ﬁbrates on the glucuronidation of statins, resulting in higher levels of statin metabolites with concomitant gemﬁbrozil use. The risk of myopathy with this combination can be minimized with appropriate patient selection and education (50). The American Heart Association and the American College of Cardiology released a joint clinical advisory (24) afﬁrming the potential beneﬁts of combination therapy with statin plus ﬁbrate but suggesting caution in groups of patients at higher risk of myopathy, such as elderly (>70 years), frail patients with multiple medical problems; patients with underlying renal or hepatic insufﬁciency; or patients already taking multiple prescription medications. The ACCORD trial (discussed above) is the ﬁrst deﬁnitive test of the cardiovascular beneﬁt of adding a ﬁbrate to a statin, and its eagerly awaited results represent the largest safety data set in the setting of a randomized controlled trial. Adding niacin to statin therapy. Adding niacin to a statin is considered in a relatively high-risk patient whose remaining major lipid abnormality is HDL-C (deﬁned as <40 mg/dl, although many clinicians consider HDL-C <50 mg/dl to be low in women, and this cutpoint is used for the metabolic syndrome criteria). Several controlled trials have demonstrated the lipid efﬁcacy of adding niacin to a statin (35, 51). Some of the best controlled comparative data come from the studies performed to support the approval of a ﬁxed-dose combination tablet of lovastatin and extended-release niacin (niacin-ER). In a study of patients with hypercholesterolemia and low HDL-C levels, patients were randomized to two escalating doses of niacinER/lovastatin or escalating doses of atorvastatin (10–40 mg) and simvastatin (10–40 mg) over 16 weeks (52). At 16 weeks, the combi-

nation of 2000 mg ER-niacin and 40 mg lovastatin was superior in its effect on the TGHDL axis, lowering triglyceride by 49% and raising HDL-C by 32%. Thus the lipid efﬁcacy of this combination is clear, although, as noted above, hard outcomes are still lacking. Important concerns are tolerability and effects on glucose metabolism. The most important tolerability issue with niacin is cutaneous ﬂushing, which often deters physicians from prescribing niacin and patients from taking it. Engineering of niacin to delay its absorption helps, but does not eliminate this issue. A variety of other strategies, such as pretreating with aspirin and having a snack prior to taking niacin, can also reduce ﬂushing. Most patients experience relatively rapid tachyphylaxis to the ﬂushing effects of niacin if they are able to tolerate the ﬂushing for a few weeks after initiation and after each dose increase. New approaches to reduce niacin-associated ﬂushing are being investigated. The niacin receptor on adipocytes, called GPR109A (53), is also expressed on dendritic cells and macrophages in the skin; when activated by niacin, it is responsible for the release of vasodilatory prostaglandins such as PGD2 , which activate its receptor DP1 on vascular cells (54). Thus, one approach under development is to speciﬁcally block the PGD2 receptor that mediates ﬂushing in the skin (55). It has long been noted that niacin therapy can have adverse effects on glycemic control in patients with type 2 diabetes or metabolic syndrome, motivating more cautious use of niacin in such patients. However, a few controlled trials of niacin in diabetics suggest this may not be a major problem for most patients (56). In the Arterial Disease Multiple Intervention Trial (ADMIT), niacin had relatively minimal effect on glycemic control in patients with well-controlled type 2 diabetes (57). In the Assessment of Diabetes Control and Evaluation of the Efﬁcacy of Niaspan Trial (ADVENT), moderate-dose niacin was given to patients who had type 2 diabetes but no clinical cardiovascular disease (58). There

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was a modest increase in hemoglobin A1C values from 7.2% to 7.5% in the niacin-treated subjects. Data in nondiabetics with impaired fasting glucose are limited, but some of these patients may experience an increase in fasting glucose with niacin therapy that approaches the mild diabetes range. Thus, the decision to add niacin to a statin in patients with type 2 diabetes or impaired fasting glucose must balance the lipid beneﬁts against the possibility of slightly worsened glycemic control. With regard to the liver, although transaminases should be monitored when niacin is added to a statin, serious hepatotoxicity appears to be very rare with this combination. Some nonprescription sustained-release niacin preparations, which are classically administered twice a day, have been associated with several cases of severe hepatotoxicity (51).

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Adding omega-3 fatty acids to statin therapy. A few small studies have investigated the effects of statin–omega-3 combinations on lipids. It appears that the addition of ﬁsh oils to statin therapy has at least additive effects on the TG-HDL axis and is generally well-tolerated. For signiﬁcant triglyceride reduction, the two active omega-3 fatty acids eicosapentanoic acid (EPA) and decohexanoic acid (DHA) generally need to be dosed up to ∼4 g/day. Most ﬁsh oil preparations are sold as dietary supplements without a prescription. An FDA-approved preparation of omega-3 fatty acids is the most highly concentrated of the preparations available, with 890 mg of DHA and EPA per 1-g capsule. The addition of omega-3 fatty acids might be considered as an alternative to adding a ﬁbrate to a statin, particularly for a patient at increased risk for myopathy.

SUMMARY Lipid-lowering therapy has clearly reduced the risk of cardiovascular event and mortality, with the most abundant data generated from the statin trials. Statins are safe and generally well-tolerated. Tolerability is primarily limited by myalgias, and myopathy and rhabdomyolysis are rare. The beneﬁt of appropriately used lipid-lowering therapy far exceeds the risk for those patients at above-average risk of cardiovascular disease. Thus, detailed risk assessment is critical to guide the intensity of the lipid-lowering therapy. This includes use of the FRS (and potentially the new Reynolds Risk Score), incorporation of family history, diagnosis of the metabolic syndrome, and use of additional blood markers and/or noninvasive vascular imaging to optimally assess future cardiovascular risk. Currently, LDL-C reduction remains the cornerstone of therapy, though how low to treat, how to achieve more aggressive targets, and how early to initiate therapy continue to be debated. Often, while treatment may result in adequate LDL lowering, abnormalities of the TG-HDL axis persist, and a major lacuna in our evidence base is the lack of completed trials demonstrating the beneﬁt of adding TGHDL-modifying therapy to LDL-lowering therapy. Nevertheless, in high-risk patients, addition of a ﬁbrate, niacin, or omega-3 fatty acids can be reasonably considered after a trial of therapeutic lifestyle changes. In such patients, the beneﬁt must be weighed against the issues of tolerability and safety. The use of many different types of combination lipidmodifying therapy is likely to increase substantially over the next several years, particularly if ongoing outcome trials of combination therapy demonstrate efﬁcacy in reducing cardiovascular outcomes.

DISCLOSURE STATEMENT Dr. Rader has received research funding from, served as a consultant to, and/or received honoraria from the majority of companies that have currently marketed drugs for the treatment of lipid disorders. Dr. Rader also consults with companies involved in developing new therapeutic agents for lipid disorders and holds equity in Aegerion Pharmaceuticals. 90

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19. Lipka LJ. 2003. Ezetimibe: a ﬁrst-in-class, novel cholesterol absorption inhibitor. Cardiovasc. Drug Rev. 21:293–312 20. Garcia-Calvo M, Lisnock J, Bull HG, et al. 2005. The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1). Proc. Natl. Acad. Sci. USA 102:8132–37 21. Bays HE, Moore PB, Drehobl MA, et al. 2001. Effectiveness and tolerability of ezetimibe in patients with primary hypercholesterolemia: pooled analysis of two phase II studies. Clin. Ther. 23:1209–30 22. Davidson MH, Ballantyne CM, Kerzner B, et al. 2004. Efﬁcacy and safety of ezetimibe coadministered with statins: randomised, placebo-controlled, blinded experience in 2382 patients with primary hypercholesterolemia. Int. J. Clin. Pract. 58:746–55 23. Zema MJ. 2005. Colesevelam HCl and ezetimibe combination therapy provides effective lipid-lowering in difﬁcult-to-treat patients with hypercholesterolemia. Am. J. Ther. 12:306–10 24. Pasternak RC, Smith SC Jr., Bairey-Merz CN, et al. 2002. ACC/AHA/NHLBI clinical advisory on the use and safety of statins. J. Am. Coll. Cardiol. 40:567–72 25. Thompson GR. 2003. LDL apheresis. Atherosclerosis 167:1–13 26. Tatami R, Inoue N, Itoh H, et al. 1992. Regression of coronary atherosclerosis by combined LDL-apheresis and lipid-lowering drug therapy in patients with familial hypercholesterolemia: a multicenter study. Atherosclerosis 95:1–13 27. Thompson GR, Maher V, Matthews S, et al. 1995. Familial hypercholesterolaemia regression study: a randomised trial of low-density lipoprotein apheresis. Lancet 345:811–16 28. Sachais BS, Katz J, Ross J, et al. 2005. Long-term effects of LDL apheresis in patients with severe hypercholesterolemia. J. Clin. Apher. 20:252–55 29. Ballantyne CM, Olsson AG, Cook TJ, et al. 2001. Inﬂuence of low high-density lipoprotein cholesterol and elevated triglyceride on coronary heart disease events and response to simvastatin therapy in 4S. Circulation 104:3046–51 30. Steiner G. 2004. Fibrates in the metabolic syndrome and in diabetes. Endocrinol. Metab. Clin. North Am. 33:545–55, vi–vii 31. Frick MH, Elo O, Haapa K, et al. 1987. Helsinki Heart Study: primary-prevention trial with gemﬁbrozil in middle-aged men with dyslipidemia. Safety of treatment, changes in risk factors, and incidence of coronary heart disease. N. Engl. J. Med. 317:1237– 45 32. Rubins HB, Robins SJ, Collins D, et al. 1999. Gemﬁbrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol. N. Engl. J. Med. 341:410–18 33. 2000. Secondary prevention by raising HDL cholesterol and reducing triglycerides in patients with coronary artery disease: the Bezaﬁbrate Infarction Prevention (BIP) study. Circulation 102:21–27 34. Keech A, Simes RJ, Barter P, et al. 2005. Effects of long-term fenoﬁbrate therapy on cardiovascular events in 9795 people with type 2 diabetes mellitus (the FIELD study): randomised controlled trial. Lancet 366:1849–61 35. Meyers CD, Kamanna VS, Kashyap ML. 2004. Niacin therapy in atherosclerosis. Curr. Opin. Lipidol. 15:659–65 36. Pike NB, Wise A. 2004. Identiﬁcation of a nicotinic acid receptor: Is this the molecular target for the oldest lipid-lowering drug? Curr. Opin. Investig. Drugs 5:271–75 37. The Coronary Drug Project Research Group. 1975. Cloﬁbrate and niacin in coronary heart disease. JAMA 231:360–81

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38. Canner PL, Berge KG, Wenger NK, et al. 1986. Fifteen year mortality in Coronary Drug Project patients: long-term beneﬁt with niacin. J. Am. Coll. Cardiol. 8:1245– 55 39. Taylor AJ, Sullenberger LE, Lee HJ, et al. 2004. Arterial Biology for the Investigation of the Treatment Effects of Reducing Cholesterol (ARBITER) 2: a double-blind, placebocontrolled study of extended-release niacin on atherosclerosis progression in secondary prevention patients treated with statins. Circulation 110:3512–17 40. Brown BG, Zhao XQ, Chait A, et al. 2001. Simvastatin and niacin, antioxidant vitamins, or the combination for the prevention of coronary disease. N. Engl. J. Med. 345:1583– 92 41. Kris-Etherton PM, Harris WS, Appel LJ. 2003. Fish consumption, ﬁsh oil, omega-3 fatty acids, and cardiovascular disease. Arterioscler. Thromb. Vasc. Biol. 23:e20–30 42. Hooper L, Thompson RL, Harrison RA, et al. 2006. Risks and beneﬁts of omega 3 fats for mortality, cardiovascular disease, and cancer: systematic review. BMJ 332:752–60 43. Balk EM, Lichtenstein AH, Chung M, et al. 2006. Effects of omega-3 fatty acids on serum markers of cardiovascular disease risk: a systematic review. Atherosclerosis 189:19–30 44. 1999. Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico. Lancet 354:447–55 45. Foster GD, Wyatt HR, Hill JO, et al. 2003. A randomized trial of a low-carbohydrate diet for obesity. N. Engl. J. Med. 348:2082–90 46. Samaha FF, Iqbal N, Seshadri P, et al. 2003. A low-carbohydrate as compared with a low-fat diet in severe obesity. N. Engl. J. Med. 348:2074–81 47. Ansell BJ. 2005. Rationale for combination therapy with statin drugs in the treatment of dyslipidemia. Curr. Atheroscler. Rep. 7:29–33 48. Grundy SM, Vega GL, Yuan Z, et al. 2005. Effectiveness and tolerability of simvastatin plus fenoﬁbrate for combined hyperlipidemia (the SAFARI trial). Am. J. Cardiol. 95:462– 68 49. Jones PH, Davidson MH. 2005. Reporting rate of rhabdomyolysis with fenoﬁbrate + statin vs gemﬁbrozil + any statin. Am. J. Cardiol. 95:120–22 50. Wierzbicki AS, Mikhailidis DP, Wray R, et al. 2003. Statin-ﬁbrate combination: therapy for hyperlipidemia: a review. Curr. Med. Res. Opin. 19:155–68 51. Carlson LA. 2005. Nicotinic acid: the broad-spectrum lipid drug. A 50th anniversary review. J. Intern. Med. 258:94–114 52. Bays HE, Dujovne CA, McGovern ME, et al. 2002. Comparison of once-daily, niacin extended-release/lovastatin with standard doses of atorvastatin and simvastatin (the Advicor vs Other Cholesterol-Modulating Agents Trial Evaluation [ADVOCATE]). Am. J. Cardiol. 91:667–72 53. Tunaru S, Kero J, Schaub A, et al. 2003. PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect. Nat. Med. 9:352–55 54. Pike NB. 2005. Flushing out the role of GPR109A (HM74A) in the clinical efﬁcacy of nicotinic acid. J. Clin. Invest. 115:3400–3 55. Cheng K, Wu TJ, Wu KK, et al. 2006. Antagonism of the prostaglandin D2 receptor 1 suppresses nicotinic acid-induced vasodilation in mice and humans. Proc. Natl. Acad. Sci. USA 103:6682–87 56. Shepherd J, Betteridge J, Van Gaal L. 2005. Nicotinic acid in the management of dyslipidaemia associated with diabetes and metabolic syndrome: a position paper developed by a European Consensus Panel. Curr. Med. Res. Opin. 21:665–82 www.annualreviews.org • Lipid Management and Cardiovascular Events

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57. Elam MB, Hunninghake DB, Davis KB, et al. 2000. Effect of niacin on lipid and lipoprotein levels and glycemic control in patients with diabetes and peripheral arterial disease. JAMA 284:1263–70 58. Grundy SM, Vega GL, McGovern ME, et al. 2002. Efﬁcacy, safety, and tolerability of once-daily niacin for the treatment of dyslipidemia associated with type 2 diabetes: results of the Assessment of Diabetes Control and Evaluation of the Efﬁcacy of Niaspan Trial. Arch. Intern. Med. 162:1568–76

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237

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Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

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Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez Diabetes Unit (Department of Medicine) and Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts; the Program in Medical and Population Genetics, Broad Institute of Harvard and MIT, Cambridge, Massachusetts; and the Department of Medicine, Harvard Medical School, Boston, Massachusetts; email: jcﬂ[email protected]

Annu. Rev. Med. 2008. 59:95–111

Key Words

First published online as a Review in Advance on October 15, 2007

MODY, TCF7L2, KCNJ11, PPARG, pharmacogenetics, single-nucleotide polymorphism, genome-wide association

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.090706.135315 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0095$20.00

Abstract Despite major advances in our knowledge of glycemic pathophysiology and the availability of multiple therapeutic options to confront type 2 diabetes, unraveling the complex link between genetic risk and environmental factors in this burgeoning epidemic has proven difﬁcult. Linkage approaches have clariﬁed the etiology of monogenic diabetic syndromes and congenital lipodystrophies, and candidate gene association studies have identiﬁed a number of common variants implicated in type 2 diabetes. This year we have witnessed the advent of genome-wide association scanning: As many as nine genetic loci have now been reproducibly associated with type 2 diabetes in ﬁve genome-wide scans. Of particular interest are preliminary explorations of the connections between genetic risk and pharmacologic response. An improved understanding of genetic mechanisms should allow us to test whether behavioral or pharmacologic therapies can be tailored and thus the tremendous disease burden inﬂicted by type 2 diabetes alleviated.

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MODY: maturity-onset diabetes of the young

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INTRODUCTION

MONOGENIC DIABETES

Diabetes mellitus affects more than 150 million people worldwide, and this number is expected to double within the next two decades. One in three Americans born in the year 2000 will develop the disease. Its complications include renal failure, blindness, amputation, myocardial infarction, and stroke. We have gained signiﬁcant insight into the many advantages of tight blood glucose control, but the genetic susceptibility to the disease and the interaction between potential therapies and an individual’s genetic signature remain incompletely understood. The abundant evidence that supports the genetic basis of type 2 diabetes (T2D) is based on population, family, and twin studies. The prevalence of T2D varies widely across ethnic groups, with incidence rates as low as 1% in some rural Asian populations while Pima Indians and Polynesians have among the highest diabetic incidence rates in the world, reaching the 30%–50% range. Moreover, in the United States and the United Kingdom, rates of diabetes in minority ethnic groups are at least twofold higher than those of their white counterparts, and this difference is not eliminated when environmental factors are controlled for. Familial aggregation studies further bolster the notion of genetic susceptibility: The offspring of a diabetic parent has a 40% chance of developing diabetes in contrast to a population risk of ∼7%, and if both parents are affected, the risk rises to 70% (1). Similarly, a sibling of a diabetic patient has an approximate relative risk of 3 of developing diabetes later in life. Perhaps the strongest genetic evidence is derived from twin studies, which reveal concordance rates of 0.20 to 0.91 in monozygotic twins and 0.10 to 0.43 in dizygotic twins (2, 3). Despite this convincing evidence for an inborn susceptibility to T2D, the concordance rates are not 100%; the environment and chance contribute as well.

Some of the most compelling evidence that inherited variation can cause glycemic dysregulation comes from the clinical and genetic description of monogenic diabetes, including maturity-onset diabetes of the young (MODY), insulin resistance syndromes, mitochondrial diabetes, and neonatal diabetes. Although rare, these syndromes provide a framework for understanding and investigating the complex genetics of T2D.

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Maturity-Onset Diabetes of the Young MODY was ﬁrst described in 1960 as a unique type of non–insulin-dependent, autosomal dominant diabetes in thin, young adults (usually <25 years of age) who are not prone to ketoacidosis (4). It is thus distinguished from T2D by family history, younger onset, and absence of obesity. This syndrome accounts for approximately 1%–2% of diabetes cases, although MODY has been reported as responsible for as many as 5% of European diabetic cases (5). By using both linkage and candidate gene approaches, investigators have identiﬁed six different MODY genes to date, although ∼10% of MODY patients have no identiﬁable genetic mutation (a condition called MODY X). Despite differences in clinical presentation and treatment response, all six MODY syndromes share three features: (a) They result from mutated genes that are expressed in the pancreatic β cell; (b) these genes encode nuclear transcription factors (with the important exception of glucokinase); and (c) these mutations result in a signiﬁcant loss of pancreatic β cell function and subsequent insulin production. MODY1 was identiﬁed through a linkage study of an American family (6). The syndrome results in severe, progressive β cell dysfunction and insulin dependence. MODY1 results from mutations in HNF4A on

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chromosome 20, which affects the development and function of human pancreatic β cells. MODY1 is the third most common MODY subtype, with 31 mutations in 40 families described to date. MODY2 accounts for ∼20% of MODY cases. Whereas the other MODY syndromes are caused by mutations in transcription factors, MODY2 results from mutations in the glucokinase gene (GCK ) on chromosome 7p. Glucokinase catalyzes the phosphorylation of glucose and controls the ﬁrst rate-limiting step of glycolysis. This enzyme functions as the glucose sensor of pancreatic β cells and regulates insulin production. Inactivating mutations of GCK result in mild, stable, lifelong fasting hyperglycemia because those affected lack an effective glucose sensor. Levels of insulin secretion in MODY2 patients are similar to levels seen in noncarriers of the mutation, although these levels are seen at higher serum glucose levels (7). Treatment is rarely required, and microvascular disease is unusual. As predicted by the pathophysiology, patients with GCK mutations do not respond to insulin or oral hypoglycemic agents because they decrease their endogenous insulin production in response to exogenous therapy. Some positive preliminary animal studies using GCK activators are emerging (8). MODY3 is the most common MODY subtype, with more than 193 mutations identiﬁed in 373 families (9). Diabetes usually develops in early adulthood but can develop later in life. Patients lose β cell function progressively with minimal insulin requirements at ﬁrst, but eventual insulin dependence is the rule. The syndrome results from mutations in HNF1A (TCF1), which impair key steps of glucose transport, metabolism, and mitochondrial metabolism. Retinopathy and nephropathy are common in MODY3; however, macrovascular disease is not. MODY4 results from heterozygous mutations in the gene that encodes insulin promoter factor 1 (IPF1 or PDX1), a tran-

scription factor that regulates insulin gene transcription as well as islet and pancreatic development. Homozygous mutations in exon 1 of IPF1 give rise to pancreatic agenesis (10). MODY5, a more common MODY variant than originally suspected, is unique in that it is associated with renal anomalies. The syndrome was identiﬁed through the genetic screening of a Japanese cohort, which identiﬁed a nonsense mutation in HNF1B (TCF2) (11). HNF1B is highly expressed in the pancreas, liver, and kidney. Diabetes results from both hepatic insulin resistance and β cell loss (12). β cell dysfunction is more severe in MODY5 than in MODY3 and ketoacidosis has been reported (13). These patients typically require insulin therapy and do not respond to sulfonylureas. HNF1B mutations have been associated with renal dysfunction, genitourinary problems, abnormal liver function, and hyperuricemia (14). Finally, MODY6 has been shown to result from mutations in NEUROD1 (BETA2), which is important for pancreatic development and insulin gene transcription (15). Patients develop moderately severe and progressive β cell dysfunction. MODY6 is extremely rare, with only a few cases reported in the literature (16).

Insulin Resistance Syndromes and Lipodystrophies Insight into the genetic basis of diabetes has also been culled from several rare insulin resistance syndromes. These syndromes are characterized biochemically by hyperinsulinemia and insulin resistance in fat, muscle, and liver. The insulin receptor was a logical initial candidate gene for the severe hyperinsulinemia described in index patients. The most severe form of insulin resistance is leprechaunism (Donahue syndrome), named for the dysmorphic appearance of the affected infants. Patients may also show intrauterine growth retardation. The syndrome results www.annualreviews.org • Pharmacogenetics in Type 2 Diabetes

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PPARG: gene encoding peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor that acts as a lipid sensor and induces a cascade of transcriptional events that lead to increased insulin sensitivity and adipocyte differentiation TNDM: transient neonatal diabetes mellitus PNDM: permanent neonatal diabetes mellitus DEND: developmental delay, epilepsy, neonatal diabetes

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from homozygous mutations in the insulin receptor. Those affected rarely survive into the second year of life (17). The RabsonMendenall syndrome is characterized by pineal hyperplasia, acanthosis nigricans, accelerated growth, and dental dysplasia. Mutations in the insulin receptor allow incomplete receptor activation (18). The lipodystrophies are characterized by dysregulation of fat storage. Adipose tissue is deposited inappropriately in muscle and liver rather than the usual subcutaneous compartment. Congenital-generalized lipodystrophy (Berardinelli-Seip syndrome) results in loss of subcutaneous fat and hepatomegaly secondary to abnormal fat and glycogen deposition. Two genes have been implicated thus far: BSCL2 on chromosome 11, which encodes the protein seipin, an integral component of the endoplasmic reticulum; and AGPAT2, which encodes an enzyme in the synthesis of glycerophospholipids (19). Other forms of lipodystrophy result from mutations in the LMNA and PPARG genes. LMNA, initially identiﬁed through linkage analysis, is spliced to produce two proteins, lamin A and lamin C, which form part of the nuclear envelope responsible for nuclear trafﬁcking (20). Perhaps the best known of the laminopathies is familial partial lipodystrophy (Dunnigan syndrome). Interestingly, adipose tissue is lost in the extremities as with other lipodystrophies but is preserved in the neck and face. Associated endocrinopathies include polycystic ovaries, hyperandrogenism, and hyperlipidemia. The peroxisome proliferatoractivated receptor γ (PPARγ) is a nuclear hormone receptor highly expressed in adipose tissue that acts as a lipid sensor and induces a cascade of transcriptional events that lead to increased insulin sensitivity and adipocyte differentiation. Mutations in its gene, PPARG, produce a truncated receptor protein that is unable to bind signaling ligands appropriately. Homozygous mutations result in severe insulin resistance, whereas heterozygotes have a mild phenotype.

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Mitochondrial Diabetes Mitochondrial diabetes is another rare variant of monogenic diabetes. Maternally inherited diabetes and deafness (MIDD) is caused by an alanine-to-guanine mutation in the gene encoding the tRNA for leucine. This defect leads to ineffective oxidative phosphorylation.

Neonatal Diabetes Neonatal diabetes, which usually presents in the initial days or months of life, can be transient (resolving at a median of 12 weeks) or permanent. Most cases of transient neonatal diabetes mellitus (TNDM) result from imprinting mutations of the ZAC and HYNAI genes on chromosome 6q (21). This type of neonatal diabetes usually remits in childhood but is associated with an increased risk of T2D in adulthood. Permanent neonatal diabetes mellitus (PNDM), traditionally treated with insulin, has recently been associated with activating mutations in the KCNJ11 gene, which encodes the islet ATP-sensitive potassium channel Kir6.2 (22). Activating mutations in KCNJ11 result in constitutive opening of the inwardly rectifying potassium channel, hyperpolarization of the β cell membrane, and subsequent hypoinsulinemic diabetes. Conversely, loss-of-function polymorphisms have been associated with persistent hyperinsulinemia of infancy (23). Several novel heterozygous missense activating mutations in KCNJ11 have now been identiﬁed in TNDM. The severity of the mutation correlates with the clinical phenotype—some KCNJ11 defects result in PNDM, and even more severe genetic mutations result in the DEND syndrome (developmental delay, epilepsy, neonatal diabetes) (24). Mutations in this gene appear to be the most common cause of neonatal diabetes and account for about one third of PNDM cases (25). The closely associated sulfonylurea receptor (SUR1) also plays an important role in infant diabetes. Such a role was ﬁrst proposed by Huopio et al. (26), who reported that the

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missense mutation E1506K in ABCC8, the gene that encodes SUR1, resulted in congenital, autosomal dominant hyperinsulinemia in a Japanese family. The dominant mutation results in neonatal hyperinsulinemia and subsequent hypoglycemia, and the heterozygous form may increase adult susceptibility to common T2D. Consistent with these expectations, Babenko et al. (27) recently described a number of activating mutations in ABCC8 that affect SUR1 function and cause neonatal diabetes. The pharmacogenetic implications of these ﬁndings are discussed below.

COMMON TYPE 2 DIABETES Monogenic diabetes, as described above, results from single mutations that lead to profound phenotypes. The complex individual portfolio of susceptible and protective alleles in polygenic T2D is more difﬁcult to discern, given the limited individual impact of a single genetic locus. Indeed, a full understanding of the complex gene-gene and gene-environment interactions at play in this disesase has proven quite challenging thus far.

MODY Genes To identify a connection between monogenic diabetes and common T2D, a natural starting point is to test whether common but less severe variants of monogenic diabetes might play a role in the pathogenesis of T2D. This hypothesis has been fairly comprehensively tested for the six known MODY genes, with suggestive but often conﬂicting results (28– 36). For example, common variants of HNF4A (MODY1) have been associated with T2D in both Finnish and Ashkenazi Jewish populations (28, 29). In addition, variants in GCK (MODY3) have been associated with elevated fasting blood glucose levels and possibly with common T2D, although the evidence here is somewhat weaker (36). Given the small effect of GCK mutations on fasting blood sugar (approximately 2 mmol/L), the mutation is un-

likely to have a substantial impact on T2D risk.

CAPN10 One of the earliest diabetes genes identiﬁed was CAPN10, which encodes the intracellular cysteine protease Calpain 10. A linkage signal in chromosome 2q in a Mexican-American cohort (LOD score 4.1) (37) eventually led to the identiﬁcation of an adenosineto-guanosine polymorphism in intron 3 in CAPN10, which was associated with T2D. Although subsequent studies have not produced a consistent relationship between variation in CAPN10 and metabolic phenotypes, recent meta-analyses indicate that variants in CAPN10 may indeed confer a modest increase in T2D susceptibility (38–40). Interestingly, Calpain inhibitors increase insulin secretion and increase the metabolism of muscle glucose to glycogen in animal models (41).

Pharmacogenetics: the study of the interaction of therapies with target genes

PPARG A proline-to-alanine change in codon 12 of PPARG (P12A) was the ﬁrst genetic variant to be deﬁnitively implicated in the common form of T2D (42). Deeb et al. (43) initially reported that the alanine allele was associated with increased insulin sensitivity and protection from T2D in a Finnish and secondgeneration Japanese cohort. Since this initial work, the preponderance of evidence has conclusively supported PPARG’s association with T2D, with an odds ratio (OR) of ∼1.2 (44). The risk of T2D conferred by this singlenucleotide polymorphism (SNP) has been studied prospectively in the Finnish Diabetes Prevention Study and the larger Botnia Prevention Study. In the Finnish study, >500 subjects with impaired glucose tolerance were randomized to placebo or lifestyle modiﬁcation. The relative risk of developing diabetes was doubled in alanine carriers in the placebo arm, contradicting the prior evidence that the alanine allele was protective. In the larger Botnia study, comprising >2000 subjects, www.annualreviews.org • Pharmacogenetics in Type 2 Diabetes

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proline homozygotes were 1.7 times more likely to develop diabetes than alanine carriers (45). Our study of 3548 participants enrolled in the Diabetes Prevention Program (DPP) also revealed a modest risk for the homozygous proline genotype (46). Interestingly, we uncovered a signiﬁcant interaction between genotype and body mass index (BMI): The protective effect of the alanine allele was lost in subjects with a BMI greater than 35 kg/m2 . It is possible that this ﬁnding may explain some of the heterogeneity of results noted in the literature regarding this variant.

KCNJ11 The KCNJ11 gene, described above in the context of neonatal diabetes, encodes the β cell potassium channel and is functionally closely related to the sulfonylurea receptor SUR1, encoded by ABCC8. These genes are adjacent to each other on chromosome 11. Work on this locus has conﬁrmed that a less drastic change in a gene implicated in a rare, monogenic subtype of diabetes can indeed contribute to its more common form: SNP E23K of KCNJ11 has now been convincingly associated with T2D. Although initial smaller studies failed to replicate the association of the E23K polymorphism with T2D, largescale studies and meta-analyses have consistently associated the lysine variant with T2D, with an OR of ∼1.15 (35, 47–52).

group of diabetes genetics investigators (reviewed in Reference 54). All of these studies demonstrate robust and convincing statistical evidence of association with diabetic risk and consistent effect sizes. The accumulated evidence from >50,000 subjects reveals a remarkable overall p value of <10−80 . The effect of the risk allele appears to be additive; one allele confers ∼40% risk whereas two copies confer 80% risk of diabetes (54). The precise mechanisms of this increased risk are poorly understood. TCF proteins are transcription factors that affect cell proliferation and differentiation via the Wnt signaling pathway. Tcf7l2-null mice have marked deﬁciencies in intestinal cells including enteroendocrine cells, which are responsible for incretin production. Incretins, including glucagon-like peptide 1 (GLP-1), inﬂuence gut mobility, satiety, and energy regulation, thus providing a plausible biological explanation for the role they may play in T2D. The TCF7L2 risk allele may result in a defective or poorly expressed protein that leads to decreased insulin secretion and consequent hyperglycemia. We tested the clinical implications of this hypothesis in the multiethnic cohort of the DPP and found that the risk allele was associated with impaired insulin secretion but not insulin resistance (55). These ﬁndings have been recently conﬁrmed in a general population (56).

PHARMACOGENETICS TCF7L2 Investigators at deCODE Genetics in Iceland recently reported a strong association of a common allele in the gene of transcription factor 7–like 2 (TCF7L2) with an increased risk of T2D in three white populations (53). In this case-control study of 3774 subjects from Iceland, Denmark, and the United States, the estimated allelic risk was 1.56 ( p = 4.7 × 10−18 ). The effect size was greater than that of any diabetes gene identiﬁed to date. These results have subsequently been conﬁrmed in multiple ethnic groups by virtually every other 100

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Much of the work described above is motivated by the prospect that a clearer genetic understanding may lead to more effective strategies for diabetes prevention and treatment. Pharmacogenetics, the study of the interaction of therapies with target genes, is emerging as a fruitful discipline with testable hypotheses (57). The recently described drugresponse interaction between warfarin and variation in VKORC1 illustrates this tantalizing paradigm (58). In metabolic disease, treatment of leptin-deﬁcient humans with recombinant leptin causes a dramatic reduction

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in adiposity and normalization of appetite (58a). T2D is a promising disease exemplum in which pharmacogenetics can have a potentially tremendous impact, given the reasonably well-understood pathophysiology of glucose metabolism and the heavy burden of disease. Heterogeneity in response to diabetic medications, including thiazolidinediones (TZDs) and sulfonylurea therapy, is well known to many physicians who treat patients with T2D, and these variable response rates may result from heterogeneity at the genetic level. Approximately 5%–7% of diabetic patients treated with sulfonylurea monotherapy convert to insulin annually as a result of treatment failure (59). Although many genes have now been reproducibly associated with T2D, much less is known about gene-drug interactions. Similarly, the putative genetic predisposition of selected individuals to the development of side effects is presently unexplored.

Pharmacogenetics of Monogenic Diabetes Perhaps the most compelling evidence suggesting the validity of a pharmacogenetic approach in diabetes comes from elegant studies conducted in the newly described forms of PNDM. Even though mutated Kir6.2 β cell potassium channels are refractory to endogenous ATP, they can often respond to sulfonylureas, medications that bind to the SUR1 receptor and prompt channel closure. Sagen et al. (60) showed in 2004 that PNDM patients with Kir6.2 mutations could be effectively managed on a sulfonylurea without insulin therapy, with improved hemoglobin A1C levels. Two subsequent studies were published simultaneously in 2006 conﬁrming this ﬁnding. Babenko et al. (27) identiﬁed ABCC8 mutations in nine families with neonatal diabetes and conﬁrmed that sulfonylurea blockade was preserved. Pearson et al. (61) demonstrated that 44 out of 49 PNDM patients with Kir6.2 mutations could be successfully transitioned from

insulin to sulfonylurea therapy. Sulfonylurea treatment improved insulin secretion, and glycemic control was sustained at one year. A similar phenomenon can be observed in some MODY subtypes. MODY3 patients are uniquely sensitive among the MODY subtypes to sulfonylurea medications because the affected β cell potassium channel is downstream of the physiologic effects known to result from genetic mutations. In a randomized crossover trial in which adult responses to a sulfonylurea and metformin were compared between patients with HNF1A mutations and matched type T2D patients, the HNF1A group demonstrated a ﬁvefold greater response to sulfonylurea than metformin. Their response to sulfonylurea was fourfold higher than that of the T2D patients (62).

TZD: thiazolidinedione

Pharmacogenetics of Polygenic Diabetes An early focus of T2D pharmacogenetic studies has been the common, functional P12A PPARG variant, since this nuclear receptor is the known drug target of TZD medications (63). TZDs are known to decrease glucose levels in T2D (64); however, clinical experience and multiple clinical trials indicate that there is a large percentage of nonresponders. In vitro studies reveal decreased binding of the variant receptor to the PPAR response element in the presence of increasing concentrations of TZDs (65). Four published studies have examined the effect of PPARG P12A on the response to ¨ TZDs. Bluher et al. (66) found that when 131 diabetic subjects were treated for 26 weeks with pioglitazone, the percentage of responders (deﬁned as >20% decrease in fasting glucose or >15% decrease in hemoglobin A1C ) did not differ between proline homozygotes and alanine carriers. Similarly, in the TRIPOD (Troglitazone in Prevention of Diabetes) study, one third of subjects showed no increase in insulin sensitivity in response to troglitazone (400 mg daily); however, the P12A variant did not explain this failure (67). www.annualreviews.org • Pharmacogenetics in Type 2 Diabetes

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These same investigators continued their study on the same subjects by genotyping 131 common variants of PPARG and investigating their relationship to treatment response. Eight different polymorphisms were associated with response to troglitazone therapy (67a). In contrast with the above ﬁndings, when Kang et al. (68) examined the response of 198 T2D patients to rosiglitazone, they found that 15 carriers of the P12A polymorphism had a better response to TZD therapy than Pro12Pro homozygotes. Patients with the alanine allele had a larger reduction in fasting glucose and hemoglobin A1C than those without the allele, although the sample size was small. On the other hand, we were unable to show an effect of PPARG P12A or ﬁve of the other PPARG polymorphisms implicated by Snitker et al. (67) on response to troglitazone therapy in the 340 participants randomized to troglitazone in the DPP (46). Thus, knowledge of allelic variation at this locus does not yet offer a rationale for therapeutic choices. The impact of the KCNJ11 genetic variant E23K on the effectiveness of sulfonylurea therapy is also unclear. Sesti et al. (69) genotyped KCNJ11 in 525 white T2D patients and investigated whether failure to respond to sulfonylurea therapy (deﬁned as fasting plasma glucose >300 mg/dl despite combined sulfonylurea-metformin therapy and appropriate diet) was due, in part, to the risk allele. The authors found carriers to have a relative risk of failure of 1.45 compared to E23E homozygotes. Also, the risk allele was associated with an earlier onset of diabetes and worse metabolic control in nonresponders. This group also performed in vitro experiments of human islet secretory function in response to glucose and glibenclamide, a second-generation sulfonylurea. After 24-h exposure to high glucose concentration followed by stimulation with glibenclamide, insulin release was 30% lower in K allele carriers. These results stand in contrast to those of the UK Prospective Diabetes Study (UKPDS), in which the authors found no

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signiﬁcant association of the E23K variant with response to sulfonyurea therapy in 364 newly diagnosed T2D patients (47). The disparity between these results may follow from different deﬁnitions of treatment failure, durations of therapy, speciﬁc sulfonylureas used, patient clinical characteristics, or statistical ﬂuctuations. In the DPP, although metformin was effective in lowering the risk of diabetes for E23E homozygotes, it failed to protect carriers of the risk lysine allele. In addition, K23K homozygotes randomized to metformin did not show the expected improvement in insulin sensitivity at one year (70). This effect is poorly understood and will require validation with additional physiologic studies in an independent cohort. Although the precise molecular mechanism by which TCF7L2 contributes to T2D is still under investigation, it is apparent that this variant confers a defect in insulin secretion, possibly by affecting GLP-1 metabolism (55, 56, 71–73). In regard to a possible drug × genotype interaction, the recently published Go-DARTS study genotyped 6516 UK participants for TCF7L2 and found the T allele overrepresented in individuals requiring insulin treatment and underrepresented in the patients being managed by diet alone. The authors concluded that TCF7L2 variants may be associated with increased disease severity and therapeutic failure (74). Another recent publication from the same group reported the effect of TCF7L2 genotypes on therapeutic response in 901 diabetic patients treated with sulfonylurea and 945 patients treated with metformin. Carriers of the risk TCF7L2 variants were more likely to fail to sulfonylurea but not metformin therapy as measured by a hemoglobin A1C > 7% within 3–12 months after treatment initiation (74a). This ﬁnding further supports the hypothesis that TCF7L2 variants are important in β cell function. Finally, studies from the DPP on participants with impaired glucose tolerance and elevated fasting glucose reveal that the lifestyle preventive intervention was effective

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in reducing the genetic risk conferred by the high-risk homozygous genotype to the level of their wild-type counterparts (55). A possible interaction between this locus and insulin secretagogues, incretin mimetics, or dipeptidyl peptidase IV inhibitors has not yet been investigated. One exciting new area of active research involves variability in medication transport and metabolism. Recent work by Shu et al. (75) has introduced the hepatic transport of metformin as yet another area in which genetic variability may play a signiﬁcant role in therapy response . This group showed that the organic cation transporter 1 (OCT1), which participates in the hepatic uptake of metformin, may contribute to variation in response to metformin. These authors reported a reduced effect of metformin on AMP kinase phosphorylation in Oct1-deﬁcient mouse hepatocytes and poor absorption of metformin in Oct1-deﬁcient mice. They also showed that the OCT1 reduced-function allele in healthy human subjects is predictive of higher glucose levels during an oral glucose tolerance test. Thus, this complementary area of pharmacogenetic investigation holds great promise in explaining the human variability in drug response.

GENOME-WIDE ASSOCIATION SCANS The most dramatic recent advance in T2D genomics is the availability of data from genome-wide association scans (GWASs), enabled by the development and affordability of high-throughput genotyping platforms, the compilation of SNPs in public databases, the assembly of large patient cohorts, the completion of the HapMap, and the generation of sophisticated analytical tools. GWASs query the genome while remaining agnostic to biological plausibility, and these surveys currently provide 70%–80% coverage of human common genomic variation. Five GWASs, as well as a partial metaanalysis that encompasses three of them, have

been published in the past year (Table 1). The scans have both conﬁrmed known T2D loci and introduced at least six novel diabetes genes. Of note, TCF7L2 has been replicated as the genetic locus with unequivocally the strongest signal. The ﬁrst GWAS was performed on a French population in February 2007. It reproduced the powerful impact of TCF7L2 (OR 1.65, p < 1.0 × 10−7 ) and also identiﬁed HHEX (OR 1.21, p = 9.1 × 10−6 ) and SLC30A8 (OR 1.26, p = 5.0 × 10−7 ) as potential novel T2D loci. SLC30A8, a zinc transporter gene initially cloned and sequenced in 2004, is expressed exclusively in pancreatic β cells, where it transports zinc from the cytoplasm into insulin secretory vesicles (76). This is a critical step in the ﬁnal biosynthetic pathway of insulin production and secretion. HHEX is thought to be involved in β cell development or function (77). Two other loci reported by these investigators have not yet been replicated in subsequent work. Following this initial report, four wellpowered GWAS surveys were published simultaneously. In a Nature Genetics report, the deCODE group and collaborators conﬁrmed again the strong signal of TCF7L2 (OR 1.38, p = 1.9 × 10−10 ) and replicated the HHEX and SCL30A8 ﬁndings. CDKAL1 (CDK5 regulatory subunit associated protein 1–like 1) was also identiﬁed by these investigators (OR 1.2, p = 18 × 10−4 ). This gene affects CDK5/CDK5R1 activity, and in so doing, may lead to β cell degeneration. Further, they found that the insulin response of homozygotes was ∼20% lower than that of heterozygotes or noncarriers, conﬁrming the biological plausibility of this ﬁnding (78). Simultaneously with the deCODE paper, the Diabetes Genetics Initiative (DGI) (79), the Wellcome Trust Case Control Consortium (WTCCC), (80) and the Finland– United States Investigation of NIDDM Genetics (FUSION) (81) each published a GWAS in Science. The studies represent, in aggregate, more than 32,000 samples and have www.annualreviews.org • Pharmacogenetics in Type 2 Diabetes

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Genetic loci associated with type 2 diabetes that have achieved genome-wide significance Sladek et al. (77) (n = 6794)

Markera

Chr

Position

Nearest gene

Notes

Risk allele

deCODE (78) (n = 10,056)

OR (95% CI)

p value

OR (95% CI)

p value

1.38

1.9×10−10

1.2

7.7 × 10−9

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Previously known rs7903146

10

114748339

TCF7L2

Transcription factor; risk allele impairs insulin secretion

T

1.65

3.3 × 10−10

rs5219

11

17366148

KCNJ11

Kir6.2 potassium channel; risk allele impairs insulin secretion

T

1.34

0.074

rs1801282

3

12368125

PPARG

Nuclear C hormone receptor; target for thiazolidinediones

1.22

0.11

GWAS-identified rs4402960

3

186994389

IGF2BP2

Growth factor binding protein; pancreatic development

T

rs10811661

9

22124094

CDKN2B Cyclindependent kinase inhibitor and p15 tumor suppressor; islet development

T

rs7754840

6

20769229

CDKAL1

Homologous to CDK5RAP1, CDK5 inhibitor; islet glucotoxicity sensor

C

rs1111875

10

94452862

HHEX

Pancreatic transcription factor; pancreatic development

C

1.21

8.6 × 10−6

1.17

0.001

rs13266634

8

118253964

SLC30A8 β cell zinc transporter ZnT8; insulin biosynthesis

C

1.18

5.0 × 10−7

1.19

0.001

a b

Proxies include: for rs7754840, rs10946398; for rs7903146, rs7901695; and for rs5219, rs5215. Meta-analysis of DGI, WTCCC, and FUSION.

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Table (Continued ) DGI (79) (n = 13,781)

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OR (95% CI)

p value

WTCCC (80) (n = 13,965) OR (95% CI)

p value

FUSION (81) (n = 4808) OR (95% CI)

p value

Meta-analysisb (n = 32,554) OR (95% CI)

p value

1.38 (1.31–1.46)

2.3 × 10−31

1.37 (1.25–1.49)

6.7 × 10−13

1.34 (1.21–1.49)

1.4 × 10−8

1.37 (1.31–1.43)

1.0 × 10−48

1.15 (1.09–1.21)

1.0 × 10−7

1.15 (1.05–1.25)

1.3 × 10−3

1.11 (1.02–1.21)

0.014

1.14 (1.10–1.19)

6.7 × 10−11

1.09 (1.01–1.16)

0.019

1.23 (1.09–1.41)

1.3 × 10−3

1.20 (1.07–1.33)

1.4 × 10−3

1.14 (1.08–1.20)

1.7 × 10−6

1.17 (1.11–1.23)

1.7 × 10−9

1.11 (1.05–1.16)

1.6 × 10−4

1.18 (1.08–1.28)

2.4 × 10−4

1.14 (1.11–1.18)

8.9 × 10−16

1.20 (1.12–1.28)

5.4 × 10−8

1.19 (1.11–1.28)

4.9 × 10−7

1.20 (1.07–1.36)

2.2 × 10−3

1.20 (1.14–1.25)

7.8 × 10−15

1.08 (1.03–1.14)

2.4 × 10−3

1.16 (1.10–1.22)

1.3 × 10−8

1.12 (1.03–1.22)

9.5 × 10−3

1.12 (1.08–1.16)

4.1 × 10−11

1.14 (1.06–1.22)

1.7 × 10−4

1.13 (1.07–1.19)

4.6 × 10−6

1.10 (1.01–1.19)

0.025

1.13 (1.08–1.17)

5.7 × 10−10

1.07 (1.00–1.16)

0.047

1.12 (1.05–1.18)

7.0 × 10−5

1.18 (1.09–1.29)

7.0 × 10−5

1.12 (1.07–1.16)

5.3 × 10−8

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been combined in an initial meta-analysis of the best results. These GWASs conﬁrmed the known TCF7L2, KCNJ11, and PPARG loci as well as the recently published HHEX and SCL30A8 associations. They independently discovered the CDKAL1 association and also identiﬁed the novel diabetes loci IGF2BP2 (OR 1.14, p = 8.9 × 10−16 ) and CDKN2B (OR 1.2, p = 7.8 × 10−15 ). Taken together, these studies provide strong evidence for the potential power of GWASs. The following considerations emerge from this sizeable advancement in our knowledge of the genetic etiology of T2D:

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1. All of the genetic effects are modest, with a ceiling of effect (produced by the combination of allele frequency and genetic risk) hovering around TCF7L2. 2. The collaborative integration of multiple datasets and consequent very large sample sizes will be essential to conﬁrm or refute individual ﬁndings, extend the search for statistical associations further down the p-value distribution, explore phenotypic covariates, and assess epistatic interactions. 3. Novel biology remains to be discovered in relation to the effects of inherited DNA variation on human phenotypic diversity in general and metabolic traits in particular.

4. Association studies, if adequately powered, can indeed help characterize the genetic architecture of complex diseases. 5. This rapid progress has been greatly facilitated by the generous and disinterested posting of anonymized genomic data and analytical tools in the public domain.

CONCLUSIONS Genetic studies of T2D have been extremely productive. Multiple genes have been identiﬁed in the past ten years, and a series of GWAS surveys were released in the ﬁrst months of 2007. This growing ensemble captures only a small fraction of the genetic risk of T2D. Moreover, the precise metabolic consequences of these novel genetic discoveries are yet to be determined, and whether these results will affect clinical practice must be tested empirically. Understanding the complex interactions among genetic proﬁles, individual lifestyles, and environmental factors lies at the core of effective diabetes treatment. Thus, although this body of work represents a tremendous accomplishment, it is but a springboard. The hope is that improved genetic understanding may one day enhance the health and life span of the diabetic patients we treat.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

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74a. Pearson ER, Donnelly LA, Kimber CH, et al. 2007. Variation in TCF7L2 inﬂuences therapeutic response to sulfonylureas: a GoDARTs study. Diabetes 56:2178–82 75. Shu S, Sheardown SA, Brown C, et al. 2007. Effect of genetic variation in the organic cation transporter 1 (OCT1) on metformin action. J. Clin. Invest. 117:1422–31 76. Chimienti F, Devergnas S, Favier A, et al. 2004. Identiﬁcation and cloning of a β-cell speciﬁc zinc transporter, Zn T8, localized into insulin sectretory granules. Diabetes 53:2330– 37 77. Sladek R, Rocheleau G, Rung J, et al. 2007. A genome-wide association study identiﬁes novel risk loci for type 2 diabetes. Nature 445:828–30 78. Steinthorsdottir V, Thorleifsson G, Reynisdottir I, et al. 2007. A variant in CDKAL1 inﬂuences insulin response and risk of type 2 diabetes. Science 39:770–75 79. Diabetes Genetics Initiative of Broad Institute of Harvard and MIT, Lund University and Novartis Institutes for BioMedical Research. 2007. Genome-wide association analysis identiﬁes loci for type 2 diabetes and triglyceride levels. Science 316:1331–36 80. Zeggini E, Weedon MN, Lindgren CM, et al. 2007. Replication of genome-wide association signals in U.K. samples reveals risk loci for type 2 diabetes. Science 316:1336–41 81. Scott LJ, Mohlke KL, Bonnycastle LL, et al. 2007. A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants. Science 316:1341–45

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:95-111. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030; email: [email protected], [email protected]

Annu. Rev. Med. 2008. 59:113–29

Key Words

First published online as a Review in Advance on October 25, 2007

microarray, chromosomal imbalance, genotyping, mutation analysis, DNA sequencing

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.012907.101800 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0113$20.00

Abstract Advances in the fabrication of DNA microarrays as well as transformations in detection chemistries have vastly increased the throughput for genotyping, DNA sequencing, and array-based copy number analysis (ABCNA). Rapid changes in technology are not only affecting research but also revolutionizing DNA diagnostics. Here we focus on the application of high-throughput ABCNA and genotyping. Targeted and genome-wide ABCNA has led to the discovery of extensive DNA copy number variation in the population and the delineation of many previously unrecognized submicroscopic chromosomal aberrations (genomic disorders). High-throughput singlenucleotide polymorphism (SNP) genotyping is being widely applied in genome-wide association studies (GWASs) with recent successes in identiﬁcation of common variants that confer risk for common adult diseases. Future applications of high-throughput genotyping and array-based DNA sequencing technology will undoubtedly involve research and diagnostic analyses of rare mutations and perhaps ultimately enable full individual genome sequencing.

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HIGH-THROUGHPUT ANALYSIS OF COPY NUMBER VARIATION Feature: a spot on an array, whether BAC, oligonucleotide, PCR product, or other

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Array CGH: array comparative genomic hybridization ABCNA: array-based copy number analysis Chromosomal microarray analysis (CMA): a clinically friendly term to encompass copy number analysis, including array CGH but also non-CGH platforms such as Affymetrix and Illumina when used to determine copy number BAC: bacterial artiﬁcial chromosome Copy number variant (CNV): alternative structure in genomic DNA that typically includes deletions (reduced copy number) and duplications (more copies) in adjacent segments of DNA. The presence of these deleted/ duplicated segments can vary among individuals; they are, therefore, a type of polymorphism

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Microarrays of increasing complexity are becoming available at decreasing cost per feature. This has made it feasible to perform genome-wide analysis of copy number variation for research purposes and for clinical diagnosis. Two distinct platforms are available: Some methods use array comparative genomic hybridization (array CGH), in which direct comparison to a control is essential, whereas others determine relative copy number for a region quantitatively within a single genome without the use of an internal control in a cohybridization. All of these methods quantify copy number differences within a human sample for the purposes of research or clinical diagnosis. As a group, they all offer quantitative array-based copy number analysis (ABCNA), and in a clinical setting, they can be described as chromosomal microarray analysis (CMA). Some commercial platforms for array CGH use large insert clones such as bacterial artiﬁcial chromosomes (BACs) (Perkin-Elmer and BlueGnome), but platforms using oligonucleotides (Agilent Technologies and Nimblegen) are increasingly preferred (1). The two major commercial platforms that do not use CGH (Affymetrix and Illumina) grew out of arrays that were originally designed for typing single-nucleotide polymorphisms (SNPs). These platforms now offer combined SNP genotyping and copy number analysis.

GENOME ARCHITECTURE The enormous extent of copy number variation in the human genome has only recently been appreciated. Although copy number variation in the human genome has been known for decades for loci such as the αglobin and color pigment genes, the 1991 discovery that genomic duplications encompassing the PMP22 gene are the cause of the most common form of Charcot-MarieTooth disease (2) opened the much broader Beaudet

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vista of what have been called genomic disorders (3, 4). Genomic disorders are clinical phenotypes caused by abnormal dosage or dysregulation of one or more genes resulting from rearrangement of the genome. It has become clear that low copy repeats (LCRs) mediate deletions, duplications, and inversions. The extent of LCRs in the genome was extensively documented by Eichler and colleagues using bioinformatic methods (5). These genomic LCRs are genomic sequences of substantial length that are typically 98–99% identical in sequence. Copy number variants (CNVs) often occur within a region ﬂanked by LCRs, but many CNVs occur independently of LCRs. LCRs can contain one or more intact functional genes within the repeat, although this is often not the case. The location of LCRs has been used to identify regions of the genome that will be particularly susceptible to deletion and duplication (6). A CNV whose less common form accounts for at least 1% of the alleles in the population can be deﬁned as a copy number polymorphism by analogy to the standard deﬁnition of a polymorphism.

FUNCTIONAL SIGNIFICANCE OF COPY NUMBER VARIANTS From current data, it is clear that some CNVs represent single-region genomic disorders with very high or complete penetrance. These may involve a single gene within the CNV that mediates most or all of the phenotype (e.g., Charcot-Marie-Tooth disease type 1A and duplication of PMP22) or multiple genes contributing to the phenotype (as for Williams syndrome). It also seems likely, but is not proven, that the majority of CNVs in the genome are benign and have minimal or no detectable effect on the phenotype. However, it is also clear that some CNVs mediate a single gene/region disorder with incomplete penetrance [e.g., deletions of PMP22 causing HNPP (hereditary neuropathy with liability to pressure palsies), in which many individuals with the genotype are asymptomatic

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throughout life]. Of enormous interest at this moment is the role of CNVs in common diseases of complex etiology, in which the CNV may be only one of a group of genetic variations contributing to the phenotype. One can also imagine that CNVs might contribute to phenotypic variation within the normal population, affecting skin pigmentation, height, behavioral differences, musicality, mathematical ability, and a myriad of other human traits. It is known that deletion of the P locus in association with Prader-Willi syndrome and Angelman syndrome can cause hypopigmentation. It is known that deletion of the imprinted IGF2 locus on the paternal chromosome can lead to short stature and that duplication can lead to increased stature or gigantism.

METHODOLOGY As noted above, many different methods are available for analysis of copy number variation, some of which utilize the principles of array CGH (Figure 1). Much of the early work with array CGH has utilized large insert BAC arrays. Unlike oligonucleotide arrays, which for the most part have been produced by large commercial entities, large insert BAC arrays can be produced by academic labs and small companies. Another advantage of BAC arrays is that they can obtain a result from a miniscule amount of starting DNA because the hybridization signal can come from tens of kilobases (kb) of DNA. Oligonucleotide arrays have the great advantage that they can be chosen to be free of repetitive sequences, and they interrogate a very speciﬁc region. Oligonucleotides used in the various platforms typically range from 20 to 80 nucleotides. Oligonucleotides offer the possibility of strand-speciﬁc detection, which can be useful in certain circumstances. A disadvantage of BAC arrays is that they contain repetitive human sequences and require the use of Cot-1 DNA to suppress hybridization of repetitive sequences, and even then the signal-to-background ratio is

not as robust as for oligonucleotide arrays. Except for the Illumina platform, which attaches oligonucleotides to microspheres, the BACs or oligonucleotides are attached to a ﬂat surface of glass or silica. The oligonucleotide platforms that offer SNP detection have potential advantages in terms of detection of uniparental disomy (UPD) and loss of heterozygosity (LOH). SNP detection offers the possibility to determine the parent of origin for a de novo deletion or duplication, and to determine paternity, in addition to detection of UPD and LOH. Detection of LOH is particularly important for analysis of tumors. The platforms that combine copy number quantitation and SNP detection offer the opportunity to do genome-wide studies that might detect disease susceptibilities related either to a CNV or a SNP. It is obvious that CNVs have a much higher mutation rate than SNPs. This has substantial implications for linkage and association studies that go far beyond what can be reviewed here, but the linkage disequilibrium (LD) and heritability found with CNVs has been subjected to an initial evaluation (7). The availability of wholegenome ampliﬁcation (WGA) allows an enormous increase in the amount of research and diagnostic analysis that can be done with a small clinical sample. WGA is particularly useful for large collaborative studies analyzing a collection of samples that is available to multiple investigators. WGA can be applied to single-cell analysis, a strategy that potentially can be used for preimplantation genetic diagnosis.

LD: linkage disequilibrium WGA: whole-genome ampliﬁcation

RESEARCH APPLICATIONS Current research has focused on simply deﬁning the extent of copy number variation in the human genome. A number of publications have analyzed a series of normal individuals with generally similar results (5, 8, 9). One of the most extensive studies of 270 individuals identiﬁed 1447 CNVs covering 360 megabases (Mb) or 12% of the human genome. These CNVs encompass www.annualreviews.org • Array Diagnostics

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a.

Label patient DNA Label control DNA with Cy3 with Cy5

Mix

Hybridize DNA to genomic clone microarray

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Analyze Cy3/Cy5 fluorescence ratio of patient to control

Cy3/Cy5 ratio >1 Cy3/Cy5 ratio <1 Deletion Duplication

Oligo array no dye reversal

b. BAC array with dye reversal -1.5

-1

-0.5

0

0.5

1

Oligos within a BAC region 1.5

RP11-487A2

0 Figure 1 Array CGH methodology and a comparison of BAC and oligonucleotide arrays. (a) For array comparative genomic hybridization, a patient’s DNA is labeled with green dye (left) and a control DNA is labeled with red dye (right). The DNA preparations are mixed and cohybridized to an array of BACs or oligonucleotides on a glass slide. The DNA bound to each spot is quantiﬁed using a laser scanner. In the patient’s DNA, normal regions will be indicated by a yellow balanced color; regions of duplication will be identiﬁed by greener spots, and regions of deletion will be identiﬁed by redder spots. (b) A patient sample with a small deletion affecting one BAC is shown on a BAC array (left) and on an oligonucleotide array simulating a BAC conﬁguration (middle). Data for individual oligonucleotides within the BAC are also shown (right). The deviation of the abnormality relative to background is greater for the oligonucleotide array than for the BAC array.

hundreds of functional genes, and as many as 2908 RefSeq genes were identiﬁed using multiple platforms. A substantial fraction of these CNVs are very rare and 116

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were seen on only one chromosome in 270 individuals. The immediate future promises to be very exciting for research on the role of CNVs

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in human evolution and diversity as well as research related to disease susceptibility and normal human variation. Although the population genetics of copy number variation is largely unexplored, one early report has shown extensive differences among African, European, and Asian populations (8). The extent to which CNVs contribute to complex disease traits and to normal human variation is virtually unexplored at this time. Any gene located within a CNV might be completely insensitive to copy number or dosage with either deletion or duplication. This is thought generally to be true for heterozygous deﬁciency of many genes encoding enzymes associated with inborn errors of metabolism, where heterozygous individuals are generally assumed to have a normal phenotype, although subtle phenotypic variations may be more common than is widely appreciated. There is very extensive knowledge of medical disorders in which haploinsufﬁciency for a gene generates a distinct phenotype with very high penetrance and some amount of variation in expression. These genes are extremely dosage-sensitive. There is less information about dosage sensitivity related to duplication, although the experience with CMT-1A makes it clear that duplications can affect phenotypes on the basis of sensitivity to excessive dosage. It seems quite likely that many genes would have intermediate levels of sensitivity to dosage variation, that the effect would depend on the genotype at other loci and on the environment (i.e., the mutations have incomplete penetrance), and that there would be extensive phenotypic variability among individuals symptomatic from a given deletion or duplication (i.e., the mutations show variation in expression).

CLINICAL APPLICATIONS Copy number analysis is already in use for identiﬁcation of genomic mutations causing mental retardation, developmental delay, dysmorphic features, and other syndromic abnor-

malities. The majority of studies have used array CGH, but some have used oligonucleotide platforms that detect both SNPs and copy number independent of a CGH strategy. This literature has been reviewed recently (10). There are already two reports summarizing the experience with 1000–3000 clinical pediatric samples (11, 12), and the unpublished experience amounts to tens of thousands of clinical samples. It is overwhelmingly clear that ABCNA can detect a wide range of genomic abnormalities that are not detectable by conventional Giemsa banded karyotype. ABCNA methods are particularly advantageous for detection of duplications, which are difﬁcult to detect even with metaphase ﬂuorescence in situ hybridization (FISH). ABCNA methods provide the equivalent of hundreds or even thousands of FISH tests in a single clinical assay, with enormous cost savings compared to performing each FISH test individually. The frequency with which abnormalities of diagnostic signiﬁcance are detected in various patient populations is quite variable, depending on the extent of previous cytogenetic evaluation and on the heterogeneity of the populations studied. The rate of discovery of causative abnormalities also depends on the complexity of the array platform being used, and there has been considerable debate about the advantages and disadvantages of targeted arrays versus tiling path arrays. We have estimated that ABCNA will detect a causal abnormality in ∼12%–18% of children with multiple congenital anomalies and/or developmental delay–mental retardation, with 3%–5% of these abnormalities being detected by a banded karyotype and 9%–13% detected by ABCNA but not by karyotype (10). The question of whether to use targeted arrays or tiling arrays in the clinic has been hotly debated. Targeted arrays typically focus on regions of known disease pathology, such as all known deletion and duplication syndromes. Targeted arrays additionally often have relatively dense coverage of subtelomeric www.annualreviews.org • Array Diagnostics

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and pericentromeric regions. These targeted arrays can establish an etiologic abnormality in a great number of patients who have a normal karyotype. The targeted arrays typically focus on CNV regions that are known to give phenotypic abnormalities with near 100% penetrance while avoiding regions of common CNVs where the clinical signiﬁcance is unknown. Often, clinicians using ABCNA encounter a CNV on which there is minimal previous information. At least four sources of information bear on the clinical interpretation of such CNVs. First, if the CNV is de novo in the child and not present in either parent, it is statistically much more likely to be the cause of the child’s disabilities. Second, if the CNV is in a region known to be highly polymorphic in the normal population, it is less likely to be of clinical signiﬁcance. Databases of CNVs are currently available on most genome browsers. Third, the size of the deletion or duplication matters. In general, the presumably benign CNVs are smaller than the disease-causing CNVs; the median sizes of CNVs from two platforms in one study were 228 kb and 81 kb (8), whereas disease-causing mutations are often many megabases in size. Finally, the gene content of a region can help to determine the clinical signiﬁcance. If it is known that point mutations causing haploinsufﬁciency produce a phenotype with high penetrance, a deletion can be presumed to be the cause of that phenotype, even if a genomic deletion of the region has not been reported previously. Deletions or duplications encompassing large numbers of genes are statistically more likely to be disease-causing, since there is a probability that at least one gene in the region is dosagesensitive. In contrast to targeted arrays, tiling arrays offer much greater density of analysis and a greater chance of detecting a clinically causative abnormality if any is present. However, high-density tiling arrays typically detect 20–30 CNVs in a normal individual compared to a consensus genome. The majority of these CNVs will be common variants that are very likely to be benign on the basis of cur-

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rent databases. However, some CNVs, perhaps 2–10, will be rare, and analysis of parents will be indicated to determine whether any of the variants are de novo rather than inherited. The use of targeted arrays for study of children with various disabilities can now be considered an advanced standard of care, although only ∼10% of chromosomal studies will include ABCNA in 2007. This proportion is increasing rapidly and will soon be the majority. It is probably most cost-effective to start with targeted arrays and regard tiling arrays as the next generation of technology. In most cases, parents should be studied for abnormalities detected by targeted arrays. A reasonable case can be made that all cases with normal results on targeted arrays could go on to tiling arrays. The clinician might well go directly to the highest-density tiling array that is available within an acceptable cost, and assume that it will be necessary to study parents in all cases. The use of ABCNA in the clinical evaluation of autism and in autism research deserves special mention. It is well known that chromosomal deletions and duplications involving virtually every chromosome can cause an autism phenotype, most often in combination with mental retardation and dysmorphic features (13). Some recent investigations using ABCNA to study autism populations have found higher rates of diagnostic abnormalities than previously expected, namely 28% in a study of dysmorphic patients (14) and 10% in a population of which presumably only a portion were dysmorphic (15). It has been argued (16) that these results predict that a substantial fraction of all autism is caused by classical mutations (both genomic and point mutations), most of which are de novo and associated with substantially reduced reproductive ﬁtness. The use of ABCNA rather than karyotypic analysis is attractive for prenatal diagnosis because ABCNA can detect many more abnormalities. The potential advantages of such an approach have been reviewed (17), but substantial disagreement has been expressed (18).

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We believe that it is inevitable that ABCNA will replace karyotype for routine analysis of prenatal samples and that invasive prenatal diagnosis will be offered to all pregnant patients, at least until such analysis might become possible using a noninvasive sample from the mother (17).

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HIGH-THROUGHPUT GENOTYPING OF COMMON POLYMORPHISMS Microarray genotyping is making possible a new generation of powerful genetic association studies that escape the need for selection of candidate genes. In genetic association studies, cases are compared with either population- or family-based controls. The principles underlying genetic association studies have been reviewed recently (18a) and are not repeated here. Direct tests of association assess whether a particular variant plays a causal role in disease by comparing the frequency of the variant in a set of cases to that in the controls. Even in the absence of a direct test of association, the correlation between adjacent polymorphisms—called linkage disequilibrium (LD)—allows a subset of common markers to serve as surrogates for the variants that alter gene functions. The International HapMap Project has provided a very high-resolution LD map that has allowed selection of an efﬁcient set of SNPs to capture a high fraction of genetic correlations (these are called tag SNPs). Given some knowledge of the LD patterns in the genome, sets of hundreds of thousands of markers can be selected that “cover” the most common variants in the population. The availability of the LD map and the technical capacity to genotype the necessary markers has now made genomewide association studies (GWASs) feasible. These new studies allow an unbiased search for associations that do not depend on any pre-existing knowledge of the genes or pathways involved.

GENOTYPING PLATFORMS Several technologies now allow SNPs to be genotyped rapidly, with high accuracy and very low cost. Robust chemistries that can analyze standard sets of up to 1.8 million SNPs and nonpolymorphic positions in a single assay are now available (Figure 2). These assays require scalable, multiplex assay chemistries coupled to an array-based address system to allow readout. The detection chemistry must have extremely high speciﬁcity for single-nucleotide variations, i.e., very low error rates in detection of both homozygous and heterozygous positions in SNPs. The very low molar concentration of unique DNA sequences in the diploid human genome requires coupled ampliﬁcation systems to obtain useful signal-to-noise ratios. An important general principle for achieving singlebase speciﬁcity is the detection of physically coincident events (19). This is exactly the route taken by the polymerase chain reaction (PCR) assay, in which speciﬁc annealing of both primers is required for the reaction to take place. More complex multistep speciﬁcity and universal ampliﬁcation strategies are employed in highly parallel genotyping assays to allow both single-base speciﬁcity and the sensitivity necessary for picking out the locus-speciﬁc signal from the complex mixture. Whole-genome sampling and ampliﬁcation (WGSA) (Affymetrix; Figure 2a) allows parallel ampliﬁcation of short DNA segments (100–1100 bp) that are then labeled and hybridized to oligonucleotide probe arrays (20). The oligonucleotides are speciﬁc for each of the two SNP alleles and the resulting hybridization intensities can be used to derive the genotypes. These assays are ﬁxed in the sense that the investigator uses the information from a standardized array but cannot add SNPs for particular purposes. An alternative approach, incorporated in the Inﬁnium assays (Illumina; Figure 2b,c) involves detection of allele-speciﬁc primer extension or single-base extension products on a

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a.

Restriction digest target DNA StyI

StyI

Sty1

gDNA

Adaptor ligation Genomic Sampling PCR

Hybridize to allele-specific 25-mer probe oligos, wash, and stain with fluorescent tagged streptavidin

End label b b b

b

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Fragment size 100-1100 bp

b.

Target DNA gDNA

Immunostaining Whole-genome amplification

b

CG

T

A

B

Denature

Anneal

b

G

T

A

B

CG

T

A

B

Allele specific primer extension

c.

Whole-genome amplify target DNA

AA A-DNP SNP1

CC C-bio SNP2

AC P A-DN C-bio SNP3

Detect hapten-labeled bases by immunostaining Figure 2 High-throughput genotyping of known variants. (a) Whole-genome sampling assay. This chemistry is used by the Affymetrix whole-genome products. (b) Allele-speciﬁc primer extension with immunodetection of incorporated label (Inﬁnium I). (c) Single-base extension with hapten-labeled nucleotides (Inﬁnium II). Inﬁnium I and II chemistries are used by the Illumina genotyping platform. 120

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self-assembling high-density microbead array (21). In the Inﬁnium assays, genomic DNA is ﬁrst subjected to WGA, and those products are hybridized to an array of locus-speciﬁc 50-mer capture probes. Either primer extension or single-base extension reactions on the array surface are used for allele discrimination. Signal ampliﬁcation by means of antibody methods familiar to users of ELISA assays are used to enhance the sensitivity. A distinct advantage of this class of assays is that the “assay conversion” (percentage of SNPs that actually allow inference of the genotype in the ﬁnal assay mixture) is very high.

RECENT SUCCESSES IN GENOME-WIDE ASSOCIATION STUDIES There has been deserved skepticism among geneticists about the likelihood that association studies will identify the genetic loci underlying common diseases. The validity of case-control study results has often been undermined by inadequate sample sizes, mismatching of cases and controls, inadequate understanding of the variation in patterns of LD across the genome, and the necessity to select candidate genes. This very discouraging picture began to change a few years ago with the publication of association studies that tested genomic regions ﬁrst identiﬁed in linkage analyses. In these cases, the preliminary linkage studies provided crucial information not only about the possible location of the disease loci but also about the likely magnitude of locus-speciﬁc effects and other features of the genetic architecture in the speciﬁc diseases being studied. The successful identiﬁcation of genetic variants underlying type I diabetes and inﬂammatory bowel disease provides some timely examples. This route was also helpful in identifying genes for less common but complex diseases such as Hirschsprung anomaly and nonsyndromic cleft lip and palate.

With the dramatic increase in the number of SNPs identiﬁed in targeted and shotgun resequencing efforts (>15 million recorded in the dbSNP database), it has become possible to test direct associations to large numbers of SNPs that alter protein function. There are about 58,500 known SNPs whose positions in the protein-coding exons lead to nonsynonymous amino acid substitutions (nsSNPs). Two recent genome-wide surveys of nsSNPs illustrate how this approach can be successful. Smyth et al. (22) demonstrated that variants in the innate immunity viral RNA receptor gene (IFIH1) on chromosome 2q24.3 confer risk for type 1 diabetes. The identiﬁcation of this locus reinforced our previous understanding that autoimmune responses following viral infection are central pathways in that disease. In another example of genome-wide testing of possibly functional polymorphisms, Hampe et al. (23) screened for association of Crohn’s disease with almost 20,000 nsSNPs. This led to the discovery of an important role for the ATG16L1 locus, which encodes a component of a large protein complex essential for autophagy. Autophagy is the intracellular process in which membrane-bound autophagosomes deliver cytoplasmic elements to lysosomes for degradation. A unifying theme in inﬂammatory bowel disease research appears to be impairment of the handling of intracellular bacterial pathogens. The availability of genotyping platforms that can inexpensively analyze 100,000 to one million SNP markers in a single DNA sample allows studies to take an “agnostic” approach to genomic association. Without any prior information about genes or pathways, it is possible to ask whether any gene shows strong association with a disease. Since 2005 there have been several studies that did not rely on linkage analysis to focus attention on a single genomic region. Examples of disorders studied in this way include age-related macular degeneration, obesity, coronary artery disease, type I diabetes, and type II diabetes. In perhaps the earliest example of success with www.annualreviews.org • Array Diagnostics

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the GWAS strategy, several groups independently demonstrated a large risk effect for age-related macular degeneration caused by a common variant in the complement factor H gene (24). This gene was previously unsuspected, and its exact role in the disorder is not yet clear. Very large studies, perhaps best exempliﬁed by the Wellcome Trust Case Control Consortium (WTCCC), have shown that an integration of epidemiological studies with genetic analysis can identify the most important common variants contributing to disease. In the WTCCC studies, ∼2000 DNA samples from each of seven common diseases—bipolar disorder, coronary artery disease, Crohn’s disease, rheumatoid arthritis, types I and II diabetes, and hypertension— were compared to 3000 common control individuals. The study yielded strong conﬁrmations of several genes that had been previously associated, showing a total of 24 associations that reached genome-wide signiﬁcance ( p < 5 × 10−7 ). In addition to those associations, 58 more had suggestive signiﬁcant associations (10−5 < p < 5 × 10−7 ). Some of these have been validated in powerful replication studies (25–27). This multistage process, in which the initial genome screen is used to identify the most promising loci for further testing, is expected to be very useful in the future. The WTCCC and several just-published studies using genome-wide association analysis have now provided strong evidence that GWAS can identify the variants that increase risk of disease with relatively small locus-speciﬁc effects. Table 1 is not meant to be a comprehensive list of diseases and loci but merely summarizes some of the largest and highest-impact studies (22–44). Several broad conclusions can be inferred from these studies. First, as previously suspected, there appear to be certain key pathways in each disease, and genetic variants af-

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fecting a pathway can interact to increase risk. Second, the locus-speciﬁc risks are typically very low. This requires that both primary and replication studies be very large in order to achieve the statistical power necessary to detect and validate associations. Third, most of the risk genes identiﬁed were not strong functional candidates, highlighting the necessity for an unbiased genome-wide approach. Fourth, the functional consequences of SNP variants identiﬁed to date are often regulatory, so simply scanning for amino acid substitutions or sequencing of exons will have only limited effectiveness.

HIGH-THROUGHPUT GENOTYPING AND ANALYSES OF KNOWN MUTATIONS Genotyping platforms that are useful for testing SNPs can also be adapted to testing for much rarer genetic variants that have strong causal roles in disease. The literature is not yet extensive but points toward much greater use of these assays in the future. The molecular inversion probe (MIP) assay (45, 46), array-based primer extension (APEX), and GoldenGate assays (47) are examples that exploit a combination of linked speciﬁcity steps with a universal PCR. MIP uses padlock probes whereas GoldenGate uses allele-speciﬁc primer extension for singlebase interrogation. Both MIP and GoldenGate use universal PCR to amplify the diverse locus-speciﬁc assays, a process that allows multiplexing at a level in which thousands of genomic loci are interrogated in parallel in the same reaction. The amplicons in this PCR reaction all contain short, unique “address tags” that are used as indicators for the individual assays. Both MIP and GoldenGate are read out through hybridization of the multiplex PCR products to a standardized array of oligonucleotides that are complementary to the address sequences. Arrayed mutation

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Table 1

Findings of some genome-wide association studies

Disease

Genotyping strategy

Gene

Function

References

breast cancer

500K tag SNP

FGFR2

increased expression of receptor tyrosine kinase

29

coronary heart disease

500K tag SNP

9p21

unknown

28, 30

dry age-related macular degeneration

100K tag SNP

CFH

inﬂammation

24

exudative age-related macular degeneration

100K tag SNP and positional tag SNP

HTRA1

increased expression of a secreted serine protease

31, 32

inﬂammatory bowel disease (Crohn’s)

300K tag SNP

IL23R

IL23 signaling

28, 33

nonsynonymous SNPs

ATG16L1

autophagosome pathway

23

positional cloning and 500K tag SNP

CARD15/NOD2

caspase recruitment

28, 34

300K tag SNP and 500K tag SNP

5p13.1

gene desert modulates expression of PTGER4

28, 35

Positional tag SNPs and 500K tag SNP

5q31

cytokine gene cluster

28, 36

500K tag SNP and replication

IRGM

GTP-binding protein that induces autophagy

25, 28

MST1

phagocytosis

25, 28

NKX2–3

intestinal lymphoid development

25, 28

PTPN2

T cell phosphatase regulator of inﬂammatory responses

25, 28

100K tag SNP

INSIG2

insulin-induced gene 2

37

replication

FTO

unknown

38, 39

prostate cancer

300K tag SNPs

8q24

unknown

40, 41

rheumatoid arthritis

500K tag SNP

HLA-DRB1

antigen presentation

28

500K tag SNP

PTPN22

signal transduction

28

nonsynonymous SNPs, 500K tag SNP, replication

IF1H1

innate immunity viral RNA receptor

22, 26, 28

500K tag SNP, replication

12q24, SH2B3

signal transduction via pleckstrin homology domain

26, 28

500K tag SNP, replication

ERBB3

growth factor receptor

26, 28

500K tag SNP, replication

16p13

unknown

26, 28

500K tag SNP, replication

PTPN2

T cell protein tyrosine phosphatase

26, 28

candidate, 500K tag SNP

INS

insulin

28

candidate, 500K tag SNP

PTPN22

signal transduction

28

candidate, 500K tag SNP

CD25

IL2 receptor subunit

28

candidate, 500K tag SNP

CTLA4

T cell activation

28

obesity

type I diabetes

(Continued )

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(Continued ) Genotyping strategy

Gene

Function

References

type II diabetes

500K tag SNP, replication

CDKAL1

cyclin-dependent kinase 5 (CDK5) regulatory subunit associated protein 1–like 1

27, 28, 42, 43

500K tag SNP, replication

CDKN2A/ CDKN2B

regulator of pancreatic beta cell replication

27, 28, 42, 43

500K tag SNP, replication

IGF2BP2

insulin-like growth factor binding protein

27, 42, 43

500K tag SNP, replication

HHEX IDE

homeobox transcription factor, insulin degrading enzyme

27, 28, 42–44

100K and 300K (Illumina) tag SNP, replication

SLC30A8

zinc transporter

27, 42–44

500K tag SNP, replication

TCF7L2

transcription factor

27, 28, 42, 44

300K tag SNP

FTO

unknown

42

300K tag SNP

PPARG

peroxisome proliferator-activated receptor-gamma

42

300K tag SNP

KCNJ11

potassium channel

42

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Disease

assays have been developed for mutations in speciﬁc genes, such as ABCA4 (48), for disorders with extensive locus heterogeneity, exempliﬁed by Leber’s congenital amaurosis (49) and nonsyndromic congenital deafness (50). We believe that such assays could play a major role in clinical diagnosis and carrier testing for the majority of known Mendelian diseases.

NEXT-GENERATION DNA SEQUENCING ON ARRAYS New technologies that promise to dramatically increase the throughput and reduce the cost of DNA sequencing are being intensely investigated (51). Unlike Sanger sequencing, which produces sequence data by controlled termination of the polymerase, the new techniques derive the sequence as nucleotides are sequentially added by the polymerase. Two of these chemistries have now been developed into early-stage high-throughput sequencing

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instruments (Figure 3). The ﬁrst of these uses pyrosequencing chemistry. Pyrosequencing works by detecting the release of pyrophosphate as nucleotides are added to a DNA primer hybridized to template DNA (52). DNA polymerase can extend the primer when there is a complementary dNTP available but stops when only a noncomplementary base is present. DNA synthesis is reinitiated following the addition of the next complementary dNTP. Pyrophosphate is released as the base is added to the growing DNA chain and detected by enzyme ampliﬁcation. The sequencing instrument controls the order of dNTP addition to the reaction. The order of base addition combined with the light emission is then decoded as the sequence. The 454 Life Sciences Corporation exploits pyrosequencing on an array of ∼300,000 microscopic (40 μm) wells (Figure 3a). The wells each contain a single sepahrose bead on which are coupled the reaction products of a single molecule of template DNA, which itself has been ampliﬁed in

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an oil-water emulsion (53). An advantage of clonal ampliﬁcation is that it neatly deals with the problem of heterozygosity. The pyrosequencing reaction is monitored in a ﬁber optic system. Read lengths of ∼250 nucleotides are feasible. Another general method of sequencing is called cyclic reversible termination (CRT). CRT uses cycles of base addition, imaging, and deprotection to follow the sequence of DNA synthesized on an unknown template. Fluorescence CRT exploits ﬂuorescentdye-labeled reversible terminator nucleotides (Figure 3b). These specialized nucleotides have a protecting group that terminates DNA synthesis. The ﬂuorescent label can then be imaged. Catalytic removal of the protecting group allows another cycle of base addition and imaging. An instrument and reagents that exploit this method have been introduced by Solexa, Inc. Solexa’s method combines this chemistry with in situ ampliﬁcation of large numbers of single DNA molecules. Two recent studies are among the ﬁrst to exploit next-generation sequencing methods. In a survey of mutations in epidermal growth factor receptor (EGFR), an oncogene known to be important in a variety of cancers, speciﬁc mutations were identiﬁed in tumors and archived parafﬁn block samples using 454 pyrosequencing (54). This method holds promise for very sensitive detection of low-level genetic mosaicism that may be important in many neoplastic diseases. In another recent study, array sequencing on the Solexa instrument was used to identify methylation patterns in the genome (55). Those investigators systematically surveyed the distribution of 20 methylations of histone, lysine, and arginine across the genome, introducing a very powerful way to obtain a genome-wide view of chromatin control of gene expression. Simultaneous sequence analysis of >1 million molecules per assay is possible. The general goal of a “$1000 genome” articulated by the

National Human Genome Research Institute no longer seems like a pipedream. Although there will be some lag in clinical application, it is highly likely that mutation scanning of large numbers of genes or even complete genome sequencing will be feasible in the near future.

a.

Beads with capture oligos

Distribution of beads to picoliter wells

Emulsion PCR

PPi

dNMP

light

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PPi + APS Sulfurylase

Read out with fiber optic coupling to individual wells

b.

• Attach single molecules to surface • Amplify on surface to form clusters • 32M clusters per run

Pyrosequencing reaction

anchor primers

incorporation 4-color fluorescent-labeled dNTPs

imaging

deprotection Figure 3 Next-generation sequencing methods. (a) Pyrosequencing microfabricated picoliter reactors. This technology has been developed by 454 Life Sciences Corporation. (b) Sequencing by synthesis. Clusters of ampliﬁed DNA products are formed by a bridge PCR method using paired anchor oligos. Sequencing is accomplished by cycles of incorporation, imaging, and deprotection. This method is used by the Solexa/Illumina instrument.

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SUMMARY POINTS 1. Array-based analysis of genomic DNA has now entered clinical practice as a method to test for submicroscopic chromosomal aberrations. 2. Array methods for high-throughput genotyping of known genetic variants are being extensively used to identify genetic associations with complex common diseases. 3. Unbiased genome-wide association studies (GWASs), in which very large numbers of SNPs serve as proxies for the most common genetic variants in the population, are now technically feasible. They are proving to be a very powerful way to identify and replicate robust associations of variants with diseases such as breast and prostate cancer, autoimmune diseases, and cardiovascular disease. Annu. Rev. Med. 2008.59:113-129. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

4. Next-generation methods for DNA sequencing rely on scaling new and established sequencing chemistries in highly parallel array formats. Array-based sequencing should lead to research and clinical applications in both inherited and neoplastic diseases.

DISCLOSURE STATEMENT The authors are faculty members in the Department of Molecular and Human Genetics at Baylor College of Medicine (BCM), which generates revenue by offering extensive genetic laboratory testing including use of arrays for genomic copy number analysis. BCM has a collaborative marketing agreement with Athena Diagnostics and currently uses arrays manufactured by Agilent Technologies.

LITERATURE CITED 1. Ylstra B, van den Ijssel P, Carvalho B, et al. 2006. BAC to the future! or oligonucleotides: a perspective for micro array comparative genomic hybridization (array CGH). Nucleic Acids Res. 34:445–50 2. Lupski JR, de Oca-Luna RM, Slaugenhaupt S, et al. 1991. DNA duplication associated with Charcot-Marie-Tooth disease type 1A. Cell 66:219–32 3. Lupski JR, Stankiewicz P. 2006. Genomic Disorders—The Genomic Basis of Disease. Totowa, NJ: Humana Press. 427 pp. 4. Lupski JR, Stankiewicz P. 2005. Genomic disorders: molecular mechanisms for rearrangements and conveyed phenotypes. PLoS Genet. 1:e49 5. Sharp AJ, Locke DP, McGrath SD, et al. 2005. Segmental duplications and copy-number variation in the human genome. Am. J. Hum. Genet. 77:78–88 6. Sharp AJ, Hansen S, Selzer RR, et al. 2006. Discovery of previously unidentiﬁed genomic disorders from the duplication architecture of the human genome. Nat. Genet. 38:1038–42 7. Locke DP, Sharp AJ, McCarroll SA, et al. 2006. Linkage disequilibrium and heritability of copy-number polymorphisms within duplicated regions of the human genome. Am. J. Hum. Genet. 79:275–90 8. Redon R, Ishikawa S, Fitch KR, et al. 2006. Global variation in copy number in the human genome. Nature 444:444–54 9. Komura D, Shen F, Ishikawa S, et al. 2006. Genome-wide detection of human copy number variations using high-density DNA oligonucleotide arrays. Genome Res. 16:1575– 84 126

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10. Stankiewicz P, Beaudet AL. 2007. Use of array CGH in the evaluation of dysmorphology, malformations, developmental delay, and idiopathic mental retardation. Curr. Opin. Genet. Dev. 17:182–92 11. Lu X, Shaw CA, Patel A, et al. 2007. Clinical implementation of chromosomal microarray analysis: summary of 2513 postnatal cases. PLoS ONE 2:e327 12. Shaffer LG, Kashork CD, Saleki R, et al. 2006. Targeted genomic microarray analysis for identiﬁcation of chromosome abnormalities in 1500 consecutive clinical cases. J. Pediatr. 149:98–102 13. Vorstman JA, Staal WG, van Daalen E, et al. 2006. Identiﬁcation of novel autism candidate regions through analysis of reported cytogenetic abnormalities associated with autism. Mol. Psychiatry 11(1):18–28 14. Jacquemont ML, Sanlaville D, Redon R, et al. 2006. Array-based comparative genomic hybridisation identiﬁes high frequency of cryptic chromosomal rearrangements in patients with syndromic autism spectrum disorders. J. Med. Genet. 43:843–49 15. Sebat J, Lakshmi B, Malhotra D, et al. 2007. Strong association of de novo copy number mutations with autism. Science 316:445–49 16. Beaudet AL. 2007. Autism: highly heritable but not inherited. Nat. Med. 13:534–36 17. Van den Veyver IB, Beaudet AL. 2006. Comparative genomic hybridization and prenatal diagnosis. Curr. Opin. Obstet. Gynecol. 18:185–91 18. Shuster E. 2007. Microarray genetic screening: a prenatal roadblock for life? Lancet 369:526–29 18a. Hirschhorn JN, Daly MJ. 2005. Genome-wide association studies for common diseases and complex traits. Nat. Rev. Genet. 6:95–108 19. Fan JB, Chee MS, Gunderson KL. 2006. Highly parallel genomic assays. Nat. Rev. Genet. 7:632–44 20. Kennedy GC, Matsuzaki H, Dong S, et al. 2003. Large-scale genotyping of complex DNA. Nat. Biotechnol. 21:1233–37 21. Peiffer DA, Le JM, Steemers FJ, et al. 2006. High-resolution genomic proﬁling of chromosomal aberrations using Inﬁnium whole-genome genotyping. Genome Res. 16:1136–48 22. Smyth DJ, Cooper JD, Bailey R, et al. 2006. A genome-wide association study of nonsynonymous SNPs identiﬁes a type 1 diabetes locus in the interferon-induced helicase (IFIH1) region. Nat. Genet. 38:617–19 23. Hampe J, Franke A, Rosenstiel P, et al. 2007. A genome-wide association scan of nonsynonymous SNPs identiﬁes a susceptibility variant for Crohn disease in ATG16L1. Nat. Genet. 39:207–11 24. Klein RJ, Zeiss C, Chew EY, et al. 2005. Complement factor H polymorphism in agerelated macular degeneration. Science 308:385–89 25. Parkes M, Barrett JC, Prescott NJ, et al. 2007. Sequence variants in the autophagy gene IRGM and multiple other replicating loci contribute to Crohn’s disease susceptibility. Nat. Genet. 39:830–32 26. Todd JA, Walker NM, Cooper JD, et al. 2007. Robust associations of four new chromosome regions from genome-wide analyses of type 1 diabetes. Nat. Genet. 39:857–64 27. Zeggini E, Weedon MN, Lindgren CM, et al. 2007. Replication of genome-wide association signals in U.K. samples reveals risk loci for type 2 diabetes. Science 316:1336–41 28. Wellcome Trust Case Control Consortium. 2007. Genome-wide association study of 14000 cases of seven common diseases and 3000 shared controls. Nature 447:661–78 29. Hunter DJ, Kraft P, Jacobs KB, et al. 2007. A genome-wide association study identiﬁes alleles in FGFR2 associated with risk of sporadic postmenopausal breast cancer. Nat. Genet. 39:870–74 www.annualreviews.org • Array Diagnostics

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30. McPherson R, Pertsemlidis A, Kavaslar N, et al. 2007. A common allele on chromosome 9 associated with coronary heart disease. Science 316:1488–91 31. Dewan A, Liu M, Hartman S, et al. 2006. HTRA1 promoter polymorphism in wet agerelated macular degeneration. Science 314:989–92 32. Yang Z, Camp NJ, Sun H, et al. 2006. A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration. Science 314:992–93 33. Duerr RH, Taylor KD, Brant SR, et al. 2006. A genome-wide association study identiﬁes IL23R as an inﬂammatory bowel disease gene. Science 314:1461–63 34. Hugot JP, Chamaillard M, Zouali H, et al. 2001. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 411:599–603 35. Libioulle C, Louis E, Hansoul S, et al. 2007. Novel Crohn disease locus identiﬁed by genome-wide association maps to a gene desert on 5p13.1 and modulates expression of PTGER4. PLoS Genet. 3:e58 36. Rioux JD, Daly MJ, Silverberg MS, et al. 2001. Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease. Nat. Genet. 29:223–28 37. Herbert A, Gerry NP, McQueen MB, et al. 2006. A common genetic variant is associated with adult and childhood obesity. Science 312:279–83 38. Frayling TM, Timpson NJ, Weedon MN, et al. 2007. A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science 316:889–94 39. Dina C, Meyre D, Gallina S, et al. 2007. Variation in FTO contributes to childhood obesity and severe adult obesity. Nat. Genet. 39:724–26 40. Yeager M, Orr N, Hayes RB, et al. 2007. Genome-wide association study of prostate cancer identiﬁes a second risk locus at 8q24. Nat. Genet. 39:645–49 41. Gudmundsson J, Sulem P, Manolescu A, et al. 2007. Genome-wide association study identiﬁes a second prostate cancer susceptibility variant at 8q24. Nat. Genet. 39:631–37 42. Scott LJ, Mohlke KL, Bonnycastle LL, et al. 2007. A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants. Science 316:1341–45 43. Saxena R, Voight BF, Lyssenko V, et al. 2007. Genome-wide association analysis identiﬁes loci for type 2 diabetes and triglyceride levels. Science 316:1331–36 44. Sladek R, Rocheleau G, Rung J, et al. 2007. A genome-wide association study identiﬁes novel risk loci for type 2 diabetes. Nature 445:881–85 45. Hardenbol P, Baner J, Jain M, et al. 2003. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat. Biotechnol. 21:673–78 46. Hardenbol P, Yu F, Belmont J, et al. 2005. Highly multiplexed molecular inversion probe genotyping: over 10000 targeted SNPs genotyped in a single tube assay. Genome Res. 15:269–75 47. Oliphant A, Barker DL, Stuelpnagel JR, et al. 2002. BeadArray technology: enabling an accurate, cost-effective approach to high-throughput genotyping. Biotechniques 56 (Suppl. 8):60–61 48. Jaakson K, Zernant J, Kulm M, et al. 2003. Genotyping microarray (gene chip) for the ABCR (ABCA4 ) gene. Hum. Mutat. 22:395–403 49. Yzer S, Leroy BP, De Baere E, et al. 2006. Microarray-based mutation detection and phenotypic characterization of patients with Leber congenital amaurosis. Invest. Ophthalmol. Vis. Sci. 47:1167–76 50. Gardner P, Oitmaa E, Messner A, et al. 2006. Simultaneous multigene mutation detection in patients with sensorineural hearing loss through a novel diagnostic microarray: a new approach for newborn screening follow-up. Pediatrics 118:985–94

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51. Metzker ML. 2005. Emerging technologies in DNA sequencing. Genome Res. 15:1767–76 52. Ahmadian A, Ehn M, Hober S. 2006. Pyrosequencing: history, biochemistry and future. Clin. Chim. Acta 363:83–94 53. Margulies M, Egholm M, Altman WE, et al. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376–80 54. Thomas RK, Nickerson E, Simons JF, et al. 2006. Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing. Nat. Med. 12:852–55 55. Barski A, Cuddapah S, Cui K, et al. 2007. High-resolution proﬁling of histone methylations in the human genome. Cell 129:823–37

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:113-129. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

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Inherited Mitochondrial Diseases of DNA Replication∗ William C. Copeland Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; email: [email protected]

Annu. Rev. Med. 2008. 59:131–46

Key Words

First published online as a Review in Advance on September 24, 2007

DNA polymerase γ, nucleotide pools, mitochondrial DNA depletion syndrome, progressive external ophthalmoplegia, ataxia-neuropathy

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.053006.104646 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0131$20.00 ∗ The U.S. Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

Abstract Mitochondrial genetic diseases can result from defects in mitochondrial DNA (mtDNA) in the form of deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These mutations may be spontaneous, maternally inherited, or a result of inherited nuclear defects in genes that maintain mtDNA. This review focuses on our current understanding of nuclear gene mutations that produce mtDNA alterations and cause mitochondrial depletion syndrome (MDS), progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). To date, all of these etiologic nuclear genes fall into one of two categories: genes whose products function directly at the mtDNA replication fork, such as POLG, POLG2, and TWINKLE, or genes whose products supply the mitochondria with deoxynucleotide triphosphate pools needed for DNA replication, such as TK2, DGUOK, TP, SUCLA2, ANT1, and possibly the newly identiﬁed MPV17.

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INTRODUCTION mtDNA: mitochondrial DNA pol γ: DNA polymerase γ, encoded by POLG gene

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MDS: mitochondrial DNA depletion syndrome PEO: progressive external ophthalmoplegia TWINKLE: gene encoding mitochondrial helicase dNTP: deoxynucleotide triphosphate

Mitochondrial diseases can be caused by genetic defects in mitochondrial DNA (mtDNA) or in nuclear genes that encode proteins that function in the mitochondria (1). The mitochondrial genome contains 37 genes, all of which are directly or indirectly involved in the production of ATP. Thirteen of these genes encode protein subunits involved in electron transport to carry out oxidative phosphorylation. The remaining 24 genes encode the transfer RNAs (22 genes) and ribosomal RNAs (2 genes) required for mitochondrial protein synthesis. The mtDNA copy number is high; a cell contains between 1000 and 10,000 copies. MtDNA is replicated by the concerted action of DNA polymerase γ (pol γ), its accessory subunit p55 (encoded by POLG2), and replication factors, such as the mitochondrial single-stranded DNA binding protein and the Twinkle helicase. Pol γ is the only known DNA polymerase found in mammalian mitochondria and thus bears the burden of DNA replication and DNA repair functions (2). Since 1999, there has been a ﬂurry of discoveries involving nearly a dozen genes linked to mitochondrial depleTable 1

tion syndrome (MDS) and related disorders (Table 1). MDS includes not only commonly known disorders such as progressive external ophthalmoplegia (PEO) and ataxia but also some very rare tricarboxylic acid (TCA) cycle abnormalities. Mutations in POLG, POLG2, TWINKLE, and ANT1 are associated with PEO, and mutations in several nuclear genes encoding enzymes involved in mitochondrial nucleotide metabolism can cause point mutations, deletions, or depletion in mtDNA, resulting in mitochondrial syndromes. Mitochondria depend heavily on either mitochondrial transport proteins or salvage pathway enzymes to supply deoxynucleotide triphosphates (dNTPs) required for mtDNA replication.

DNA POLYMERASE γ, THE POLG GENE Diseases resulting from mutations in POLG, the gene encoding the catalytic subunit of pol γ, are highly heterogeneous (2–5). Mutations in POLG are associated with such diverse disorders as PEO, parkinsonism, premature menopause, Alpers syndrome,

Nuclear loci that affect the stability of mitochondrial DNA Chromosome locus

Gene

Disordera

POLG

PEO/Alpers/ataxia

15q25

mitochondrial DNA polymerase

POLG2

PEO

17q23–24

pol γ accessory subunit

TWINKLE (PEO1)

PEO/ataxia

10q24

mitochondrial DNA helicase

ANT1

PEO

4q34–35

adenine nucleotide translocator

TP (ECGF1)

MNGIE

22q13.32

thymidine salvage

DGUOK

MtDNA depletion

2p13

deoxyguanosine kinase

TK2

MtDNA depletion

16q22

mitochondrial thymidine kinase

DNCb

MCPHA

17q25.3

deoxynucleotide carrier (thiamine pyrophosphate transport)

MPV17

mtDNA depletion

2p21–23

mitochondrial inner membrane protein

SUCLA2

mtDNA depletion

13q12.2–q13.3

succinate-CoA ligase

RRM2B

mtDNA depletion

8q23.1

ribonucleotide reductase

Function

a Abbreviations: PEO, progressive external ophthalmoplegia; MNGIE, mitochondrial neurogastrointestinal encephalomyopathy; MCPHA, Amish lethal microcephaly. b DNC mutations have not been associated with mitochondrial DNA depletion syndrome, and recent evidence suggests the gene product functions to transport thiamine pyrophosphate.

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Figure 1 Schematic diagram of human pol γ protein showing locations of disease-causing amino acid substitutions and polymorphisms. The protein is arranged with the exonuclease domain in the ﬁrst third, followed by a long linker region, with the polymerase domain in the C-terminal third. The disease substitutions are represented by boxes: Light blue boxes, autosomal dominant PEO; light green boxes, autosomal recessive mutations; gray boxes, sporadic PEO; pink boxes, Alpers syndrome; yellow boxes, ataxia-neuropathy-like syndromes; and orange box, male infertility. Striped boxes represent disease substitutions found in more than one disease. Red arrows depict the nonsynonymous polymorphic amino acid changes.

mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), and sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO) (2, 4–6). Nearly 90 pathogenic mutations have been found in POLG (Figure 1) (6) (see also the Human Polymerase Gamma Mutation Database, http://dir-apps.niehs.nih.gov/polg).

POLG and Progressive External Ophthalmoplegia Mutations in POLG were ﬁrst identiﬁed as a locus for PEO in 2001 (7). PEO is a mitochondrial disorder associated with mtDNA depletion and/or accumulation of mtDNA mutations and deletions (7–9). PEO is characterized by late onset (between 18 and 40 years of age), bilateral ptosis, and progressive weakening of the external eye muscles, resulting in blepharoptosis and ophthalmoparesis. Proximal muscle weakness and

wasting, as well as exercise intolerance, are also associated with PEO. Skeletal muscles of PEO patients have decreased respiratory chain enzyme activity and show ragged red ﬁbers pathologically. Multiple large-scale deletions of mtDNA isolated from muscle biopsies were ﬁrst demonstrated in Italian families with heritable autosomal dominant PEO (adPEO) (8). To date, with one exception, all autosomal dominant POLG mutations responsible for PEO are in the polymerase domain of pol γ. Two of the substitutions, R943H and Y955C, change side chains that interact directly with the incoming dNTP (10). These enzymes retain <1% of the wild-type polymerase activity and display a severe decrease in processivity. The extremely low catalytic activities and resultant stalling of DNA synthesis are the two most likely causes of the severe clinical presentation in R943H and Y955C heterozygotes (10). The Y955C substitution

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MNGIE: mitochondrial neurogastrointestinal encephalomyopathy SANDO: sensory ataxic neuropathy, dysarthria, and ophthalmoparesis ad: autosomal dominant

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also increases nucleotide misinsertion errors 10–100-fold in the absence of exonucleolytic proofreading (11). In a yeast model developed to evaluate the homologous mutation in the yeast MIP1 gene, we found that a Y757C mutant (Y955C in humans) demonstrated mtDNA abnormality indicative of oxidative damage and very high petite frequency (12). In a related study, Barufﬁni et al. showed that this high petite frequency could be rescued by treatment with antioxidants or upregulation of ribonucleotide reductase (13). In a mouse transgenic model in which the Y955C POLG was targeted to the heart, the mice developed cardiomyopathy, loss of mtDNA, and increased levels of 8-oxo-dG in their mtDNA (14). Collectively, these phenotypes suggest that patients harboring the Y955C mutation may have elevated oxidative damage and may beneﬁt from antioxidant therapy. Co-occurrence of parkinsonism and adPEO was described in two individuals with mutations in POLG (15). Parkinsonism manifested several years after initial PEO symptoms. Women with PEO from POLG mutations may experience premature menopause and suffer from high gonadotropin and low estrogen concentrations indicative of premature ovarian failure (15, 16). Most POLG mutations are associated with autosomal recessive PEO (arPEO), and patients with PEO are often compound heterozygotes with two different mutant PEO alleles. For example, the A467T mutation has been found in trans with other POLG missense mutations in PEO, ataxia-neuropathy, and Alpers syndrome (17, 18). The A467T mutation was found in two pedigrees as a homozygous mutation and associated with severe ataxia in mid-life (19). Biochemical analysis indicates that the A467T mutant pol γ possesses only ∼4% of the wild-type polymerase activity with only a modest effect on the exonuclease (20). Additionally, the A467T pol γ protein fails to interact with its accessory subunit, p55, which is normally required for highly processive DNA synthesis (20). Nevertheless, A467T is a common mu-

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ar: autosomal recessive

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tation, present in 0.6% of the Belgian population (17).

POLG and Alpers Syndrome Alpers syndrome is a rare but severe autosomal recessive MDS disease that afﬂicts young children. Within the ﬁrst few years of life, patients exhibit progressive spastic quadriparesis, progressive cerebral degeneration leading to mental deterioration and seizures, cortical blindness, deafness, liver failure, and eventual death. Naviaux et al. reported an Alpers patient with reduced electron transport chain function, dicarboxylic aciduria, fulminant hepatic failure, refractory epilepsy, and lactic acidosis that resulted in death at 42 months (21). Skeletal muscle biopsy indicated a reduction of mtDNA content to 30% of wild-type levels with no detectable pol γ activity (21). Sequencing of the POLG gene in these pedigrees revealed a heterozygous G-to-T nonsense mutation in POLG that converts Glu873 (GAG) to a stop codon and the heterozygous A467T substitution just after the exonuclease domain in the linker region (18). Pol γ mRNAs with the E873 stop mutation are removed from the pool of mRNAs by nonsense-mediated decay, resulting in mono-allelic expression of POLG containing only the A467T mutation (22). To date, the number of reported Alpers-associated POLG mutations has risen to >35, from 46 different probands (23–25). In all cases, the POLG mutations in Alpers patients are recessive, and many of these same mutations may also cause arPEO. The A467T mutation commonly found as a compound mutation in arPEO is the most frequent Alpers mutation and is found as either a homozygous or a heterozygous mutation combined with other mutations.

POLG and Ataxia-Neuropathy Mutations in POLG can also cause ataxianeuropathy syndrome with onset in the early teens to late thirties. This ataxia, also termed mitochondrial-associated ataxia syndrome

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(MIRAS) (26), spino-cerebellar ataxiaepilepsy syndrome (SCAE), or SANDO (19), is caused by autosomal recessive mutations in POLG producing multiple mtDNA deletions in the affected individuals. Symptoms include peripheral neuropathy, dysarthria, mild cognitive impairment, involuntary movements, psychiatric symptoms, myoclonus, and epileptic seizures. Ataxic patients who are homozygous for the A467T mutation present with symptoms in their early to late teens (19, 27). SANDO patients have also been found to have compound heterozygous mutations with the A467T mutation in one POLG allele and R3P, L304R, or R627W in the other (17). One patient with ataxia-myopathy syndrome was shown to have the A467T mutation in one POLG allele and R627Q and Q1236H mutations in the other POLG allele (28). Other patients with ataxia were found to be heterozygous, with the A467T mutation in one allele and W748S in cis with the E1143G mutation in the other allele. The E1143G mutation was originally identiﬁed as a single-nucleotide polymorphism (SNP) in 4% of the general population (6). However, the accumulated reports of this mutation with other POLG mutations in mitochondrial disease suggest that E1143G may augment the disease process (5, 29). The W748S mutation in combination with E1143G has been found to be a frequent cause of ataxia (26, 27). Haplotype analysis of the Finnish population demonstrates a carrier frequency of 1:125 for the W748S mutation, with a common-ancestor origin of this allele in the ancient European population (26). We have found that the W748S mutation alone causes the polymerase to have a low catalytic activity and a severe DNA-binding defect (30). The E1143G substitution partially rescues the deleterious effects of the W748S mutation and appears to modulate the effects of disease mutations in POLG.

POLG and Male Infertility The human POLG gene contains a 10-unit CAG trinucleotide tract encoding a poly-

glutamine stretch near the N terminus of the mature protein (31). Although deletion of the CAG repeat has no detectable effect on mitochondrial function in tissue culture cells (32), some studies suggest that alteration of the CAG repeat is associated with loss of sperm quality and contributes to 5%–10% of the male infertility cases in the European population (33, 34). In contrast to these studies, two independent studies failed to conﬁrm a relationship between the polymorphic CAG repeat in POLG and male infertility (35, 36).

POLG2, THE ACCESSORY SUBUNIT OF POL γ Recently, a single mutation in the gene encoding the accessory subunit, POLG2, was reported in a patient with adPEO (37). This mutation results in G451E substitution in a loop region not involved in p55 dimerization. Characterization of the recombinant G451E mutant of p55 demonstrates that the mutant accessory subunit is defective in binding with the pol γ catalytic subunit and fails to stimulate processive DNA synthesis. The failure to enhance processivity in the catalytic subunit would cause the complex to stall during DNA replication and is consistent with the accumulation of mtDNA deletions detected in PEO.

THE MITOCHONDRIAL DNA HELICASE, THE TWINKLE OR PEO1 GENE The mtDNA helicase encoded by the TWINKLE gene, also known as PEO1 is related to the phage T7 gp4 helicase-primase. This gene was ﬁrst isolated as a locus for PEO on chromosome 10, C10orf2 (38). The derived amino acid sequence has signiﬁcant sequence homology to the C-terminal end of T7 gp4 helicase but lacks critical primaseassociated sequences found in T7 gp4 (38, 39). Screening of the TWINKLE gene in individuals with adPEO, associated with multiple mtDNA deletions, identiﬁed 11 different coding-region mutations that cosegregated

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with the disorder in 12 affected families (38). The majority of these mutations reside in a linker region between the helicase domain in the C terminus and the N-terminal region. This region is thought to be involved in subunit interaction between the monomers to form a functional hexamer. Mutations in TWINKLE are mainly associated with adPEO, but one report has described a recessive TWINKLE mutation as a cause of SANDO (40). Mouse transgenic models that overexpressed several of the PEO mutations in TWINKLE recapitulated many of the characteristics of human PEO, including multiple mtDNA deletions, progressive respiratory dysfunction, and cytochrome c oxidase deﬁciency (41).

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TP: thymidine phosphorylase

ADENINE NUCLEOTIDE TRANSLOCATOR, ANT1 ANT1 is one of three adenine nucleotide translocator proteins found as innertransmembrane mitochondrial proteins and is the most abundant protein in the mitochondria. ANT1 functions as a homodimer composed of 30-kDa monomers and is highly expressed in heart, kidney, liver, and skeletal muscle. Its principal function is to transport ATP out of the mitochondrial matrix in exchange for ADP. Kaukonen et al. (42) used positional cloning in one adPEO family to narrow the locus for adPEO to 4q, which includes ANT1 and 64 other genes. Sequence analysis of ANT1 in ﬁve families and one patient with sporadic adPEO identiﬁed two heterozygous missense mutations in ANT1 (43). The autosomal mutation A114P and the sporadic mutation V289M are both found within transmembrane domains in the structure of the protein. The analogous A114P mutation in the yeast ortholog, AAC2, caused respiratory deﬁciency (43). A heterozygous T293C ANT1 mutation was found in a Greek family with adPEO (44), a heterozygous V289M mutation in an Italian patient with sporadic PEO (45), and a heterozygous A90D mutation in a German 136

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family with adPEO. In the German family, although microsatellite markers showed that the allele was dominant and inherited from the mother, the patient did not carry the mutation in blood, indicating germ-line mosaicism (46). Expression of several other equivalent mutations in the yeast gene AAC2 in an aac2defective haploid strain of yeast resulted in a marked growth defect on nonfermentable carbon sources, as well as a reduction in cellular respiration (47). The AAC2 pathogenic mutants showed a signiﬁcant defect in ADP versus ATP transport compared to wild type.

THYMIDINE PHOSPHORYLASE, ECGF1 Thymidine phosphorylase (TP) is part of the pyrimidine salvage pathway required for the conversion of thymidine and phosphate to thymine and deoxyribose-1-phosphate (Figure 2). A defect in ECGF1, the gene encoding TP, causes the accumulation of thymidine and uracil in the blood. Because mitochondria rely heavily on salvage pathways for generating intramitochondrial dNTP pools, the mitochondria take up the excess thymidine, which in turn stimulates the synthesis of excess deoxythymidine triphosphate (dTTP) by thymidine kinase 2 in the mitochondria. The resulting unbalanced mitochondrial deoxynucleotide pools cause mtDNA depletion, multiple deletions, and point mutations. MNGIE is an autosomal recessive disorder caused by mutations in ECGF1 (48, 49). To date, more than 30 mutations in ECGF1 are known to be associated with MNGIE (49). Like PEO, the disease is associated with multiple deletions and depletion of mtDNA (9). Onset is usually between the second and ﬁfth decades of life, and typical clinical features include ptosis, PEO, gastrointestinal dysmotility, cachexia, peripheral neuropathy, myopathy, and leukoencephalopathy (50, 51). TP deﬁciency leads to increased concentrations of circulating deoxythymidine (52) and deoxyuridine (53). These increases result in

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Figure 2 Schematic diagram of the mitochondrion, showing enzyme pathways that cause mtDNA mutations or depletion when disrupted. Gene products associated with MDS or mtDNA mutations are in green boxes.

imbalanced mitochondrial deoxyribonucleoside triphosphate pools, the effect of which can increase mtDNA mutagenesis (54). More than 80% of mtDNA mutations found in tissues from MNGIE patients are T-to-C transitions preceded by a short run of As (55). This signature mutation suggests a “next-nucleotide effect” caused by the more common misinsertion T:dGMP (deoxyguanosine monophosphate) (56), which is quickly extended by the elevated dTTP concentration resulting from TP deﬁciency in the mitochondria of MNGIE cells (55). Additionally, elevated concentrations of dTMP (deoxythymidine monophosphate) derived from the increased thymidine can inhibit the exonuclease activity of pol γ (57). HeLa cells grown in media supplemented with 50 μM thymidine demonstrated mtDNA deletions and elevated mitochondrial pools of dTTP and dGTP, a result that recapitulated many of the genetic effects seen in MNGIE (54). These results support a mutagenic mechanism involving competition between dGTP

and dATP for incorporation opposite to template T (55). Several exciting avenues of research are showing potential for the treatment of MNGIE. Hemodialysis has been shown to transiently reduce thymidine levels in blood (58). Allogeneic stem cell transplantation has had some success in restoring TP activity and lowering plasma thymidine levels (59). In addition, repeated platelet infusions can reduce thymidine levels in blood in MNGIE patients (60).

MITOCHONDRIAL THYMIDINE KINASE, TK2 In a study of four unrelated patients with fatal myopathy during infancy and mitochondrial depletion syndrome, Saada et al. (61) found reduced mitochondrial respiratory chain function and reduced amounts of mtDNA in muscle. Sequence analysis of the thymidine kinase 2 (TK2) gene identiﬁed either of two homozygous mutations, H90R

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or I181D. Activity analysis in mitochondrial lysates from these patients revealed reduced TK2 activity (61). DNA sequences by other groups have identiﬁed a total of 14 missense mutations found either as recessive homozygous or as compound heterozygous mutations and one stop codon from a collection of 12 probands with muscle weakness and hypotonia (62–67). Mitochondrial dNTP pools arise either through active transport of cytosolic dNTPs or through salvage pathways by the action of two mitochondrial deoxyribonucleoside kinases, TK2 and deoxyguanosine kinase (Figure 2). In nondividing cells, cytosolic TK1 and dNTP synthesis is downregulated, forcing the burden of mitochondrial dNTP pool synthesis on the two mitochondrial deoxyribonucleoside kinases. TK2 mutations primarily affect muscle tissue with no effect on liver, brain, heart, or skin. Quantitation of TK2 activity in various tissues relative to mtDNA or cytochrome c oxidase activity helps to explain the tissue speciﬁcity of TK2 deﬁciency (68).

with these substitutions. They found that the R142K variant had very low activity with deoxyguanosine (dG) and no activity with deoxyadenosine (dA). The E227K protein had normal afﬁnity for dG and dA but low catalytic efﬁciency, indicating that this mutation disrupts catalysis without affecting substrate binding. The C-terminal truncated variants were inactive. Further analysis found that one patient with MDS, muscular weakness, and exercise intolerance due to a severe mitochondrial myopathy harbored the L250S mutation in DGUOK (65). Examination of the recombinant L250S-dGuoK protein revealed <1% activity compared to the wild-type enzyme with differential competition between deoxycytidine (dC) and deoxythymidine (dT) as substrates (65). Freisinger et al. (72) also identiﬁed ﬁve new mutations and two previously described mutations in six children with infantile hepatoencephalopathies and MDS. To date, 13 mutations have been described in the DGUOK gene, most presented as homozygous mutations from 14 probands (67, 72).

DEOXYGUANOSINE KINASE, DGUOK

DEOXYNUCLEOTIDE CARRIER, SLC25A19

Deoxyguanosine kinase is the other mitochondrial deoxyribonucleoside kinase that phosphorylates the purine nucleosides into nucleotide monophosphates (Figure 2). Homozygosity mapping in three consanguineous kindreds with hepatocerebral MDS identiﬁed a region on chromosome 2p13 that included the deoxyguanosine kinase gene, DGUOK (69). Sequence analysis of this gene identiﬁed a nucleotide deletion (204delA) that segregated with the disease (69). In a screen of 21 patients with MDS, Salviati et al. (70) identiﬁed three patients (14%) with DGUOK mutations. Phenotypes of these three patients were highly variable, including one patient who developed liver failure but responded well to liver transplant (70). To conﬁrm that the DGUOK mutations did indeed disrupt enzymatic function, Wang et al. (71) characterized recombinant deoxyguanosine kinase

In 2002, Kelley et al. (73) described a metabolic disorder among the Old Order Amish of Lancaster County, Pennsylvania, characterized by severe congenital microcephaly, severe 2-ketoglutaric aciduria, and death usually within the ﬁrst year. Amish lethal microcephaly (MCPHA), an autosomal recessive disorder, has an unusually high incidence of at least 1 in 500 births in this population (73). By using a pedigree analysis in 23 ancestrally related families combined with whole-genome scanning, Rosenberg et al. (74) localized the gene for MCPHA to chromosome 17q25. Contained within this region is the mitochondrial deoxynucleotide carrier gene (DNC or SLC25A19), which was found to harbor a missense mutation that alters Gly177 to Ala and was not found in 252 control chromosomes. Functional analysis of the G177A mutant DNC conﬁrmed that the

Copeland

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mutant protein was defective in transport activity (74). A DNC knockout in mouse causes 100% prenatal lethality by day 12 with neural-tube closure defects and elevated α-ketoglutarate (75). However, mouse DNC−/− cells did not show any reduction in mtDNA levels. Furthermore, mitochondrial ribo- and deoxyribonucleoside triphosphate levels were normal, suggesting that nucleotide transport may not be the primary role of DNC. A protein with similar amino acid homology in yeast, Tpc1p, was found to transport thiamine pyrophosphate (ThPP) into mitochondria in exchange for thiamine monophosphate (76). Indeed, in vitro transport assays conﬁrmed that the G177A DNC did not cause a defect in ribonucleotide or deoxynucleotide transport into the mitochondria. Furthermore, the mitochondria from the mouse knockout had no detectable ThPP and mitochondria from MCPHA cells had reduced levels of ThPP. Thus, reduction of ThPP levels causes the inability of the α-ketoglutarate dyhydrogenase complex to function properly, which leads to the high levels of α-ketoglutaric acid in these patients.

SUCCINYL-COA SYNTHETASE, SUCLA2 A small Muslim pedigree with autosomal recessive encephalomyopathy associated with mtDNA depletion was reported in 2005 (77). Genome-wide linkage mapping identiﬁed a 20-Mb region on chromosome 13, which was narrowed down to three genes by mitochondrial import prediction programs. DNA sequencing of one of these, the SUCLA2 gene, revealed a homozygous deletion of 43 nt at the 3 end of exon 6. This mutation results in deletion of exon 6 and part of exon 7 in the mRNA of the affected patients (77). Succinyl-CoA synthetase is a mitochondrial matrix enzyme that catalyzes the reversible synthesis of succinate and ATP (or GTP) from succinyl-CoA and ADP in the tricarboxylic acid (TCA) cycle. SUCLA2 encodes

the β-subunit of succinyl-CoA synthetase. The reverse reaction occurs in the Krebs cycle, whereas the forward reaction may produce succinyl-CoA for activation of ketone bodies and heme synthesis. Following this initial ﬁnding, 12 patients with autosomal recessive mitochondrial encephalomyopathy and elevated methylmalonic acid were identiﬁed in the Faroe Islands population (78). Elevated methylmalonic acid is characteristic of methylmalonic acidemia (MMA), a heterogeneous group of disorders with symptoms that include vomiting, dehydration, lethargy, seizures, failure to thrive, progressive encephalopathy, and developmental delays. The increased level of methylmalonic acid impairs the conversion of vitamin B12 into its two metabolically active forms. The accumulated succinyl-CoA inhibits the metabolism of methylmalonyl-CoA to succinyl-CoA, causing the accumulation of methylmalonyl-CoA and MMA. Mutation analysis identiﬁed a novel splice site mutation in SUCLA2 leading to skipping of exon 4. A founder effect caused the high incidence (1 in 1700) in the Faroe Islands population, with a carrier frequency of 1 in 33 (78). In a related study, DNA sequencing of 14 patients from southern Italy and the Faroe Islands conﬁrmed SUCLA2 mutations in all patients and led to the identiﬁcation of three novel mutations (79). The frequency of these mutated alleles in the Faroe Islands population was 2%, corresponding to an estimated homozygote frequency of 1:2500 (79). How a defect in the citric acid cycle and accumulation of methylmalonic acid cause depletion of mtDNA is not clear. However, immunoprecipitation experiments have found succinyl-CoA synthetase in complex with mitochondrial nucleotide diphosphate kinase (80). Elpeleg et al. (77) propose that the defect in SUCLA2 disrupts association with the nucleotide diphosphate kinase, causing a defect in the last step of mitochondrial dNTP salvage by nucleotide diphosphate kinase, which leads to decreased dNTPs

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and subsequent mtDNA depletion. Mutations in the succinyl-CoA gene should be considered in patients with early/neonatal-onset encephalomyopathy, dystonia, deafness, and Leigh-like MRI abnormalities mainly affecting the putamen and the caudate nuclei (79).

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One of the latest genetic loci to be added to the list of mtDNA instability disorders is the MPV17 gene (81). Using enhanced computational algorithms designed to identify mitochondrial targeted gene products, Calvo et al. (82) identiﬁed 1080 gene products, including 368 that were not previously predicted to localize to mitochondria. Eight of these new genes, including MPV17, were identiﬁed as candidate loci for mitochondrial disorders (82). In an accompanying article, Spinazzolla et al. (81) reported several families with MDS in which patients suffered hepatic failure early in life. Genome-wide linkage analysis mapped this gene to the same region as MPV17, and DNA sequence analysis identiﬁed a common mutation at Arg50, which was mutated to either Gln or Trp in these patients. The protein MPV17 was originally presumed to be localized to peroxisomes, but confocal immunoﬂuorescence in human and monkey cells demonstrates colocalization of MPV17 with mitochondria (81). A parallel study identiﬁed the same R50Q mutation in Navajo neurohepatopathy in six patients (83). Navajo neurohepatopathy is an autosomal recessive multisystem disorder found in the Navajo of the southwestern United States. It is characterized by liver failure, severe sensory neuropathy, corneal anesthesis and scarring, cerebral leukoencephalopathy, failure to thrive, and acidosis (83 and references therein). The MPV17 homolog in yeast, SYM, is localized to the inner mitochondrial membrane. SYM1 deletion or mutations in SYM1 that produce the R51Q or R51W substitution (equivalent to R50Q or R50W in human) cause respiration deﬁciency and mtDNA re140

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arrangement (81). MPV17−/− mice develop age-dependent hearing loss and glomerular sclerosis (84), along with a decrease of mtDNA in liver, muscle, brain, and kidney, and decreased respiratory function (81). Although the function of MPV17 remains to be elucidated, the ﬁnding of this protein through integrative genomic analysis paves the way for the identiﬁcation of other genes involved in MDS. Like the defect in SUCLA2, the defect in MPV17 may result in a destabilized protein complex with a nucleotide metabolism protein, or destabilization of the membraneassociated protein-DNA nucleoid complex where mtDNA replication is expected to take place.

P53-INDUCIBLE RIBONUCLEOTIDE REDUCTASE, RMM2B The nucleotide precursors required for DNA replication can be directly obtained by reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates by ribonucleotide reductase. Ribonucleotide reductase is made up of two subunits: a large catalytic subunit, R1, and the smaller R2 subunit. Cells have two forms of the R2 subunit: a cell cycle regulated form that is maximally expressed in S-phase, and a p53-inducible form known as p53R2. The p53R2 form is required for a basal level of DNA repair and mtDNA synthesis in nonproliferating cells. Recently, Bourdon et al. (85) identiﬁed the RRM2B gene encoding p532B as a candidate disease gene from a genome-wide linkage analysis in a family with severe muscle mtDNA depletion. Sequence analysis of RRM2B in this family and three other affected families identiﬁed nonsense, missense, and splice-site mutations and inframe deletions within the RRM2B gene that were not found in control chromosomes. Severe mtDNA depletion from RRM2B disruption was also conﬁrmed in Rrm2b−/− mice, demonstrating the essential role of this gene in mtDNA nucleotide metabolism and mitochondrial disease (85).

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CONCLUSION Diseases of mtDNA stability have been traced to core proteins of mtDNA replication or genes involved in supplying the mitochondrial nucleotide precursors needed for mtDNA replication (Figure 2). Mutations in the POLG gene are responsible for several mitochondrial disorders, including fatal childhood diseases such as Alpers syndrome, PEO, ataxia-neuropathy, and possibly male infertility. Although most mutations probably result in inactive and/or truncated proteins, the dominant mutations in POLG and

TWINKLE commonly found in PEO produce “dominant-negative” proteins that interfere with their wild-type counterparts. The fact that many genes involved in nucleotide salvage pathways and nucleotide transport are responsible for mitochondrial diseases suggests that imbalanced nucleotide pools are detrimental to mtDNA replication. Mouse models, yeast genetics, and in vitro biochemical analyses of mutant proteins have become invaluable for understanding the in vivo consequences of heritable mutations in these genes.

SUMMARY POINTS 1. Disease of mtDNA replication falls primarily into two genetic categories, those that directly affect DNA replication proteins, and those that affect the mitochondrial nucleotide pools. 2. These diseases can affect a variety of organs, with variable ages of onset. The speciﬁc manifestations of mtDNA replication diseases are usually determined by the types of mutations within these sets of genes. 3. There is generally a correlation with age of onset and tissue speciﬁcity for many symptoms. For example, the hepatopathy usually occurs early in life whereas skeletal muscle myopathy presents later in life. However, neuropathy appears to occur any time in these patients. 4. POLG is frequently mutated in this collection of diseases.

FUTURE ISSUES 1. A better understanding of the pathogenesis of these diseases and their tissue speciﬁcity. 2. The need to develop suitable animal models that emulate diseases of mtDNA replication to elucidate tissue presentation and pathology. 3. Development of therapies that may slow the progression of these diseases.

DISCLOSURE STATEMENT The author is not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS The author thanks Rajendra Prasad, Jeffrey Stumpf, and Rajesh Kasiviswanathan for critical reading of this manuscript. This review was supported by intramural funds from the National Institute of Environmental Health Sciences, NIH. www.annualreviews.org • Mitochondrial Diseases of DNA Replication

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60. Lara MC, Weiss B, Illa I, et al. 2006. Infusion of platelets transiently reduces nucleoside overload in MNGIE. Neurology 67:1461–63 61. Saada A, Shaag A, Mandel H, et al. 2001. Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat. Genet. 29:342–44 62. Mancuso M, Salviati L, Sacconi S, et al. 2002. Mitochondrial DNA depletion: mutations in thymidine kinase gene with myopathy and SMA. Neurology 59:1197–202 63. Mancuso M, Filosto M, Bonilla E, et al. 2003. Mitochondrial myopathy of childhood associated with mitochondrial DNA depletion and a homozygous mutation (T77M) in the TK2 gene. Arch. Neurol. 60:1007–9 64. Carrozzo R, Bornstein B, Lucioli S, et al. 2003. Mutation analysis in 16 patients with mtDNA depletion. Hum. Mutat. 21:453–54 65. Wang L, Limongelli A, Vila MR, et al. 2005. Molecular insight into mitochondrial DNA depletion syndrome in two patients with novel mutations in the deoxyguanosine kinase and thymidine kinase 2 genes. Mol. Genet. Metab. 84:75–82 66. Galbiati S, Bordoni A, Papadimitriou D, et al. 2006. New mutations in TK2 gene associated with mitochondrial DNA depletion. Pediatr. Neurol. 34:177–85 67. Alberio S, Mineri R, Tiranti V, et al. 2007. Depletion of mtDNA: syndromes and genes. Mitochondrion 7:6–12 68. Saada A, Shaag A, Elpeleg O. 2003. mtDNA depletion myopathy: elucidation of the tissue speciﬁcity in the mitochondrial thymidine kinase (TK2) deﬁciency. Mol. Genet. Metab. 79:1–5 69. Mandel H, Szargel R, Labay V, et al. 2001. The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA. Nat. Genet. 29:337–41 70. Salviati L, Sacconi S, Mancuso M, et al. 2002. Mitochondrial DNA depletion and dGK gene mutations. Ann. Neurol. 52:311–17 71. Wang L, Eriksson S. 2003. Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome. FEBS Lett. 554:319–22 72. Freisinger P, Futterer N, Lankes E, et al. 2006. Hepatocerebral mitochondrial DNA depletion syndrome caused by deoxyguanosine kinase (DGUOK) mutations. Arch. Neurol. 63:1129–34 73. Kelley RI, Robinson D, Puffenberger EG, et al. 2002. Amish lethal microcephaly: a new metabolic disorder with severe congenital microcephaly and 2-ketoglutaric aciduria. Am. J. Med. Genet. 112:318–26 74. Rosenberg MJ, Agarwala R, Bouffard G, et al. 2002. Mutant deoxynucleotide carrier is associated with congenital microcephaly. Nat. Genet. 32:175–79 75. Lindhurst MJ, Fiermonte G, Song S, et al. 2006. Knockout of Slc25a19 causes mitochondrial thiamine pyrophosphate depletion, embryonic lethality, CNS malformations, and anemia. Proc. Natl. Acad. Sci. USA 103:15927–32 76. Marobbio CM, Vozza A, Harding M, et al. 2002. Identiﬁcation and reconstitution of the yeast mitochondrial transporter for thiamine pyrophosphate. EMBO J. 21:5653–61 77. Elpeleg O, Miller C, Hershkovitz E, et al. 2005. Deficiency of the ADP-forming succinyl-CoA synthase activity is associated with encephalomyopathy and mitochondrial DNA depletion. Am. J. Hum. Genet. 76:1081–86 78. Ostergaard E, Hansen FJ, Sorensen N, et al. 2007. Mitochondrial encephalomyopathy with elevated methylmalonic acid is caused by SUCLA2 mutations. Brain 130:853–61 79. Carrozzo R, Dionisi-Vici C, Steuerwald U, et al. 2007. SUCLA2 mutations are associated with mild methylmalonic aciduria, Leigh-like encephalomyopathy, dystonia and deafness. Brain 130:862–74 www.annualreviews.org • Mitochondrial Diseases of DNA Replication

61. First report describing TK2 as a locus for mitochondrial DNA depletion myopathy.

69. First report describing deoxyguanosine kinase gene involved in mitochondrial DNA depletion.

77. The discovery that a citric acid cycle gene can also cause mitochondrial DNA depletion syndrome.

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80. Kowluru A, Tannous M, Chen HQ. 2002. Localization and characterization of the mitochondrial isoform of the nucleoside diphosphate kinase in the pancreatic beta cell: evidence for its complexation with mitochondrial succinyl-CoA synthetase. Arch. Biochem. Biophys. 398:160–69 81. Spinazzola A, Viscomi C, Fernandez-Vizarra E, et al. 2006. MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion. Nat. Genet. 38:570–75 82. Calvo S, Jain M, Xie X, et al. 2006. Systematic identiﬁcation of human mitochondrial disease genes through integrative genomics. Nat. Genet. 38:576–82 83. Karadimas CL, Vu TH, Holve SA, et al. 2006. Navajo neurohepatopathy is caused by a mutation in the MPV17 gene. Am. J. Hum. Genet. 79:544–48 84. Meyer zum Gottesberge AM, Reuter A, Weiher H. 1996. Inner ear defect similar to Alport’s syndrome in the glomerulosclerosis mouse model Mpv17. Eur. Arch. Otorhinolaryngol. 253:470–74 85. Bourdon A, Minai L, Serre V, et al. 2007. Mutation of RRM2B, encoding p52-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nat. Genet. 39:776–80

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81. The use of integrative genomics to identify a new gene, MPV17, involved in a mitochondrial DNA depletion syndrome and hepatopathy.

RELATED RESOURCES Human DNA polymerase mutation database: http://dir-apps.niehs.nih.gov/polg/ United Mitochondrial Disease Foundation: http://www.umdf.org Mitomap, a human mitochondrial DNA mutation database: http://www.mitomap.org

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:131-146. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe1,2 and Edward R.B. McCabe1,2,3,4 1

Departments of Human Genetics and Pediatrics, David Geffen School of Medicine at the University of California, Los Angeles; 2 UCLA Center for Society and Genetics; 3 California Nanosystems Institute; 4 Department of Bioengineering, Henry Samueli School of Engineering and Applied Science, Los Angeles, California 90095; email: [email protected], [email protected]

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Key Words

The Annual Review of Medicine is online at http://med.annualreviews.org

continuous quality improvement (CQI), tandem mass spectrometry (MS/MS), two-tiered screening

This article’s doi: 10.1146/annurev.med.59.110106.132016 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0163$20.00

Abstract Newborn screening (NBS) represents the largest volume of genetic testing. The 45-year history of NBS has demonstrated its beneﬁts, as well as the importance of an evidence base. The recent addition of tandem mass spectrometry (MS/MS) resulted in a ﬁvefold increase in the number of tests. Experience with MS/MS also showed that laboratory tests are just one part of the NBS system. The lessons learned from NBS will provide important insights as we move into the predictive, preventive, and personalized era of genomic medicine.

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INTRODUCTION Newborn screening (NBS): system that involves testing infants before they leave the maternity ward to determine who are at a higher risk of developing a disorder with signiﬁcant morbidity and/or mortality, and who should receive diagnostic testing to determine whether they should be treated. The system includes preanalytic, analytic, and postanalytic phases Tandem mass spectrometry (MS/MS): testing method in which two sequential mass spectrometers identify and quantify signature metabolites in a newborn’s blood sample

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Newborn screening (NBS) currently and in the foreseeable future represents the venue for the largest volume of genetic testing. Each year 25%–30% of the world’s 133 million neonates, or 33–40 million babies, undergo genetic testing (B.L. Therrell, Jr., personal communication). In the United States, nearly all of the ∼4.2 million neonates born every year undergo NBS, and multiple tests are performed on each baby’s sample. The American College of Medical Genetics recommends a uniform screening panel for every neonate that includes a core group of 29 disease targets and a secondary group of 25 targeted conditions to be considered in the differential diagnosis of the core panel targets (1). Among these 54 core and secondary targets, 43 (80%) are amenable to identiﬁcation by a single testing platform, tandem mass spectrometry (MS/MS). If this recommended testing panel were implemented throughout the United States, the total number of NBS tests would exceed 225 million per year. The current magnitude of NBS, as well as the 45 years of experience behind it, establish it as a model that must be examined as we proceed with the implementation of genomic medicine (2). The mantra of genomic medicine is that it will be predictive, preventive and personalized, and NBS pioneered a medical model featuring these characteristics (2–4). The goals of NBS are (a) prediction—identifying patients before they manifest disease, (b) prevention— initiation of therapeutic interventions to forestall the course of the disorders, and (c) personalization—individualizing patients’ therapies to optimize their outcomes. Therefore, it is desirable to analyze NBS, and particularly the recent experience with expanded NBS; to identify the structural features that evolved to permit the NBS system to achieve its mission; and to consider its beneﬁts and challenges. Consideration of the lessons learned from NBS will permit us to accelerate the implementation of genomic medicine and will permit this new discipline to achieve its

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promise in a much more efﬁcient and efﬁcacious manner.

INTRODUCTION TO NEWBORN SCREENING The principles underlying NBS are often attributed (e.g., 4) to the 1968 World Health Organization (WHO) report on population screening (14). The principles included consideration of (a) the public health signiﬁcance of the disease, (b) the availability of a validated screening test for the disease, (c) evidence that the consequences of the disease are preventable if it is identiﬁed early, and (d ) the costs of the testing for the disease. In 1975, the National Academy of Sciences published an extremely inﬂuential volume that, by deﬁning genetic screening, also established principles for it (15). This US report stated that genetic screening in a population should serve one or more of the following purposes:

Identify individuals with genetic characteristics known to cause disease or predispose to disease. A positive test would result in diagnosis before disease onset, enabling therapy to prevent the disease. NBS is an example. Identify carriers, i.e., individuals whose genotypes may result in disease among their descendents. Those who test positive would receive genetic counseling to educate them about reproductive options that would allow their descendants to avoid the disease. Identify genetic variations that are of interest speciﬁcally because they are not known to be associated with disease. Test results would increase our knowledge of the impact of the variation on the population being screened, including its frequency and ethnic distribution. Such pilot screening should inform planning for future screening programs.

NBS has evolved from a “heel-stick” test for phenylketonuria (PKU) (5, 6) into a public

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health system that targets multiple disorders and includes education, laboratory and clinical follow-up, management, and system continuous quality improvement (CQI) (1–4, 7–11). We have previously described the history of NBS in three phases: early, middle, and expanded (2, 11). We discuss the ﬁrst two here, and the third in the next major section, “Expanded Newborn Screening.” The ﬁrst phase of heel-stick NBS began in the early 1960s with the development of a method to measure elevated phenylalanine concentrations in dried blood specimens from neonates prior to nursery discharge (5, 6). This permitted the identiﬁcation of patients with PKU and intervention in the form of a phenylalanine-restricted diet to prevent mental retardation in these patients. The members of the Parents of Mentally Retarded Children, now called the Association for Retarded Citizens (ARC), were mobilized as a political force. Their efforts contributed to the passage of legislation in many states mandating NBS for PKU, despite the absence of evidence to support the efﬁcacy of this testing at that time (2, 10). This early phase of NBS continued until the mid-1970s. It was recognized that screening for PKU (and occasionally other disorders) was a matter of public health (2, 11). This led to the passage of laws in most of the remaining states, under the public health mandate of state government, requiring NBS for PKU in all infants. In addition, fragmentation of testing in private and hospital laboratories was eliminated in most states; the screening activities were consolidated in the states’ public health laboratories. This consolidation is generally considered to have improved the quality and reliability of testing. With early NBS, it was also recognized that not all patients with hyperphenylalaninemia had PKU; some had benign hyperphenylalaninemia. Similar observations have been repeated whenever NBS or other laboratory diagnostic methods supersede clinical evaluation in deﬁning a phenotype. The phenotype

deﬁned by the laboratory test is generally much broader than that described by the clinical evaluation and will often include individuals who are clinically normal, such as those with benign hyperphenylalaninemia. The middle phase of NBS lasted from the mid-1970s until the early 1990s and was characterized most notably by addition of new tests to target new diseases, such as congenital hypothyroidism and hemoglobinopathies (2, 11). This expansion was fueled by improved knowledge of the value of early intervention for patients with these diseases and by technological advances, including automated sample preparation. Testing for congenital hypothyroidism utilized a new two-tiered strategy involving quantitative assay of thyroxine (T4 ) and thyroid-stimulating hormone (TSH), generally in this order (9). In most patients (1–3 days old) with congenital hypothyroidism, the T4 is low and TSH is elevated. Two-tiered screening of the same dried blood specimen was found to be more cost-effective than performing both tests simultaneously, and it improved sensitivity (lowered the false negative rate) without sacriﬁcing speciﬁcity (holding the false negative rate relatively constant or even somewhat improved). Two-tiered testing permitted NBS for additional disorders that otherwise might have been considered too costly. Effective implementation of two-tiered testing for congenital hypothyroidism led to other two-tiered NBS tests. For example, hemoglobinopathy NBS utilized a two-tiered strategy that tested protein phenotype followed by DNA genotype (2, 11). In 1986, a multicenter randomized double-blinded clinical trial of penicillin prophylaxis in patients with sickle-cell anemia demonstrated overwhelming efﬁcacy of penicillin to prevent death from sepsis (12). In 1987, an NIH Consensus Development Conference concluded that the data supported universal NBS (i.e., NBS offered to all neonates in the United States) for hemoglobinopathy, and required hemoglobinopathy screening programs to assure that penicillin was initiated prior to www.annualreviews.org • Expanded Newborn Screening

Continuous quality improvement (CQI): ongoing program to determine each laboratory’s ability with blinded samples sent by a professional organization or government agency. Improvement efforts are based on the results obtained Two-tiered screening: if the result of one test is positive, then the other test is performed

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4 months of age for babies diagnosed with sickle cell disease (13). The federal government invested approximately $12 million to establish state hemoglobinopathy screening programs between 1987 and 1990 (2, 11). Despite the evidence base and federal investment, it was not until 2005 that all 50 states and the District of Columbia required universal NBS for sickle cell disease and the other hemoglobinopathies (2). Expansion of NBS programs was managed differently in each state (9). Some expanded by political ﬁat driven by government ofﬁcials, often inﬂuenced by citizens who had family members with the disease that was added. Other programs used advisory committees composed of experts and interested citizens that recommended addition of tests and targets in a more deliberate, evidencebased manner. As a result, the menu of NBS targets varied from state to state. Several lessons can be drawn from these ﬁrst two phases of NBS that should guide NBS expansion and the implementation of “genomic medicine”:

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1. The public, including patient advocacy organizations, has more political power than the medical community, and may impose testing without an evidence base. 2. Fragmented laboratory testing, particularly for rare disorders, creates challenges for CQI. 3. Phenotypes are invariably broadened when deﬁned by laboratory criteria rather than clinical evaluation. Therefore, one should be cautious about treating everyone with a therapy that was developed for the clinically deﬁned cohort. The test-deﬁned cohort may contain normal individuals who do not require the therapy and may even be harmed by it. 4. Two-tiered screening strategies may maintain sensitivity without sacriﬁcing speciﬁcity and are often cost-effective. 5. The clinical evidence base should be the driver for inclusion of targets and test166

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ing technologies in screening. When there is strong evidence for inclusion, then advocacy and political will may be required for implementation. 6. Expansion should be carried out in a well-planned and rational manner, building on the prior experience of others, with an understanding that uniformity will facilitate implementation and CQI. All parts of the screening system must be fully integrated so that samples and information will not be lost (9, 16). This integration should encompass the preanalytic, analytic, and postanalytic aspects of NBS, all of which should include CQI (Table 1). Critical for an effectively integrated system are processes at the interfaces of these three aspects that might be considered analogous to “chain of custody” procedures in forensics. For example, some states require samples to be barcoded in the nursery; each day the barcodes of samples sent to the laboratory are transmitted to the follow-up agency. Software then compares the barcodes, and if a sample is not received in the laboratory or a report is not received by those responsible for follow-up, then the system identiﬁes this as a problem so that it will not lose track of the patient at this interface. Such accounting is critical in any large-scale population screening program. Similarly, it is critical that all screening results are transmitted to the baby’s physician, and that the physician has a reliable method to account for receipt of the NBS results on each of his or her neonatal patients (Table 1) (16). This has been a challenge in NBS, because frequently the physician of record in the hospital is the obstetrician, who will not see the neonate again and may not forward the results to the pediatrician or other practitioner who will follow the child. It is imperative for screening programs to recognize the potential discontinuities at the various interfaces to prevent the loss of samples and information. Follow-up of all positive screens is essential to assure that affected patients (true positives)

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Table 1 Components of the preanalytic, analytic and postanalytic aspects of the NBS system (adapted from Reference 16) Aspect of the NBS system Preanalytic

Components Education of parents and professionals Informed consent for participation Collection of sample Transport of sample to laboratory Transmission of identifying information to laboratory and perhaps other agencies (e.g., follow-up agency)

Analytic

Registration of sample in the laboratory and perhaps in other agencies

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Testing for the analyte(s) in the laboratory Transmission of test results to: the responsible physician/medical home possibly other state agencies, such as the agency responsible for follow-up possibly parents and others as prescribed by laws, regulations, and/or practice Postanalytic

Receipt of test results Follow-up on all test results Appropriate management of a positive test result Diagnostic testing: If negative, then NBS result was false positive, and family should be counseled and reassured that neonate is normal If positive, then (a) the family should be counseled about the implications for the baby, other family members, and reproductive decision making; and (b) the affected newborn should be referred to the appropriate subspecialist for management, which may be life-long

receive appropriate management and unaffected individuals (false positives) do not receive inappropriate therapy (Table 1) (16). In a screening program, false positives usually outnumber true positives, because screening attempts to maximize sensitivity to avoid missing affected patients and will tolerate lower speciﬁcity to achieve this aim (16, 17). Although the medical priority is the true positives, the larger number of families with newborns who are false positives must be reassured that their babies are normal so that they will not be stigmatized permanently by the initial positive screen. Resources for counseling those with false positive test results should be budgeted into all screening programs.

EXPANDED NEWBORN SCREENING Experience in the ﬁrst two phases of NBS led to proposals for the expansion of NBS based

on four major principles (20): eliminating the inconsistency among states regarding the conditions screened; greatly expanding the number of conditions screened; broadening the concept of beneﬁt from NBS to include the family; and educating the public and medical professionals regarding positive NBS results. Education is imperative, as increased numbers of disorders tested for lead to increased absolute numbers of positive results. When an infant has a positive NBS, the parents and the infant’s physician need to keep in mind that NBS is a screen, not a diagnostic test, and hence that most positive results are false positives (17). Conﬁrmatory testing using the original NBS specimen should be pursued, so that in the future, an infant’s family and physician could be contacted with a conﬁrmed diagnosis rather than merely a positive NBS result (21). Traditionally, NBS has emphasized prevention of morbidity and mortality through www.annualreviews.org • Expanded Newborn Screening

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early treatment. MS/MS detects some disorders for which there currently is no effective treatment. However, even in these cases the family derives beneﬁts from an early and accurate diagnosis: immediate supportive treatment for the infant, information to aid reproductive decision making, and the possibility of the infant participating in research on the disorder (20).

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A German pilot program using MS/MS to screen for 23 disorders doubled the detection rate (22). MS/MS had a lower false positive rate for PKU (0.05%) than enzymatic phenylalanine determination (0.23%), a proportion similar to the false positive rate for MS/MS screening for all 23 disorders (0.33%). These authors cited the retrospective and prospective detection of affected siblings and parents as other advantages of MS/MS NBS. North Carolina’s pilot program (23) led to a modiﬁcation of cutoffs in order to minimize false positives and false negatives. Each analyte had a “borderline” and a “diagnostic” cutoff. Any newborn with results above the diagnostic cutoff was referred to the metabolic specialist, who contacted the infant’s primary care physician and parents. These infants were seen by the metabolic specialist within 24– 48 h after the initial contact with their physician. Newborns with a borderline result had a second test run on their initial sample, requiring an average of 7.7 days. Conﬁrmed borderline results were mailed to the submitting hospital and the primary care physician, along with a request for a repeat sample, and parents were contacted if the repeat sample was not received in two weeks. The goal was to begin treatment by 10–14 days of age. There were six false negative cases out of 944,078 tests. These six infants were not ill as newborns but were symptomatic later. These results led to lower cutoffs for glutaric acidemia type I and for methylmalonic acidemia. Seven infants died in the ﬁrst days of life with over168

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whelming metabolic decompensation and/or prematurity. Their disorders included citrullinemia (two), isovaleric acidemia, glutaric acidemia type II, methylmalonic acidemia, very-long-chain acyl-CoA dehydrogenase deﬁciency, and long-chain hydroxyacyl-CoA dehydrogenase deﬁciency. For the families of these infants, the diagnosis led to genetic counseling regarding prenatal testing and/or treatment from birth for subsequent pregnancies. North Carolina has a web-based reporting system that enables primary care physicians to determine whether NBS was performed, whether a repeat was requested, and what the results were. California’s pilot MS/MS NBS program for 30 acylcarnitines and 14 amino acids (24) noted variable hospital participation. Many hospitals refused to participate because of the requirement for informed consent. No hospital had 100% participation of newborns, and 20% of hospitals did not offer the test to a single newborn. Statewide, 52% of all newborns were offered MS/MS screening and 47% of all families agreed to the testing while 5% declined. Based on the MS/MS screening results, the authors estimated that there were 61 affected newborns in the unscreened group, but only 14 were reported as diagnosed by clinical symptoms, meaning that 77% of affected newborns would not be identiﬁed in the absence of screening. Three cases in the screened group were not detected by MS/MS. One of these (very-long-chain acyl-CoA dehydrogenase deﬁciency) was diagnosed prenatally and was being treated. The other two (long-chain 3-hydroxyacyl-CoA dehydrogenase deﬁciency and maple syrup urine disease) would have been detected by the revised cutoff values. The overall false positive rate of MS/MS was 0.07%. There was one MS/MS-detected disorder for every 4463 infants screened.

Examples of Disorders Detected by Expanded NBS Detection of medium-chain acyl-CoA dehydrogenase deﬁciency (MCADD) has been the

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focus of expanded NBS. Grosse et al. (25) projected that in the absence of early asymptomatic detection, at least 50% of children with MCADD have a metabolic crisis, one in six die, and one in ten survivors develop serious developmental delay. Wisconsin researchers found that screening for MCADD alone was cost-effective enough to justify the adoption of MS/MS NBS (26). MCADD patients identiﬁed by MS/MS NBS were compared with clinically diagnosed patients in Australia (27). There were six deaths among the clinically diagnosed children (two at three days of age, and one each at 17 months, 19 months, 47 months, and 93 months). There was one death in the NBS group at three days of age after poor feeding. In the clinically ascertained group, 57% had a severe episode of decompensation. In the NBS group, 8% had an episode, one at three days of age and one at three years of age after recovering from gastroenteritis. A multinational study of MS/MS NBS for propionic aciduria, methylmalonic aciduria, and isovaleric aciduria (28) showed that early mortality was decreased and the symptoms at diagnosis were less severe compared with clinical diagnosis. The short-term neurodevelopmental outcome appeared to be more favorable in cases ascertained by NBS. The major advantage from NBS was that patients with milder phenotypes could begin appropriate treatment while they were presymptomatic, thus preventing severe clinical symptoms. The study found a markedly higher rate of identiﬁcation of patients with isovaleric aciduria with MS/MS, which could be due to underascertainment by clinical diagnosis or overascertainment of mild cases with MS/MS. In MS/MS NBS for primary carnitine deﬁciency, not only newborns but also their affected mothers are identiﬁed. Most affected mothers have minimal or no symptoms at diagnosis. Schimmenti et al. (29) concluded that carnitine supplementation in both mothers and infants would prevent the hypoglycemia, hepatic encephalopathy, cardiomyopathy, and

sudden death that result from primary carnitine deﬁciency.

Cost-Effectiveness of Expanded NBS California performed a cost-effectiveness analysis of MS/MS and found the cost per quality-adjusted life year (QALY) in the worst-case scenario to be between $11,000 and $19,000 (30). Economists have a standard of a cost per QALY of $50,000 or less as costeffective (30). Finland currently screens cord blood for congenital hypothyroidism only; no other NBS is performed. A group (31) modeled MS/MS NBS for ﬁve disorders (congenital adrenal hyperplasia, medium-chain acylCoA dehydrogenase deﬁciency, long-chain 3hydroxyacyl-CoA dehydrogenase deﬁciency, glutaric aciduria type 1, and phenylketonuria) in Finland and found it should cost $45 per infant to identify 5–10 cases. Early death could be prevented in 1–3 cases, and severe handicap could be prevented in 1–5 cases. The authors considered cost-effectiveness as only one factor in determining whether MS/MS NBS should be added in Finland. They decided that the expense of establishing an entirely new system would be prohibitive.

MCADD: medium-chain acyl-CoA dehydrogenase deﬁciency

Challenges to Implementation of Expanded NBS According to Carroll et al. (32), the significant start-up cost of MS/MS keeps some states from adding this technology to their NBS program. However, these authors found that MS/MS improved outcomes and reduced costs relative to not screening for every disorder studied, with the exception of congenital adrenal hyperplasia and galactosemia. MS/MS was shown to be more effective and less expensive than conventional tests. In fact, they noted that NBS was one of the rare interventions that is beneﬁcial to the patient and cost-effective. However, MS/MS is technically challenging and requires analytical and postanalytical skills for the proper interpretation of the www.annualreviews.org • Expanded Newborn Screening

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complex metabolic proﬁles (55). One proﬁle may involve 15 different markers and ratios and show considerable quantitative variability, with concentrations ranging across four orders of magnitude (0.1 to 1000 μM). Forty of the 42 conditions that MS/MS can detect and that the American College of Medical Genetics recommends are interconnected, such that considering them separately is artiﬁcial and clinically unwise. A survey of 60 NBS experts regarding MS/MS found that funding was the limiting factor for the plurality (35%) of respondents (33). Other issues included getting support within their NBS program for MS/MS, and the need for support for MS/MS from advisory boards. In determining which disorders should be included, the most important criteria were beneﬁts to the newborn and test sensitivity and speciﬁcity. Laboratory challenges for MS/MS included the high costs of equipment and supplies, the need for developing appropriate cutoffs, and the lack of trained laboratory staff. Respondents reported the following concerns about followup to MS/MS NBS: standard of care for diagnostic workups, availability of educational materials for parents and medical professionals, and funding for long-term follow-up.

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Future Expansion of NBS Lysosomal storage disorders can be added to MS/MS NBS (34, 35). Enzyme replacement therapy has improved or is expected to improve outcomes for patients with Gaucher, Fabry, mucopolysaccharidosis (MPS I), and Pompe diseases. Presymptomatic detection and umbilical stem cell transplantation may improve prognosis for patients with Krabbe and Niemann-Pick disease. Tests are being developed for other disorders using MS/MS suitable for NBS. Microarray DNA chips (20, 36) could be used in NBS for a broad range of disorders, including genetic metabolic disorders (37), immunodeﬁciency disorders (38, 39), muscular dystrophies (40), cystic ﬁbrosis (41), 170

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and hemoglobinopathies (42–45). Microarrays could also be used for molecular cytogenetic NBS using DNA from the dried blood spots (36).

Challenges Facing NBS NBS has grown dramatically from a screen for a single disease, PKU, in the 1960s, to the currently recommended panel of core and secondary targets numbering 54 (1, 11). At the same time, the number of technology platforms has increased from one to many, and their complexity has increased from the relatively simple bacterial inhibition assay (5, 6) that could be carried out with minimal equipment and training, to much more sophisticated platforms, such as enzymelinked immunosorbent assays (ELISAs), high-performance liquid chromatography (HPLC), and DNA conﬁrmatory testing, which depend on complicated instrumentation and highly trained personnel (4). The dramatic increase in technology platforms is challenging public health NBS laboratories. The addition of DNA conﬁrmatory testing has proven to be a challenge for some NBS laboratories, which have abandoned or not adopted it. DNA conﬁrmation entails the possibility of identifying misattribution of paternity (2, 4, 11, 16). At least one professional organization has recommended that unless paternity was the reason for the testing such information should not be divulged (18). Genetic discrimination in employment and insurance is a major concern of the public with respect to genetic testing (2, 3, 19). Physicians who have cared for patients identiﬁed by NBS struggle to prevent insurance companies from limiting services to those patients. At the time of this writing, legislation is in the US Congress (S.358 and H.R.493) that would prevent genetic discrimination in health insurance and employment. The passage of such legislation would reassure the public, particularly individuals already affected by genetic screening and diagnostic testing.

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ADDITIONAL LESSONS FROM EXPANDED NBS FOR GENOMIC MEDICINE

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Recognize That Genotype May Not Correlate with Phenotype NBS using MS/MS identiﬁes more individuals with biochemical abnormalities than does clinical diagnosis. This is expected, given the wide variation in symptom severity and the lack of genotype/phenotype correlation for most disorders (46–52). As genomic diagnostic testing is implemented in a prospective manner, phenotypes will therefore expand for diseases previously identiﬁed clinically with post hoc laboratory testing. As a result, new paradigms for patient segmentation and initiation of therapy must be devised.

Create a Preventive Healthcare System Based on the Laboratory Tests In genomic diagnostics, the testing platform(s) will identify mutations, polymorphisms, and rarer sequence variants. There will be tensions between discarding data of unknown value and the cost of maintaining that information. We would argue that a preventive healthcare system will be better served by cataloguing the laboratory information and correlating it with phenotypes. For example, the Mayo Clinic group (53) advocated the development of conﬁrmatory testing for all detectable conditions, rather than excluding rare conditions that are found at no additional cost.

Strive for Standardization A study of NBS for MCADD in the United States, Northern Europe, and Australia found a lack of standardization between laboratories for criteria for repeat testing, cutoffs, internal standards, and calibrations (54). Average age at screening varied from 29 h to eight days, a disparity attributed at least partly to lack of standardization. Experience from pilot projects must be shared, and health care de-

livery systems need to provide incentives for later adopters to utilize that experience. We feel that this has been a serious challenge in expanded NBS. The Mayo Clinic group (55) endorsed local review and public debate, but they were concerned that 23% of the newborns in the United States at the time of their writing were not being screened for MCADD. They endorsed quality assessment and improvement, including long-term monitoring and interlaboratory comparison of performance. They recommended uniform metrics to facilitate interlaboratory comparisons of, for example, detection rates, false positive rates, and positive predictive values. Just as in NBS, standardizing platforms and establishing uniform metrics for genomic diagnostics will improve the process for CQI in genomic medicine. Without such standardization and uniformity, it will be much more difﬁcult to establish interlaboratory comparisons and build a national genomic diagnostic infrastructure.

Recognize That Social Impediments to Implementation May Be More Important than Technological Challenges A survey of parents of patients with mucopolysaccharidosis (MPS) (56) showed that 97% supported screening for conditions for which early treatment was available and 88% supported screening for untreatable disorders. They wanted better treatment for their children and the option of prenatal diagnosis for future pregnancies. This may reﬂect a paradigm shift from the newborn being the target to the family being the target of NBS, at least in the view of parents. However, medicine is frequently conservative and resistant to change. This resistant social context has been a greater barrier to NBS expansion than any technological issues. Professionals engaged in genomic diagnostics must be sensitive to social changes that inﬂuence technology implementation. The history of www.annualreviews.org • Expanded Newborn Screening

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NBS has made it clear that when public interest groups want test expansion, and medical professionals do not work with them to implement it, then they will utilize the political process.

SUMMARY

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Although NBS is managed by each state’s health department, there have been recent attempts to standardize the methods of NBS and the disorders screened. Tandem mass spectrometry (MS/MS) presents a greater technological challenge than the early NBS testing platforms. Experience with MS/MS has highlighted the need for education of par-

ents and the medical community regarding issues such as false positives, diagnosis of disorders without effective treatments, and the broader range of phenotypes found in patients diagnosed by laboratory testing compared to those identiﬁed clinically. NBS provides important lessons for genomic medicine. The goal of “predictive, preventive and personalized” genomic medicine requires evidence bases on which cost-effectiveness analyses can be performed, recognition that genotype may not correlate with phenotype, sharing of best practices, and standardization. Finally, the understanding of genomic medicine by medical practitioners and the public will be critical (4).

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

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11. McCabe LL, Therrell BL, McCabe ERB. 2002. Newborn screening: rationale for a comprehensive, fully integrated public health system. Mol. Genet. Metab. 77:267–73 12. Gaston MH, Verter JI, Woods G, et al. 1986. Prophylaxis with oral penicillin in children with sickle cell anemia—a randomized trial. N. Engl. J. Med. 314:1593–99 13. Consensus Panel, National Institutes of Health. 1987. Newborn screening for sickle cell disease and other hemoglobinopathies. JAMA 258:1205–9 14. Wilson JMG, Jungner G. 1968. Principles and practice of screening for disease. Public Health Pap. No. 34. Geneva: World Health Org. 15. Committee for the Study of Inborn Errors of Metabolism. 1975. In Genetic Screening Programs, Principles, and Research, pp. 9, 200–13. Washington, DC: Natl. Acad. Sci. 16. McCabe LL, McCabe ERB. 2002. Newborn screening as a model for population screening. Mol. Genet. Metab. 75:299–307 17. Kotzer AM, McCabe ERB. 1988. Newborn screening for inherited metabolic disease: principles and practice. Neonatal Netw. 6:15–19 18. American Society of Human Genetics. 1996. Statement on informed consent for genetic research. Am. J. Hum. Genet. 59:471–74 19. Public Perspectives on Genetic Discrimination. 2004. http://www4.od.nih.gov/oba/ sacghs/reports/Public Perspectives GenDiscrim.pdf (Sep.–Nov.) 20. Alexander D, van Dyck PC. 2006. A vision of the future of newborn screening. Pediatrics 117:S350–54 21. Zhang Y-H, McCabe LL, Wilborn M, et al. 1994. Application of molecular genetics in public health: improved follow-up in a neonatal hemoglobinopathy screening program. Biochem. Med. Metab. Biol. 52:27–35 22. Schulze A, Lidner M, Kohlmuller D, et al. 2003. Expanded newborn screening for inborn errors of metabolism by electrospray ionization-tandem mass spectrometry: results, outcome, and implications. Pediatrics 111:1399–406 23. Frazier DM, Millington DS, McCandless SE, et al. 2006. The tandem mass spectrometry newborn screening experience in North Carolina. J. Inherit. Metab. Dis. 29:76–85 24. Feuchtbaum L, Lorey F, Faulkner L, et al. 2006. California’s experience implementing a pilot newborn supplemental screening program using tandem mass spectrometry. Pediatrics 117:S261–69 25. Grosse SD, Khoury MJ, Greene CL, et al. 2006. The epidemiology of medium chain acyl-CoA dehydrogenase deﬁciency: an update. Genet. Med. 8:205–12 26. Insinga RP, Laessig RH, Hoffman GL. 2002. Newborn screening with tandem mass spectrometry: examining its cost-effectiveness in the Wisconsin newborn screening panel. J. Pediatr. 141:524–31 27. Wilcken B, Haas M, Joy P, et al. 2007. Outcome of neonatal screening for medium-chain acyl-CoA dehydrogenase deﬁciency in Australia: a cohort study. Lancet 369:37–42 28. Dionisi-Vici C, Deodato F, Roschinger W, et al. 2005. “Classical” organic acidurias, propionic aciduria, methylmalonic aciduria and isovaleric aciduria: long-term outcome and effects of expanded newborn screening using tandem mass spectrometry. J. Inherit. Metab. Dis. 29:383–89 29. Schimmenti LA, Crombez EA, Schwahn BC, et al. 2007. Expanded newborn screening identiﬁes maternal primary carnitine deﬁciency. Mol. Genet. Metab. 90:441–45 30. Feuchtbaum L, Cunningham G. 2006. Economic evaluation of tandem mass spectrometry screening in California. Pediatrics 117:S280–86 31. Autti-Ramo I, Makela M, Sintonen H, et al. 2005. Expanding screening for rare metabolic disease in the newborn: an analysis of costs, effect and ethical consequences for decisionmaking in Finland. Acta Pediatr. 94:1126–36 www.annualreviews.org • Expanded Newborn Screening

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32. Carroll AE, Downs SM. 2006. Comprehensive cost utility analysis of newborn screening strategies. Pediatrics 117:S287–95 33. Feuchtbaum L, Faulkner L, Verghese S. 2006. Tandem mass spectrometry program implementation challenges for state newborn screening programs: national survey of barriers and issues. Pediatrics 117:S253–60 34. Meikle PJ, Ranieri E, Simonsen H, et al. 2004. Newborn screening for lysosomal storage disorders: clinical evaluation of a two-tier strategy. Pediatrics 114:909–16 35. Gelb M, Turecek F, Scott CR, et al. 2006. Direct multiplex assay of enzymes in dried blood spots by tandem mass spectrometry for the newborn screening of lysosomal storage disorders. J. Inherit. Metab. Dis. 29:397–404 36. McGhee SA, McCabe ER. 2006. Genome-wide testing: genomic medicine. Pediatr. Res. 60:243–44 37. Stanczak CM, Chen Z, Zhang Y-H, et al. 2007. Deletion mapping in Xp21 for patients with complex glycerol kinase deﬁciency using SNP mapping arrays. Hum. Mutat. 28:235–42 38. McGhee SA, Stiehm ER, Cowan M, et al. 2005. Two-tiered universal newborn screening strategy for severe combined immunodeﬁciency. Mol. Genet. Metab. 86:427–30 39. McGhee SA, Stiehm ER, McCabe ER. 2005. Potential costs and beneﬁts of newborn screening for severe combined immunodeﬁciency. J. Pediatr. 147:603–8 40. McCabe ER, Huang Y, Descartes M, et al. 1990. DNA from Guthrie spots for diagnosis of DMD by multiplex PCR. Biochem. Med. Metab. Biol. 44:294–95 41. Seltzer WK, Accurso F, Fall MZ, et al. 1991. Screening for cystic ﬁbrosis: feasibility of molecular genetic analysis of dried blood specimens. Biochem. Med. Metab. Biol. 46:105–9 42. Bhardwaj U, Zhang Y-H, Blackburn W, et al. 2002. Rapid conﬁrmation of Southeast Asian and Filipino alpha-thalassemia genotypes from newborn screening specimens. Am. J. Hematol. 71:56–58 43. Bhardwaj U, Zhang Y-H, McCabe ER. 2003. Neonatal hemoglobinopathy screening: molecular genetic technologies. Mol. Genet. Metab. 80:129–37 44. Bhardwaj U, Zhang Y-H, Lorey F, et al. 2005. Molecular genetic conﬁrmatory testing from newborn screening samples from the common African-American, Asian Indian, Southeast Asian, and Chinese beta-thalassemia mutations. Am. J. Hematol. 78:249–55 45. Bhardwaj U, McCabe ER. 2005. Multiplex-PCR assay for the deletions causing hereditary persistence of fetal hemoglobin. Mol. Diagn. 9:151–56 46. Dipple KM, McCabe ER. 2000. Phenotypes of patients with “simple” Mendelian disorders are complex traits: thresholds, modiﬁers, and systems dynamics. Am. J. Hum. Genet. 66:1729–35 47. Dipple KM, McCabe ER. 2000. Modiﬁer genes convert “simple” Mendelian disorders to complex traits. Mol. Genet. Metab. 71:43–50 48. Vockley J, Rinaldo P, Bennett MJ, et al. 2000. Synergistic heterozygosity: disease resulting from multiple partial defects in one or more metabolic pathways. Mol. Genet. Metab. 71:10– 18 49. Dipple KM, Zhang Y-H, Huang B-L, et al. 2001. Glycerol kinase deﬁciency: evidence from complexity in a single gene disorder. Hum. Genet. 109:55–62 50. Dipple KM, Phelan JK, McCabe ER. 2001. Consequences of complexity within biological networks: robustness and health, or vulnerability and diseases. Mol. Genet. Metab. 74:45–50 51. Sriram G, Martinez JA, McCabe ER, et al. 2005. Single-gene disorders: What role could moonlighting enzymes play? Am. J. Hum. Genet. 76:911–24 52. Sidransky E. 2006. Heterozygosity for a Mendelian disorder as a risk factor for complex disease. Clin. Genet. 70:275–82

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53. Rinaldo P, Tortorelli S, Matern D. 2004. Recent developments and new applications of tandem mass spectrometry in newborn screening. Curr. Opin. Pediatr. 16:427–33 54. Rhead WJ. 2006. Newborn screening for medium-chain-acyl-CoA dehydrogenase deﬁciency: a global perspective. J. Inherit. Metab. Dis. 29:370–77 55. Rinaldo P, Zafari S, Tortorelli S, et al. 2006. Making the case for objective performance metrics in newborn screening by tandem mass spectrometry. Ment. Retard. Dev. Disabil. Res. Rev. 12:255–61 56. Fletcher JM. 2006. Screening for lysosomal storage disorders—a clinical perspective. J. Inherit. Metab. Dis. 29:405–8

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:163-175. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Is Human Hibernation Possible? Annu. Rev. Med. 2008.59:177-186. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Cheng Chi Lee Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, Texas 77030; email: [email protected]

Annu. Rev. Med. 2008. 59:177–86

Key Words

The Annual Review of Medicine is online at http://med.annualreviews.org

hypothermia, 5 -AMP, torpor, hypometabolism

This article’s doi: 10.1146/annurev.med.59.061506.110403

Abstract

c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0177$20.00

The induction of hypometabolism in cells and organs to reduce ischemia damage holds enormous clinical promise in diverse ﬁelds, including treatment of stroke and heart attack. However, the thought that humans can undergo a severe hypometabolic state analogous to hibernation borders on science ﬁction. Some mammals can enter a severe hypothermic state during hibernation in which metabolic activity is extremely low, and yet full viability is restored when the animal arouses from such a state. To date, the underlying mechanism for hibernation or similar behaviors remains an enigma. The beneﬁcial effect of hypothermia, which reduces cellular metabolic demands, has many well-established clinical applications. However, severe hypothermia induced by clinical drugs is extremely difﬁcult and is associated with dramatically increased rates of cardiac arrest for nonhibernators. The recent discovery of a biomolecule, 5 -AMP, which allows nonhibernating mammals to rapidly and safely enter severe hypothermia could remove this impediment and enable the wide adoption of hypothermia as a routine clinical tool.

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INTRODUCTION

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The word hibernation is often associated with bears taking a long winter rest. However, some investigators would argue that bears do not really hibernate but rather enter a state of torpor. In mammals, the term torpor is often used when body temperature drops below 31◦ C. Hibernation is often described as deep torpor. Although both words are widely used in the scientiﬁc literature to reﬂect different degrees of a hypometabolic state, their physiological deﬁnitions are not clear. In addition, some reptiles and amphibians can undergo a hypometabolic process known as estivation to survive challenging environmental conditions. As articulated by Heldmaier et al. (1), “The physiological properties of daily torpor, hibernation and estivation are very similar. The classiﬁcation of hibernation, daily torpor or estivation simply represents gradual difference in the timing, the duration and the amplitude of physiological inhibition.” In essence, these are hypometabolic behaviors used by animals for energy conservation. On the basis of oxygen consumption measurements, it was demonstrated that the biochemical and physiological events that inhibit endogenous thermoregulation occur rapidly prior to the onset of hypothermia (1). In contrast, hypothermia is driven by heat loss from the body to the environment. This process depends on several parameters including surface/volume ratio, the amount of fur and fat insulation, and the temperature gradient between the body and the environment. The molecular and biochemical mechanisms underlying the natural shutdown of metabolic activities remain largely unknown. The goal of this article is not to recapitulate the physiology of hypometabolism that occurs naturally, as there are excellent reviews in the literature (1). Instead, I focus on recent attempts to mimic the induction of a hypometabolic state in mammals, which may have potential clinical applications. I also offer some hypotheses on how hypometabolism could occur in nonhibernat-

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ing mammals. Whether chemically induced hypometabolism has any connection with the natural process is not critical; observations from these studies can be drawn upon to reveal the underlying biochemical and molecular processes of hypometabolic behaviors. Clinically, only mild hypothermia of 32– 34◦ C can be induced safely by a cocktail of paralytic drugs and anesthesia (2, 3). Severe hypothermia (28◦ C or lower) induced by such drug cocktails results in a high cardiac arrest rate in nonhibernating mammals (4). To date, three classes of molecules will result in reversible severe hypothermia when administered to small mammals such as mice and hamsters. Two of these are metabolic inhibitors: 2-deoxyglucose and hydrogen sulﬁde (H2S). The third, recently identiﬁed in my laboratory, is the end metabolite 5 -adenosine monophosphate (5 -AMP). It is the ﬁrst natural biomolecule to have this effect (5).

2-DEOXYGLUCOSE AND HYDROGEN SULFIDE Hamsters are known to undergo daily torpor to conserve energy upon prolonged exposure to low environmental temperature and short photoperiod, which mimic seasonal changes (6). This torpor behavior of hamsters is circadian-driven. This was demonstrated by experiments in which ablation of the suprachiamastic nucleus (SCN), the central circadian structure, disrupts the animal’s temporal rhythm (7). After receiving the glycolytic inhibitor 2-deoxyglucose, hamsters readily undergo torpor even when kept in long photoperiod (8). In contrast, inhibition of fatty acid metabolism by mercaptoacetate did not induce torpor in hamsters kept in long photoperiod (6). These observations implicate impediment of glucose utilization as a key event for hypometabolism. However, the biochemical mechanism underlying 2-deoxyglucose induction of torpor remains unclear. Another metabolic inhibitor, H2S, was recently found to enable mice to enter severe

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hypothermia or suspended animation at a low dosage of 80 ppm (9). Mice given H2S could be cooled down to 15◦ C into a state of suspended animation for up to six hours. Arousal was spontaneous, and no apparent detrimental outcome was observed after recovery. It has been thought that a core body temperature below 20◦ C in nonhibernators will lead to cardiac ﬁbrillation (10). Our lowest recording of core body temperature in mice during torpor induced by fasting was about 26◦ C even when the ambient environmental temperature was maintained at 8◦ C ( J. Zhang & C.C. Lee, unpublished observations). Thus, the ability to drop a nonhibernating mammal’s core body temperature to 15◦ C is a major step forward. It suggests that nonhibernators are fully capable of withstanding extreme hypometabolism. As with 2-deoxyglucose, the mechanism underlying H2S induction of severe hypothermia remains unclear. H2S is a speciﬁc and reversible inhibitor of cytochrome c oxidase, a key component of the mitochondria respiratory complex IV (9). Inhibitors of the mitochondria respiratory chain are toxic to mammals because they disrupt ATP production by oxidative phosphorylation. Similarly, 2-deoxyglucose inhibits glycolysis, which is another biochemical process involved in the generation of ATP and NADH from glucose outside of the mitochondria. Thus, the actions of both H2S and 2-deoxyglucose affect the cellular production of ATP. In turn, the lower ATP level would downregulate biochemical reactions necessary for thermal regulation defenses. It has been observed that during hibernation, erythrocytes and organs have signiﬁcantly lower ATP levels (11, 12). In particular, the ATP level of erythrocytes from a hibernating animal is ∼50% of the level observed in a euthermic state (11). Such a large decrease in ATP level in the erythrocyte would signiﬁcantly compromise its function in regulating oxygen/carbon dioxide molecules necessary for maintaining high metabolic activity of the major organs. Under such conditions, it is highly possible that the metabolic rate of organs will slow, and heat loss from the body to

the environment will not be adequately controlled. This reasoning is consistent with the hypothesis that decreased ATP production or utilization is involved in hibernating behaviors (1, 12). How these biochemical observations can be reconciled with the widely held dogma that the preoptic area of the hypothalamus controls thermal regulatory response in mammals is not clear (13).

SEARCH FOR AN ENDOGENOUS MOLECULE FOR HYPOMETABOLISM For hibernators to achieve a severe hypothermic state, the basic principles of metabolic biochemistry must be preserved to ensure energy homeostasis (12). This raises the possibility that such biochemical processes may be retained in all mammals. Mammalian organs can be maintained in a hypothermic but highly hypoxic state for many hours (14), as we see when donor organs are transported in coolers for organ transplantation. Upon transplantation, the restoration of blood ﬂow and its rewarming revive the basic functions of the donor organ. In addition, examples from accidental hypothermia have suggested that under certain conditions, humans can recover fully from prolonged periods in severe hypothermia. Critically, these observations suggest that nonhibernators’ organs are inherently capable of withstanding extreme hypoxic stress if their metabolic demands are reduced. Thus, a project was initiated to probe the possibility of identifying genes from nonhibernating mammals that are activated in an environment encountered during hibernation. It is widely recognized that mammals enter hibernation in an environment of constant darkness (1). Using liver mRNA, gene expression in mice exposed to regular 12:12 h cycles of light:dark (LD) was compared to gene expression in mice kept in constant darkness or dark:dark (DD). From microarray analysis, a gene encoding procolipase was identiﬁed to be highly expressed in the liver of the DD mouse. Previous studies have demonstrated www.annualreviews.org • Is Human Hibernation Possible?

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Figure 1 Activation of procolipase expression by a constant-darkness environment. The Northern blot analysis shows a 4-h time course of the liver mRNA species of Gapdh and procolipase obtained from mice kept in 12:12 h light-dark (LD) cycles or in dark-dark (DD) cycles, i.e., constant darkness. Gapdh is used as an internal control. The genes were identiﬁed by radiolabeled cDNA’s probe for Gapdh and procolipase, respectively.

that procolipase gene expression is highly speciﬁc to the pancreas and gastrointestinal tract, consistent with its primary role of breaking down dietary fat into fatty acids (15). The exceptions are hibernating ground squirrels, in which procolipase was activated in other peripheral organs (16). Conﬁrming the microarray ﬁndings, an independent method of gene detection based on Northern blot analysis demonstrated that the procolipase gene was indeed activated in DD mice but not in LD mice (Figure 1). Tissue analysis revealed procolipase was activated in a circadian manner in the majority of the peripheral organs in DD mice. Exposure of these mice to white light resulted in the shutdown of procolipase expression in the various organs (5). Together, these ﬁndings suggested that the endogenous signaling mechanism is mediated by a circulatory factor.

IDENTIFICATION OF 5 -AMP AS AN ACTIVATOR OF PROCOLIPASE EXPRESSION The above observations indicate that this endogenous regulator must be a circulatory molecule that displays a circadian proﬁle in its activity. It could behave either as an activator or a repressor. If it is an activator, then injection into LD mice will induce procoli180

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pase expression in the major organs. If it acts as a repressor, then injection into DD mice will abolish procolipase expression in the peripheral organs. Through a series of careful experiments, our search for this circulatory molecule was narrowed to the soluble aqueous fractions of blood extract. These studies led to the identiﬁcation of a circulatory nucleotide displaying clear circadian oscillation that matched the hypothesized proﬁle described above (5). Characterization of this molecule based on its spectral absorption, its migration distance on high-performance liquid chromatography (HPLC) relative to known chemical nucleotide standards, and enzymatic analysis indicated the molecule was adenosine 5 monophosphate (5 -AMP). To conﬁrm this, synthetic 5 -AMP was injected into LD mice; procolipase expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in all tissues sampled with the exception of the brain (Figure 2a). These ﬁndings demonstrate that the level of circulatory 5 -AMP was involved in regulating procolipase expression. However, the induction of procolipase by 5 -AMP was not immediate. Its prolonged time course suggests that it was an indirect effect. Unexpectedly, mice that received 5 -AMP were severely hypothermic, with core body temperatures as low as 25◦ C when kept in ambient room temperatures of about 23–24◦ C (Figure 2b). The severe hypothermic period was transient; animals reentered a thermogenic period and core body temperature was restored several hours later. Further monitoring of these animals over several months revealed no apparent deﬁcit. The impact of 5 -AMP as reﬂected internally by the heart rate indicated a very rapid decline in metabolic activity. The heart rate fell from ∼600 beat/min (normal for a mouse) to ∼200 beat/min within 5 min after 5 -AMP was given (Z. Tao & C.C. Lee, unpublished observations). As core body temperature dropped from 37◦ C to 25◦ C, there was a direct correlation between this hypometabolic state and the reduced heart and respiration rates. Similarly,

POSSIBLE MECHANISMS OF 5 -AMP–INDUCED HYPOMETABOLISM Although ATP is the cellular energy currency, 5 -AMP occupies the unique position that de-

a)

Kidney Liver

as the animal aroused from the hypometabolic state, the core body temperature gradually rose toward euthermic state (∼37◦ C), along with increasing heart and respiration rates. To determine whether 5 -AMP plays a similar role in natural torpor, DD mice were fasted to induce natural torpor. Mice in fasting-induced torpor had approximately a two- to three-fold higher level of 5 -AMP in the blood than mice that did not enter torpor (5). The kinetics of natural torpor is slower than that induced by synthetic 5 -AMP; this can be accounted for by the slower buildup of natural 5 -AMP generated by fasting and the physical barriers that regulate heat loss from the body to the environment. It appears that 5 -AMP must inhibit or retard thermoregulation, as hypothermia is a result of undefended heat loss from the body to the environment. How low the core body temperature of a nonhibernator can be reduced and yet maintain viability is unclear. Hibernators such as arctic ground squirrels can withstand drops in core body temperature to several degrees below freezing (17). However, larger mammals that are known to hibernate prefer core body temperatures that are much higher. In the winter, the core body temperature of bears reduces to ∼32◦ C, and the tropical Malagasy lemur, a primate that hibernates in the dry season of the tropics, drops its core body temperature to around 25◦ C (1). These observations suggest that each mammalian species has an optimum core body temperature range for such hypometabolic activities. A temperature compensation mechanism is used to maintain the core body temperature necessary for biochemical reactions. Thus, when an animal’s core body temperature drops below the ideal range for its species, its metabolic rate increases rather than slows down further (1).

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Figure 2 Induction of procolipase expression and hypothermia by synthetic 5 -AMP. (a) RT-PCR detection of procolipase and Gapdh expression in mice kept in 12:12 h light:dark cycles given saline or 5 -AMP. (b) Hypothermic response of mice injected with various dosages of 5 -AMP.

termines salvage and catabolism of the adenine nucleotides (Figure 3). In all cells, the adenylates’ biochemical equilibrium, ATP + 5 -AMP ↔ 2ADP, which is regulated by the enzyme adenylate kinase, controls cellular energy charge (18). By and large, the ratio of ATP to 5 -AMP determines the energy state of the cell. Therefore, excess 5 -AMP can either be salvaged via ADP or is catabolized. In the catabolic pathway, 5 -AMP can be degraded via two pathways. The major pathway is via inosine 5 -monophosphate (IMP) control by the enzyme AMP deaminase. IMP is rapidly catabolized to inosine and hypoxanthine and then to uric acid, which in humans www.annualreviews.org • Is Human Hibernation Possible?

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ADA

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Figure 3 5 -AMP is pivotal in adenine nucleotide salvage and catabolism. The diagram illustrates the extracellular and intracellular roles of 5 -AMP. AdoR, adenosine receptors; CD73/ecto 5 NT, ecto 5 -nucleotidase; ADA, adenosine deaminase; AK, adenosine kinase; cyto 5 NT, cytosolic nucleotidase; AMPD, AMP deaminase; PNC, purine nucleotide cycle; 5 -IMP, inosine 5 -monophosphate; AMP, adenosine 5 -monophosphate; ADP, adenosine diphosphate; ATP, adenosine triphosphate. It is unclear how ATP gets out of the cell. Whether 5 -AMP is transported in and out of the cell is also unclear.

and other primates is excreted. In most other mammals, the uric acid is further catabolized into allantoin before being excreted. However, IMP can be salvaged to regenerate 5 AMP through the purine nucleotide cycle via an intermediate, adenylosuccinate. In the minor pathway, 5 -AMP can be dephosphorylated directly into adenosine extracellularly by ecto-5 -nucleotidase (CD73) and intracellularly by cytosolic 5 -nucleotidase. Adenosine is then degraded to inosine by the enzyme adenosine deaminase (ADA) and eventually to uric acid. Intracellularly, the rephosphorylation of adenosine to 5 -AMP is undertaken by the enzyme adenosine kinase. Extracellularly, adenosine, resulting from dephosphorylation of 5 -AMP into adenosine by CD73, can either enter the cell via transporters or bind its receptors. The latter involves binding to four known adenosine182

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activated G protein–coupled receptors: A1, A2a, A2b, and A3. These receptors have different afﬁnities for adenosine and can play a key role in either activating or inhibiting cascades regulated by adenylyl cyclase (19). Alternatively, adenosine is taken up by the cell via multiple types of transporters such as equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs) (20, 21). The ENTs are gradient-driven, whereas the CNTs are active transporters that involve exchange of a cation such as sodium. These transporters are very important to certain cells, such as erythrocytes, that are deﬁcient in organelles and cannot perform de novo synthesis of nucleoside. To maintain its cellular nucleotide pool, an erythrocyte has extremely high levels of nucleoside transporter to overcome this lack of de novo synthesis of nucleoside (20). Although the

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identiﬁcation of an AMP receptor has been reported recently (22), it remains controversial (23). The direct uptake of adenine nucleotides such as cAMP by erythrocytes (24, 25) and of 5 -AMP by intestinal cells (26) has been reported. Several genes for adenine nucleotide transporter/translocase have been identiﬁed, but to date their functions have been demonstrated only for organelles such as mitochondria and peroxisomes (27, 28). The injection of 5 -AMP and adenosine both induce procolipase expression in organs of mice (5). However, the solubility of adenosine in water is very low, and the amount required to induce procolipase expression in a mouse has to be dissolved in an organic solvent, DMSO. The solubility consideration alone would argue strongly against adenosine as the primary activator. In addition, circulating adenosine largely remains very low throughout the diurnal cycle (5). The extracellular level of 5 -AMP is regulated by the enzyme ecto-5 nucleotidase (CD73), expression of which is under circadian control (29, 30). In addition, DD mice have a signiﬁcantly dampened expression level of CD73 compared with LD animals (5). Thus, the photoperiod effect and circadian proﬁle of circulating 5 -AMP levels is linked to the endogenous clock control of CD73 expression. The induction of procolipase by 5 -AMP or adenosine was blocked in animals that had received a prior injection of dipyridamole, a broad-based transporter inhibitor (5). This observation indicates that the underlying mechanism involves an uptake of adenosine or 5 -AMP rather than being mediated through the adenosine receptors. Our studies do not exclude adenosine receptors’ role in the regulation of the adenosine transporter function. Recent studies have shown that nucleoside transporter activity can be controlled by the adenosine receptors, as for example CNT2 is controlled by the adenosine A1 receptor (31). However, once inside the cell, adenosine is rapidly phosphorylated into 5 -AMP because the Km of adenosine ki-

nase (AK) for adenosine is one to two orders of magnitude smaller than that of ADA for adenosine (32). Alternatively, 5 -AMP could be transported into cells known to take up adenine nucleotides directly, such as erythrocytes (24, 25). With either uptake mechanism, a rapid buildup of 5 -AMP inside the cell results, which in turn affects the adenylates’ biochemical equilibrium (ATP + 5 -AMP ↔ 2ADP). To counter the rise of 5 -AMP, the enzyme adenylate kinase drives the formation of ADP, which leads to a depletion of ATP. The decrease in ATP slows cellular function, which would mimic the drop in ATP levels observed in erythrocytes of hibernating mammals (11). However, ATP cannot decrease indeﬁnitely if 5 -AMP levels continue to rise, as this would result in the complete shutdown of cellular function. The AMP deaminase Km for 5 -AMP is higher than that of adenylate kinase for 5 -AMP (12). Thus, excess 5 -AMP above and beyond the Km of adenylate kinase is rapidly catabolized by AMP deaminase to IMP, thus limiting overdepletion of ATP in the cell.

ALLOSTERIC REGULATION OF METABOLIC ENZYMES BY 5 -AMP In addition to its pivotal position in the purine biochemical pathways, 5 -AMP also acts as an allosteric regulator of many rate-limiting metabolic enzymes (18) (Figure 4). One such allosteric enzyme is AMP-dependent protein kinase (AMPK). AMPK, which is activated by 5 -AMP, has been implicated as a positive regulator of glucose transport in muscle, glycolysis, and fatty acid oxidation (33). Its vast cellular role is beyond the scope of this review. Our studies showed that the 5 -AMP activation of procolipase expression in LD mice is reciprocally linked to blood glucose (5). 5 AMP is known as an allosteric regulator of two key enzymes that control glucose homeostasis: It is a positive regulator of fructose 1,6 phosphatase (FDP) and a negative reglator of www.annualreviews.org • Is Human Hibernation Possible?

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Figure 4 Allosteric regulation key metabolic enzymes by 5 -AMP. The rate-limiting enzymes for gluconeogenesis (FDP), glycolysis (PFK) and glycogenolysis (GP, glycogen phosphorylase) are allosterically regulated by 5 -AMP.

phosphofructose kinase (PFK). FDP is a ratelimiting enzyme for gluconeogenesis, and it converts fructose 1,6 phosphate to fructose 6 phosphate (18). FDP binding of 5 -AMP inhibits its enzymatic activity, thereby limiting gluconeogenesis. In the reverse direction, PFK is a rate-limiting enzyme for glycolysis. It converts fructose 6 phosphate into fructose 1,6 phosphate by utilizing an ATP molecule (18). Unlike FDP, the activity of PFK is enhanced by 5 -AMP, thereby increasing the rate of glycolysis. For both enzymes, the allosteric effects of ATP are opposite to those of 5 -AMP because these adenylate nucleotides bind competitively to the same regulatory motif (18). As proposed above, the elevated level of cellular 5 -AMP after its uptake will alter adenylate biochemical equilibrium to favor ADP production, thus reducing the cellular ATP level. The drop in ATP will slow cellular biochemical processes and

is postulated to reduce thermoregulatory activity and allow hypometabolism to set in. An animal must eventually exit from the hypometabolic state, and the catabolism of 5 AMP to IMP by AMP deaminase or by the cytosolic nucleotidase into adenosine can provide only part of the means for exit. The regeneration of ATP from ADP via glycolysis in various cells, including the erythrocytes, must occur to reestablish the euthermic adenylate biochemical equilibrium. The decline in the ratio of ATP to 5 -AMP that leads to the hypometabolism eventually activates PFK, thereby increasing glycolysis, which utilizes the accumulated ADP in the cell to regenerate ATP. This restores the adenylate biochemical equilibrium to its euthermic state, completing the metabolic events initiated by 5 -AMP.

CONCLUSION The experimental investigation of hypometabolism induced by 5 -AMP is ongoing. I have taken this opportunity to articulate some of the current hypotheses that are testable using existing biochemical and pharmaceutical tools. In addition, mouse genetics could be used to dissect the 5 -AMP–driven biochemical pathways of hypometabolism in animal models. Finally, our identiﬁcation of a natural biomolecule that allows rapid initiation of hypometabolism in mammals may eventually result in clinical applications where hypothermia has been shown to have tremendous lifesaving potential, such as trauma, heart attacks, strokes, and many major surgeries (3).

DISCLOSURE STATEMENT A patent application has been ﬁled by the University of Texas Health Science Center, Houston on behalf of the author on the potential utilities of 5 -AMP.

ACKNOWLEDGMENT This work is supported in part by an NIH Director Pioneer Award to the author. 184

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LITERATURE CITED 1. Heldmaier G, Ortmann S, Elvert R. 2004. Natural hypometabolism during hibernation and daily torpor in mammals. Respir. Physiol. Neurobiol. 141:317–29 2. Wu X, Kochanek PM, Cochran K, et al. 2005. Mild hypothermia improves survival after prolonged, traumatic hemorrhagic shock in pigs. J. Trauma 59:291–99 3. Kabon B, Bacher A, Spiss CK. 2003. Therapeutic hypothermia. Best Pract. Res. Clin. Anaesthesiol. 17:551–68 4. Moon PF, Ilkiw JE. 1993. Surface-induced hypothermia in dogs: 19 cases (1987–1989). J. Am. Vet. Med. Assoc. 202:437–44 5. Zhang J, Kaasik K, Blackburn MR, et al. 2006. Constant darkness is a circadian metabolic signal in mammals. Nature. 439:340–43 6. Stamper JL, Dark J, Zucker I. 1999. Photoperiod modulates torpor and food intake in Siberian hamsters challenged with metabolic inhibitors. Physiol. Behav. 66:113–18 7. Heller HC, Ruby NF. 2004. Sleep and circadian rhythms in mammalian torpor. Annu. Rev. Physiol. 66:275–89 8. Dark J, Miller DR, Zucker I. 1994. Reduced glucose availability induces torpor in Siberian hamsters. Am. J. Physiol. 267:496–501 9. Blackstone E, Morrison M, Roth MB. 2005. H2S induces a suspended animation-like state in mice. Science 308:518 10. Johansson BW. 1996. The hibernator heart—nature’s model of resistance to ventricular ﬁbrillation. Cardiovasc. Res. 31:826–32 11. Doherty JC, Kronon MT, Rotermund AJ Jr. 1993. The effects of short term cold storage upon ATP and 2,3-BPG levels in the blood of euthermic and hibernating thirteenlined ground squirrels Spermophilus tridecemlineatus. Comp. Biochem. Physiol. Comp. Physiol. 1104:87–91 12. English TE, Storey KB. 2000. Enzymes of adenylate metabolism and their role in hibernation of the white-tailed prairie dog, Cynomys leucurus. Arch. Biochem. Biophys. 376:91–100 13. Boulant JA. 2006. Neuronal basis of Hammel’s model for set-point thermoregulation. J. Appl. Physiol. 100:1347–54 14. Churchill TA, Simpkin S, Wang LC, et al. 1996. Metabolic effects of cold storage on livers from euthermic and hibernating Columbian ground squirrels. Cryobiology 33:34–40 15. Lowe ME. 1977. Molecular mechanisms of rat and human pancreatic triglyceride lipases. J. Nutr. 127:549–57 16. Squire TL, Lowe ME, Bauer VW, et al. 2003. Pancreatic triacylglycerol lipase in a hibernating mammal. II. Cold-adapted function and differential expression. Physiol. Genom. 16:131–40 17. Barnes BM. 1989. Freeze avoidance in a mammal: body temperatures below 0◦ C in an Arctic hibernator. Science 244:1593–95 18. Lehninger AL. 1977. Biochemistry: The Molecular Basis of Cell Structure and Function, pp. 623–57. New York: Worth. 2nd ed. 19. Ralevic V, Burnstock G. 1998. Receptors for purines and pyrimidines. Pharmacol. Rev. 50:413–92 20. Lofﬂer M, Morote-Garcia JC, Eltzschig SA, et al. 2007. Physiological roles of vascular nucleoside transporters. Arterioscler. Thromb. Vasc. Biol. 27:1004–13 21. King AE, Ackley MA, Cass CE, et al. 2006. Nucleoside transporters: from scavengers to novel therapeutic targets. Trends Pharmacol. Sci. 27:416–25 22. Inbe H, Watanabe S, Miyawaki M, et al. 2004. Identiﬁcation and characterization of a cell-surface receptor, P2Y15, for AMP and adenosine. J. Biol. Chem. 279:19790–99 www.annualreviews.org • Is Human Hibernation Possible?

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23. Abbracchio MP, Burnstock G, Boeynaems JM, et al. 2005. The recently deorphanized GPR80 (GPR99) proposed to be the P2Y15 receptor is not a genuine P2Y receptor. Trends Pharmacol. Sci. 26:8–9 24. Holman GD. 1979. Inﬁnite cis inﬂux of cyclic AMP into human erythrocyte ghosts. Biochim. Biophys. Acta 553:489–94 25. DeBari VA, Novak NA, Bennun A. 1984. Cyclic nucleotide metabolism in the human erythrocyte. Clin. Physiol. Biochem. 2:227–38 26. He Y, Chu SH, Walker WA. 1993. Nucleotide supplements alter proliferation and differentiation of cultured human (Caco-2) and rat (IEC-6) intestinal epithelial cells. J. Nutr. 123:1017–27 27. Visser WF, van Roermund CW, Ijlst L, et al. 2007. Metabolite transport across the peroxisomal membrane. Biochem. J. 401:365–75 28. Sharer JD. 2005. The adenine nucleotide translocase type 1 (ANT1): a new factor in mitochondrial disease. IUBMB Life 57:607–14 29. Frederiks WM, Marx F, Bosch KS, et al. 1987. Diurnal variation in 5 -nucleotidase activity in rat liver. A quantitative histochemical study. Histochemistry 87:439–43 30. Uchiyama Y. 1983. A histochemical study of variations in the localization of 5 -nucleotidase activity in the acinar cell of the rat exocrine pancreas over the twenty-four hour period. Cell Tissue Res. 230:411–20 31. Duﬂot S, Riera B, Fernandez-Veledo S, et al. 2004. ATP-sensitive K+ channels regulate the concentrative adenosine transporter CNT2 following activation by A1 adenosine receptors. Mol. Cell Biol. 24:2710–19 32. Arch JR, Newsholme EA. 1978. Activities and some properties of 5 -nucleotidase, adenosine kinase and adenosine deaminase in tissues from vertebrates and invertebrates in relation to the control of the concentration and the physiological role of adenosine. Biochem. J. 174:965–77 33. Lindsley JE, Rutter J. 2004. Nutrient sensing and metabolic decisions. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 139:543–59

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:177-186. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Advance Directives Linda L. Emanuel

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The Buehler Center on Aging, Health & Society, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611; email: [email protected]

Annu. Rev. Med. 2008. 59:187–98

Key Words

First published online as a Review in Advance on August 20, 2007

advance care planning, medical directives, proxy, scenarios, goals, thresholds

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.58.072905.062804 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0187$20.00

Abstract Most patients want some control over their medical care, including— or even especially—when they are too sick to participate in decisions. Clinicians who have to make decisions for patients who are unable to participate often would appreciate guidance from patients’ wishes. Advance care planning responds to these needs. The process provides for discussions about goals in different scenarios and allows inclusion of the family and physician as well as the patient. It helps to have the patient and family complete validated worksheets that walk them through the various considerations and result in expressions of preference that are clinically meaningful. For the clinician, scenariobased goals for care and personal thresholds for when desired care shifts from primarily cure-oriented to primarily palliative are the most useful features to know about. The patient and family should do most of the discussing on their own time; the physician and team should coordinate to screen for problems and ensure agreement. Ideally, this should occur over the course of regular clinical encounters, with some dedicated time for the topic at suitable intervals.

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BACKGROUND

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Two main modalities exist by which a person can express preferences about medical care in anticipation of future incapacity (1). One is to appoint a proxy to speak in the place of the principal person. The other is to write down wishes in a directive. These two modalities are usually complementary, since written statements cannot provide for all eventualities, and proxy decision makers cannot speak accurately on behalf of patients without their guidance.

Proxy Designation Physicians should be aware of three key issues on which to advise patients and proxies. First, patients, professionals, and the proxies themselves should understand the proxy’s role. Some patients want the proxy to express the patient’s own judgment (2). Others prefer that the proxy remains more independent of the patient’s stated prior wishes and tries instead to balance issues as he or she sees ﬁt. The patient should specify which type (or blend of types) of proxy judgment to apply (3). Second, studies have found that proxies often guess the prior wishes of patients inaccurately and, furthermore, proxies often imagine that the patients’ prior wishes are for more intervention than patients actually selected (4). Patients and proxies should know about these study ﬁndings. A deteriorating medical situation may be less stressful if explicit discussion has taken place well in advance (5). Third, patients should be aware that friends and family members have their own interests and issues, which may conﬂict with their role as proxy (4). Conﬂicting motivations are inevitable and need not disqualify a proxy; however, he or she may need help distinguishing different motivations and abiding by those that are most suited to the proxy role.

Instructional Directives The history of the development of instructional directives reﬂects the search for the 188

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most valid form of expressing a patient’s prior wishes. The earliest commonly used instructional directive was the Do Not Resuscitate (DNR) order, written by the physician after discussion with the patient and family (6). After its proposal in 1976, a set of studies and a culture evolved around the DNR discussion. It is still relevant and can be included in comprehensive advance planning (7). A hazard of isolated DNR discussions is that they occur too late, either missing the patients who need them or occurring so abruptly that alarmed patients make decisions without the beneﬁt of settled reﬂection (8–10). A more recent approach involves a cluster of doctors’ orders concerning life-sustaining intervention, such as the Physician Orders for LifeSustaining Treatment (POLST) form (11). This approach can optimize discussions with patients and families. An earlier, less commonly used form of instructional directive was the living will. This was introduced in 1968 by a lawyer, Louis Kutner. The living will attempted to express the widespread view that heroic levels of technological intervention should be avoided if the patient’s prognosis was hopeless. Efforts to increase the speciﬁcity of living wills began, most notably with Sissela Bok’s and Michigan’s living wills (12, 13). Thereafter, developments proceeded along two lines: One goal was to better describe the general healthrelated values of the patient (values histories) and the other was to formulate ways in which patients could make very speciﬁc treatment preference statements (treatment-speciﬁc directives) (14, 15). Empirical evidence that general statements cannot predict speciﬁc wishes has supported the more balanced view that these two modalities work best together (16–18). Importantly, empirical evidence exists that patients’ goals for care in a given situation can predict quite accurately what their speciﬁc treatment preferences would be (17). Therefore, if only a minimum of information can be elicited, physicians are now being taught to select a likely scenario and ask what the patient’s goals for treatment would be (19).

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More recently, efforts have been focused on the need to validate predrafted instructional directives, just as any other instrument intended to record subjective matters needs to be validated (20). A few forms have been validated, including at least one tailored to cancer and another to AIDS (21–23). One of the more studied forms, which is generic and adaptable and has been validated in several studies, is the Medical Directive (17, 24–27). Physicians should provide and advise patients to use validated forms, since using nonvalidated forms risks misrepresentation of patients’ true wishes and can confuse decision making. Validated forms also provide a succinct method of ensuring that patients have considered the major areas that most people need to cover. The use of a validated form as a basis for thought and discussion can be as important as its use as a recorded document.

Statutory versus Advisory Documents All states and the District of Columbia have statutes that endorse advance directives in one way or another. Some endorse the use of proxies, others endorse the use of instructional directives, and most now endorse both (28). Most state statutes specify a corresponding document, which is often available from local health care facilities or state medical organizations. These are not the only documents that carry legal standing, however. The fundamental purpose of state statutes is to allow physicians to carry out patients’ wishes without fear of liability. Because it is part of common law that competent patients have the right to accept or refuse medical intervention (including life-sustaining interventions), even casual statements have been honored as sufﬁcient evidence, and this has held true despite national political campaigns (29). Written statements have been explicitly identiﬁed as desirable evidence of patients’ preferences (30, 31). Physicians can therefore be assured that patients’ statements carry legal authority whether or

not they are recorded in a fashion speciﬁcally designed to comply with state statutes.

The Patient Self-Determination Act and JCAHO Recommendations The United States’ Patient SelfDetermination Act of 1990 requires that patients be asked about the existence of an advance directive at the time of enrollment or admission to a health care facility. The intent of the law was to increase awareness and documentation of advance directives. In addition, the Joint Commission on Accreditation of Healthcare Organizations (JCAHO) recommends that facilities have arrangements for counseling patients who wish to complete advance directives. Completion of advance directives is best done in the more stable setting of continuing outpatient care, but occasionally their completion in the inpatient setting is unavoidable. Thus, the spirit of both requirements involves thoughtful, longitudinal involvement of the physician and other members of the interdisciplinary team in discussion with the patient.

Cultural Differences Advance care planning has evolved in the context of Western cultures, in which the individual’s rights have some priority. However, the idea of planning is not predicated on individualism and is readily embraced in cultures that emphasize extended families and community responsibility. Cultural differences also invite special consideration and can usually be accommodated. When the role of deciding for others traditionally falls to a particular person in a culture, advance planning may seem threatening to that person. If those who seek to make a plan are distrusted by those who will be affected, then planning may be omitted or counterproductive unless trust is established (32). The delegation of decision making to family heads has been identiﬁed in families of Asian origin; trust concerns have been identiﬁed in www.annualreviews.org • Advance Directives

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African American patients and families; and concerns over precipitating undesired outcomes by discussing them has been identiﬁed among Navajo families (33–35). Perhaps the most problematic cultural issues have to do with inhibitions against discussing dying. This taboo has been as strong in modern Western cultures as elsewhere. Although cultural issues are powerful, generalizations are problematic. The clinician who approaches people with genuine respect and, with an open mind, inquires about and honors cultural differences should be able to accomplish advance care planning when it is appropriate in forms that suit each case and setting. Three practical tips can help with many situations. If extended family decision making may be desired, ask the patient how he or she would like decisions managed and include the relevant people in the process, perhaps suggesting designation of the decision maker in the family as the proxy. If trust may be insufﬁcient, spend more time establishing your trustworthiness by explaining the nature of your thinking as a clinician, by including other members of the family or community, and by avoiding arrogation of decision making and instead sharing information fully and carefully. If death is difﬁcult to talk about, be sure that you are comfortable with the topic and approach it with simplicity and a listening disposition. Ask if there are ways of talking about dying that might be easier and try to accommodate any requests.

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Valid Expressions An efﬁcient way for physicians to assist patients in valid expressions and recordings of prior preferences is to provide them with a validated predrafted instructional directive and go through it with them. If patients have completed such a directive, meeting standards of informed consent for the wishes they express, then the statements recorded must be considered valid. Validation of predrafted instruments for the articulation of subjective mat190

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ters is a well-developed discipline in itself. Generally speaking, instruments must meet the following standards (20): 1. Content validity: The instrument covers the relevant content. 2. Construct validity: Items in the instrument are constructed to ﬁt the concepts of the subject matter. 3. Criterion-related validity: Items bear sensible relationships to existing relevant scales and to one another. 4. Test-retest reliability: If the same items were used again at a different time, a reasonably similar set of responses would be obtained. Predrafted advance directives should meet these standards; unfortunately, most do not (36; see also http://www.medicaldirective. org).

When Patients Have No Advance Directive The estate will, a well-accepted form of planning for death, is used by only about half of all patients. It is likely that advance directives will have a similar “ceiling,” so in the attempt to match decisions with patients’ prior preferences, supplementary approaches can be helpful (38). When the preferences of a population of patients have been documented and studied, those data can help guide decisions for an individual patient in that population. For example, the proxy might ﬁnd it useful to know that the patients registered at the hospital in question declined resuscitation for the situation at hand in 85% of instances. In cases when the proxy has no idea how to speak on behalf of the patient, the physician may indicate that the greatest likelihood of matching the patient’s unarticulated prior wishes will be achieved by following the preferences of a majority of others. Naturally, these default guidelines should not be coercive or replace valid personal directives.

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HOW TO DO IT

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Five Steps in a Continuing Process The creation of recorded advance directives is one step in a longitudinal process that should be integrated into clinical care. Although ﬁve steps can be identiﬁed, they will rarely be so distinct in practice. First is raising the topic. Second, and most important, is structuring a core discussion to cover the main issues and start the patient thinking about his or her views. Third is reviewing the ﬁnal document and putting it in the medical record. Fourth is updating the directive from time to time. Fifth is ensuring its availability and use, applying it to decisions that arise after the patient has become “wishless”—a state in which cognitive function, and in particular awareness, is so compromised that wishes are not possible (39).

Raising the Topic, Providing Background Information, and Advising on Proxy Choice First, the topic must be raised. This may be the hardest part, although once expected and routinized, it is a surprisingly easy matter. With whom should the topic be raised? All patients should have the opportunity for advance care planning, whether the prognosis is excellent or poor. Sometimes, patients will have discussed advance care planning with their primary care or other physician. The clinician can build on this foundation. The topic should not be avoided on the assumption that it has been dealt with. One reason is that patients who have completed advance care planning before receiving an oncologic diagnosis may have since changed their perspective. Another is that the extent and structure of the planning may not have been as good as that which the clinician can offer. Most important, the clinician needs to know the substance of the patient’s current advance care planning. In an outpatient practice, the topic can be raised by the fourth or ﬁfth visit, depending

on the emotional and medical circumstances. Before advance care planning, time should be allowed for conﬁrmation of the diagnosis, exploration of treatment options, establishment of a solid therapeutic relationship, and adjustment by the patient to his or her diagnosis (9, 40). Earlier mention, say at the second or third visit, that this type of planning is a component of routine care—even for patients without serious illness—can facilitate the process. One fashion in which the topic can be raised is as follows: Mr. (Ms.) X, I want to talk with you about planning for future medical care. It is part of getting to know your values and being able to care for you the way you would want to be cared for, even if illness were to make communication impossible. Medical care standards recommend that people make plans whether or not they have an illness, and that doctors discuss these issues routinely with patients. There is even a federal law that we include advance planning in our care. I am not hiding bad news about your health; planning for the future simply is prudent. Is this something you have explored before?

It is helpful to be able to add truthfully: “I myself have done this as a routine matter, and I am in good health.” The role of a proxy can be explained at this point, including some issues for the patient to consider in selecting a proxy. In particular, it should be noted that being a proxy is a complex and burdensome task, and that family members and close friends may have goals that conﬂict with the patient’s. Most people prefer to select someone close to them as a proxy nonetheless, and usually for good reason, but the advantages of appointing a more distant friend or a professional such as a social worker or lawyer are worth mentioning. Some patients suggest appointing the physician as a proxy, but since the purpose of a proxy is to have someone to talk with the physician, this is not encouraged. The patient should be reassured that the physician will in any case www.annualreviews.org • Advance Directives

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be working to make the best decision for the patient and additional proxy powers are usually unnecessary. Participation by the physician in creating a written directive, perhaps without a designated proxy, can be a helpful alternative. Nonphysician health care providers can perform the ﬁrst step of raising the topic and providing background information. Interventions to improve advance care planning have been led by physicians, nurses, social workers, and combinations of these practitioners (41– 47). Brochures, predrafted forms, videos, or closed-circuit television programs can also be made available in patient information libraries and patient rooms.

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Structured Discussion Goals and treatment options. Second, the topic must be discussed. This is usually done best by the patient and family on their own time, once they have been oriented to key features of the task. The proxy should be present at this discussion whenever possible. He or she can be advised to ask for any needed clariﬁcation, in preparation for the potential role of speaking for the patient, and may even act as scribe, penciling down the patient’s statements. An efﬁcient way of structuring a discussion is to use a validated blank directive as a worksheet (36). Using a pencil rather than a pen to ﬁll in the patient’s wishes, and having the pencil in the patient’s or proxy’s hand, can help to emphasize explicitly that in this discussion the directive Table 1

is being used as a worksheet and not a ﬁnal document. Keep in mind that the simplest aspect to discuss is goals for care and that these can also be used to infer the patient’s treatment decisions quite accurately. So if discerning the patient’s goals is the only outcome of this discussion, it has been productive. Start the discussion in something like the following fashion: Let’s look at these standardized circumstances. We will go through one or two. You can then do the rest later. We will start by asking you what your goals for care would be in each. Imagine this ﬁrst case in the worksheet: Say you are in a coma with no awareness. Assume there is a chance that you might wake up, but it isn’t likely, and recovery may involve serious disability. Some people would want us to withdraw treatment and let them die, others would want us to attempt everything possible, and yet others would want us to try to restore quality of life but stop treatment if it was not working. What do you think your goals for medical care would be?

Goal options may include (a) treat everything, (b) treat but reevaluate often and be willing to withdraw what is not working, (c) noninvasive interventions only, and (d ) comfort care only (Table 1). Useful scenarios to have the patient and family go through include (a) a coma with a small chance of recovery, as above; (b) a

Discerning personal thresholds for goals using medical directives

Goal

Scenario Best prognosis

Less good prognosis

Comfort and quality of life are the primary goal

√

Treat readily curable conditions and also emphasize comfort and quality of life Treat everything but reconsider often Treat everything; longevity is the primary goal 192

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Worse prognosis

√ √

Worst prognosis √

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persistent vegetative state; and (c) a critically ill state with intermediate prognosis and waxing and waning consciousness. Approximately the ﬁrst three scenarios should be standard scenarios designed to cover the major situations commonly encountered when advance directives pertain. If the patient can manage more, two or three additional scenarios can be used that are tailored to the patient. One may be created by the physician, based on the patient’s illness and expectable circumstances (21). Another may be created by the patient if he or she wishes, based on what the patient considers to be a state worse than death that has not already been covered in previous scenarios (23). It can also be important to consider a scenario in which the patient is in his or her current health and acquires a new life-threatening illness involving incompetence. In each scenario, the patient’s goals for care are selected. If the patient is able to go further, ask him or her to consider intervention preferences. The commonly considered intervention preferences are (a) resuscitation, (b) mechanical respiration, (c) chemo- or radiation therapy, (d ) renal dialysis, (e) major surgery, and ( f ) artiﬁcial nutrition/hydration. In addition, preferences regarding (g) simple diagnostic tests and (h) antibiotics provide useful information. Thresholds. When scenario-based documents with intervention choices are used, it is possible to derive a patient’s personal thresholds for care—that is, the threshold circumstance at which the person tips from wanting primarily curative-intent treatment to Table 2

primarily comfort-oriented treatment. These thresholds can be particularly helpful when translating from scenarios in a prior statement to real situations. A person’s goals threshold can be seen by looking at a patient’s goals for care across the scenarios. A wide range of goals is possible; far from a dichotomous choice between intervention versus comfort, care can be a unique blend of both in each scenario. A patient’s goals thresholds can serve as an overall guide combining prognosis and intervention thresholds. Table 1 illustrates a hypothetical patient’s goals thresholds. Because research indicates that extrapolating from a person’s goals for care to speciﬁc interventions is reasonably accurate, it may be best to focus primarily on goals for care in advance care planning, especially if time is short (18). A threshold for interventions can be discerned in the same way. For instance, when the Medical Directive is used, scenarios are arrayed in a sequence that approximates a gradient of prognosis severity (see http://www.medicaldirective.org). For each scenario, potential interventions are arranged approximately by level of burdensomeness. Individuals tend to have thresholds regarding treatment burden and regarding prognosis that can be seen when all the options are ﬁlled in. Table 2 illustrates schematically how this can occur; the dark line represents one patient’s personal thresholds for prognosis and for treatment burden. This approach is supported by the ﬁnding that most patients are concerned about prognosis and treatment burden when they engage in advance care planning (48–50).

Discerning personal thresholds for intervention using medical directives

Intervention

Scenario Best prognosis

Less good prognosis

Worse prognosis

Worst prognosis

Least burdensome

consent

consent

decline

decline

Less burdensome

consent

consent

decline

decline

More burdensome

decline

decline

decline

decline

Most burdensome

decline

decline

decline

decline

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Once the clinician has identiﬁed the patient’s thresholds, the patient should be shown where the thresholds are so he or she can reﬂect on them, and then afﬁrm or adjust them.

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Special issues. Some sensitive issues should be speciﬁcally addressed, not least because they may not ﬁt the rest of the person’s pattern of preferences (27). One of these has to do with comfort measures, since pain control may blunt alertness. Most people can say whether pain control or being alert and unconfused to the last possible moment is more important to them. Patients can also be asked what they would consider the best to be hoped for from the dying process. Most can say, for instance, that they want enough time to settle relationships with people they are involved with, that they want to have done one particular thing, or that they want to be in a particular place. Elicitation of these images of a good dying should not be construed as promises to fulﬁll them; rather, the physician should tell the patient that the images may help in orchestrating care that ﬁts as well as possible with the patient’s wishes. There are some matters of principle that patients should be encouraged to articulate during or after their consideration of the scenarios. One is their position on withholding versus withdrawing life-sustaining intervention. It can be of critical importance to have a speciﬁc indication whether or not an existing intervention that might have become undesirable should be withdrawn. Another topic is the role of medications intended to control pain but with a known side effect of dulling consciousness and potentially hastening death. It can be helpful to clarify that pain medications can be used to treat pain without the intent of hastening death. Physician-assisted suicide, while legal only in Oregon, is receiving wide attention, and it may be helpful to clearly articulate patients’ preferences and positions on this matter. If a patient is requesting actions the physician cannot condone, the pa194

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tient should be advised of the fact at this early stage. Similarly, the proxy should raise objections at this stage if necessary. How long it should take. Even if the physician is involved in structuring most of the discussion, this step need take no more than 15 minutes once the physician has gained experience with it. The intent is not to reach ﬁnal resolution. Rather, it is to identify the key issues that patients should think about and to provide them with a good method for discussing and recording their preferences. Witnessing such expressions can give physicians a strong sense of the privilege of caring for patients, and the knowledge that emerges about how health issues ﬁt with the individual patient’s sense of meaning in life can be of great practical importance. Involvement of other members of the interdisciplinary team can increase efﬁciency and ensure that the team understands the care plan well. Reciprocally, patients can feel clearer, more understood, and conﬁdent. Patients should be encouraged to absorb the informational materials (e.g., worksheet, brochure, video) and talk over all the issues with their family, friends, pastor, lawyer, counselor, or whomever they consider relevant, until they have reached a settled view for themselves (9). The patient can then prepare a statement, preferably including both a validated advisory form and a statutory form that can be stapled together if they are not already combined.

Recording the Document Once the patient has completed the essential personal discussions outside the physician’s ofﬁce, he or she can bring the document in for ﬁnal review by the physician. The review of the document should take only a few moments. At this point, the physician can check for medical misconceptions and major changes that may need inquiry. Keep in mind that the most important things for the physician to

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know are the patient’s goals for care in relevant scenarios and their personal thresholds. It is useful to have a space where the physician can cosign the document (51). This signature should not be a requirement, but its presence fosters the physician’s involvement and carries an important implicit message of partnership between patient and physician.

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Updating the Document Documents should be reviewed at routine intervals. For a patient who has undergone a cure or for whom the prognosis is good, this review should occur every 1–5 years and after major life changes such as childbirth, marriage, divorce, signiﬁcant health status changes, bereavement, and important experiences of others’ ill health. Most reviews will be rapid. Speciﬁc treatment decisions are about as durable as other major life decisions, such as marriage (24, 52–54). When a patient shows a particularly high level of instability, this may indicate incompetence for advance decision making, and the physician should review the matter, perhaps advising simple designation of a proxy instead of instructional directives.

Applying the Document to Real Decisions Occasionally, the scenarios discussed do match the eventual circumstances, and application of the prior preferences requires little interpretation or extrapolation. More often, the ﬁt is not perfect and prior preferences can guide but not precisely direct the decisions. Interpretation and extrapolation are necessary, and it is important to know that certain patterns of decisions have high degrees of predictability:

Goal preferences predict treatment preferences well (17). Thresholds for when to shift from a primarily curative to a primarily palliative intent are helpful. If a patient has expressed speciﬁc treatment preferences but they do not cover

the decision at hand, it may well be possible to extrapolate from the preferences that are provided in the directive. Such predictions can be far more accurate than unguided judgments. Decline of less invasive interventions predicts decline of more invasive interventions, and acceptance of more invasive interventions predicts acceptance of less invasive interventions. Acceptance of intervention in poorprognosis scenarios predicts acceptance in better-prognosis scenarios, and decline in better-prognosis scenarios predicts decline in poorer prognoses.

The use of simple calculations can even provide probability estimates of speciﬁc decisions, which, when very high or very low, can be a comforting guide to proxy decision makers (25).

Pitfalls and Preventive Measures A common error is to omit advance care planning discussions altogether (55). A variation on this theme is to mistakenly suppose that a “DNR discussion” is sufﬁcient advance care planning. Discussing cardiopulmonary resuscitation and use of a Do Not Resuscitate (DNR) order without considering a range of scenarios and goals for care is often needlessly frightening for patients, focusing only on the moment of death, and often yields unstable decisions and poor guidance to providers (24, 25). If a clinician cares for a patient for whom a DNR order is written without comprehensive orders, such as are outlined in a Physician Orders for Life-Sustaining Treatment (POLST) form, this should prompt a review of advance care planning with that patient (11). As advance directives gradually come into more general use, common pitfalls are emerging (56). Noninclusion of the proxy or of family members or close friends who have divergent opinions can lead to fractured decision making. If the physician is not included, he or she may be inadequately sensitized to the patient’s thinking. A recent major study showed www.annualreviews.org • Advance Directives

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that when a nurse is the main communicator in advance planning, the physician’s understanding of the patient’s wishes is not improved (57). Poor informed consent and documentation constitute another pair of pitfalls. For example, a patient may express a strong desire to avoid perpetual dependence on a respirator, but the wording of the advance directive may indicate that a respirator should never be used. In the event of reversible pneumonia, the patient may well want temporary use of a respirator. A properly validated directive should require this kind of distinction, and physicians should ensure this minimum level of patients’ understanding. Physicians should check the document for improbable statements and ask patients to brieﬂy state their wishes in free words, checking for correspondence with medical possibilities. Perhaps the most common pitfall of all is activation of advance directives before patients have reached a wishless state, and some-

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times even before they have reached a state of incompetence for the decision(s) at hand. A widespread tendency to avoid direct communication with patients appears to exacerbate this problem. Advance directives, as distinct from their predecessor, the DNR order, have no authority when they are ﬁrst written and must not be activated until the patient is decisionally incapacitated and wishless. A ﬁnal, crucial pitfall is poor application of wishes to eventual decisions. Some studies have indicated that physicians rather commonly override advance directives (58). Whether this occurs because patients are decisionally incapacitated but not wishless, or because proxy opinions persuade physicians more than the patient’s advance directives, or for other reasons, is not well studied. Nonetheless, if a patient has a validated advance directive that uses scenarios and speciﬁc intervention choices, extrapolation to unstated decisions can be accomplished with considerable accuracy in many cases.

DISCLOSURE STATEMENT The author is not aware of any biases that might be perceived as affecting the objectivity of this review.

LITERATURE CITED 1. President’s Commission for the Study of Ethical Problems in Medicine and Biomedical Research. 1983. Deciding to forego life-sustaining treatment: a report on the ethical, medical, and legal issues in treatment decisions. Washington, DC: Gov. Print. Off. 2. Lynn J. 1991. Why I don’t have a living will. Law Med. Health Care 19:101–4 3. Seghal A, Galbraith A, Chesney M, et al. 1992. How strictly do dialysis patients want their advance directives followed? JAMA 267:59–63 4. Emanuel EJ, Emanuel LL. 1992. Proxy decision making. JAMA 267:2221–26 5. Hines SC, Glover JJ, Babrow AS, et al. 2001. Improving advance care planning by accommodating family preferences. J. Palliative Med. 4(4):481–89 6. Rabkin MT, Gillerman G, Rice NR. 1976. Orders not to resuscitate. N. Engl. J. Med. 295:364–66 7. Emanuel LL. 1989. Does the do-not-resuscitate order need life sustaining intervention? Time for advance care directives. Am. J. Med. 86:87–90 8. Ratner E, Norlander L, McSteen K. 2001. Death at home following a targeted advance-care planning process at home: the kitchen table discussion. J. Am. Geriatr. Soc. 49(6):778–81 9. Voogt E, van der Heide A, Rietjens JAC, et al. 2005. Attitudes of patients with incurable cancer toward medical treatment in the last phase of life. J. Clin. Oncol. 23:2012–19 196

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10. Bedell SE, Delbanco TL. 1984. Choices about cardiopulmonary resuscitation in the hospital: When do physicians talk with patients? N. Engl. J. Med. 310:1089–93 11. Cantor MD. 2000. Improving advance care planning: lessons from POLST. Physician Orders for Life-Sustaining Treatment. J. Am. Geriatr. Soc. 48(10):1343 12. Bok S. 1976. Personal directions for care at the end of life. N. Engl. J. Med. 295:367–69 13. Relman AS. 1979. Michigan’s sensible living will. N. Engl. J. Med. 300:1270–72 14. Doukas DJ, McCullough LB. 1991. The values history: the evaluation of the patient’s values and advance directives. J. Fam. Pract. 32:145–53 15. Emanuel LL, Emanuel EJ. 1989. The medical directive: a new comprehensive advance care document. JAMA 261:3288–93 16. Schneiderman LJ, Pearlman RA, Kaplan RM, et al. 1992. Relationship of general advance directive instructions to speciﬁc life-sustaining treatment preferences in patients with serious illness. Arch. Intern. Med. 152:2114–22 17. Fischer G, Alpert H, Stoeckle JD, et al. 1994. Relationship between goals and treatment preferences in advance directives. J. Gen. Intern. Med. 9:93 18. Doukas DJ, Gorenﬂo DW. 1993. Analyzing the values history: an evaluation of patient medical values and advance directives. J. Clin. Ethics 4:41–45 19. Quest T, Emanuel L, eds. 2007. Module: advance planning. In The Education in Palliative and End-of-life Care—Emergency Medicine (EPEC-EM) Curriculum. http://www.epec.net 20. Nunally JC. 1978. Psychometric Theory, pp. 265–70. New York: McGraw-Hill. 2nd ed. 21. Singer PA. 1994. Disease-speciﬁc advance directives. Lancet 344:594–96 22. Berry SR, Singer PA. 1998. The cancer-speciﬁc advance directive. Cancer 82(8):1570–77 23. Patrick DO, Starks HE, Cain KC, et al. 1994. Measuring preferences for health states worse than death. Med. Decision Making 14:9–18 24. Emanuel LL, Emanuel EJ, Stoeckle JD, et al. 1994. Advance directives: stability of patients’ treatment choices. Arch. Intern. Med. 154:209–17 25. Emanuel LL, Barry MJ, Emanuel EJ, et al. 1994. Advance directive: Can patients’ stated treatment choices be used to infer unstated choices? Med. Care 32:95–105 26. Alpert H, Hoijtink R, Fischer G, et al. 1996. Psychometric analysis of an advance directive. Med. Care 34:1055–63 27. Schwartz CE, Merriman MP, Reed GW, et al. 2004. Measuring patient treatment preferences in end-of-life care research: applications for advance care planning interventions and response shift research. J. Palliative Med. 7(2):233–45 28. 1994. Right to die legislation. New York: Choice in Dying (brochure) 29. Gostin LO. 2005. Ethics, the Constitution, and the dying process: the case of Theresa Marie Schiavo. JAMA 293:2403–7 30. Weir RF, Gostin L. 1990. Decisions to abate life-sustaining treatment for nonautonomous patients: ethical standards and legal liability for physicians after Cruzan. JAMA 264:1846– 53 31. Emanuel EJ. 1988. A review of the ethical and legal aspects of terminating medical care. Am. J. Med. 84:291–301 32. Perkins HS, Geppert CM, Gonzales A, et al. 2002. Cross-cultural similarities and differences in attitudes about advance care planning. J. Gen. Intern. Med. 17(1):48–57 33. Blackhall LJ, Murphy ST, Frank G, et al. 1995. Ethnicity and attitudes toward patient autonomy. JAMA 274:820–25 34. McKinley ED, Garrett JM, Evans AT, et al. 1996. Differences in end-of-life decision making among black and white ambulatory cancer patients. J. Gen. Intern. Med. 11:651–56 35. Carrese JA, Rhodes LA. 1995. Western bioethics on the Navajo reservation: beneﬁt or harm? JAMA 274:826–29 www.annualreviews.org • Advance Directives

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36. Emanuel LL. 1994. What makes a directive valid? Hastings Cent. Rep. 24(Suppl):S27–29 37. Deleted in proof 38. Emanuel LL, Emanuel EJ. 1993. When patients have no advance directives: institutional default guidelines deﬁned by communities of patients. Hastings Cent. Rep. 23:6–14 39. Emanuel LL, Danis M, Pearlman RA, et al. 1995. Advance care planning as a process. Am. Geriatr. Soc. 43:440–46 40. Knight S, Emanuel L. 2007. Processes of adjustment to end-of-life losses: a reintegration model. J. Palliative Med. 10:1190–98 41. Wenger NS, Kanouse DE, Collins RL, et al. 2001. End-of-life discussions and preferences among persons with HIV. JAMA 285(22):2880–87 42. Grimaldo DA, Wiener-Kronish JP, Jurson T, et al. 2001. A randomized, controlled trial of advanced care planning discussion during preoperative evaluations. Anasthesiology 95(1):43– 50 43. Lorenz KA, Lynn J. 2004. Oregon’s lessons for improving advance care planning. J. Am. Geriatr. Soc. 52(4):233–37 44. Henderson ML. 2004. Gerontological advance practice nurses: as end-of-life care facilitators. Geriatr. Nurs. 25(4):247–54 45. Morrison RS, Chichin E, Carter J, et al. 2005. The effect of a social work intervention to enhance advance care planning documentation in the nursing home. J. Am. Geriatr. Soc. 53(2):290–94 46. Briggs LA, Kirchhoff KT, Hammes BJ, et al. 2004. Patient-centered advance care planning in special patient populations: a pilot study. J. Prof. Nurs. 20(1):47–58 47. Pearlman RA, Starkes H, Cain KC, et al. 2005. Improvements in advance care planning in the Veterans Affairs system: results of a multifaceted intervention. Arch. Intern. Med. 165(6):667–74 48. Weeks JC, Cook EF, O’Day SJ, et al. 1998. Relationship between cancer patients’ predictions of prognosis and their treatment preferences. JAMA 279:1709–14 49. Fried TR, Bradley EH, Towle VR, et al. 2002. Understanding the treatment preferences of seriously ill patients. N. Engl. J. Med. 346:1061–66 50. Fried TR, Bradley EH. 2003. What matters to seriously ill older persons making end-of-life treatment decisions? A qualitative study. J. Palliative Med. 6(2):237–44 51. Orentlicher D. 1990. Advance medical directives. JAMA 263:2365–67 52. Silverstein MD, Stocking CB, Antel JP, et al. 1991. Amyotrophic lateral sclerosis and lifesustaining therapy: patient’s desires for information, participation in decision making, and life-sustaining therapy. Mayo Clin. Proc. 66:906–13 53. Everhart MA, Pearlman RA. 1990. Stability of patient preferences regarding life-sustaining treatments. Chest 97:159–64 54. Danis, Patrick DL, Garrett J, et al. 1994. Stability of choices about life-sustaining treatments. Ann. Intern. Med. 120:567–73 55. Lynn J, Goldstein NE. 2003. Advance care planning for fatal chronic illness: avoiding commonplace errors and unwarranted suffering. Ann. Intern. Med. 138(10):812–18 56. Emanuel LL. 1994. Appropriate and inappropriate use of advance directives. Clin. Ethics 5(4):357–59 57. The SUPPORT investigators. 1995. A controlled trial to improve decision-making for seriously ill hospitalized patients: the struggle to understand prognoses and preferences for outcomes and risks of treatments (SUPPORT). JAMA 274(20):1591–98 58. Danis M, Southerland LI, Garrett JM, et al. 1991. A prospective study of advance directives for life-sustaining care. N. Engl. J. Med. 324:882–88

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:187-198. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Genetic Determinants of Aggressive Breast Cancer Annu. Rev. Med. 2008.59:199-212. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Alejandra C. Ventura1,2 and Soﬁa D. Merajver1,2 1

Department of Internal Medicine, Division of Hematology and Oncology and Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan 48109-0948; email: [email protected]

2

Annu. Rev. Med. 2008. 59:199–212

Key Words

First published online as a Review in Advance on October 3, 2007

biomarker, genetic marker, inﬂammatory breast cancer, drug target

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.060106.184830 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0199$20.00

Abstract The development and spread of breast and other human cancers are caused by the overexpression, mutation, and/or deletion of speciﬁc genes that drive these events. Finding genetic and molecular differences between cancerous and healthy cells can reveal the genetic determinants of cancer. This knowledge results in a better understanding of the carcinogenic process and improved predictive power, with implications for identifying new drug targets, designing novel therapies, and improving preclinical and clinical studies. We review the concepts of biomarker, genetic marker, and genetic determinant in cancer, with particular focus on the most aggressive and lethal form of breast cancer, termed inﬂammatory breast cancer (IBC). Using IBC as an example, we describe in detail the approaches to identify the genes that are responsible for—and not merely associated with—this disease.

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INTRODUCTION

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BRCA gene test: blood test to check for speciﬁc mutations in the tumor suppressor genes BRCA1 or BRCA2 (“breast cancer genes”) HER-2/neu test (human epidermal growth factor receptor test): determines whether Her-2/neu gene is overexpressed in a breast cancer tumor to help guide treatment and determine prognosis CAT: computed axial tomography CA125: cancer antigen 125 Gene expression profiling: research method that measures messenger RNA levels of many different genes in various cell types or under certain conditions

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Cancer is a genetic disease (1). Although nongenetic factors play a role at many stages of tumorigenesis, the development and spread of breast and other human cancers are ultimately caused by the overexpression, mutation, and/or deletion of speciﬁc genes or groups of genes. Based on this idea, cancer research has focused on the identiﬁcation of genetic and molecular differences between cancerous and healthy cells as a general method to ﬁnd credentialed biomarkers and ﬁnally to unveil the genetic determinants that drive each cancer type.

Genetic Markers and Genetic Determinants in Cancer A biomarker is an indicator of a biological state. Knowing how that biomarker or biological feature should behave in the spectrum of normal (homeostatic) conditions, one can detect deviations from homeostasis and thus an abnormal state, such as disease. A biomarker can be a biological state that results from the integration of many genetic, psychological, and environmental variables, such as performance, or it can be a single variable, such as level of expression of one gene. Cancer biomarkers are diverse (see Reference 2 for a list), ranging from patient performance status to gene or protein expression proﬁles. Some well-known biomarkers in breast cancer are, for example, the results of a mammogram, BRCA gene test, or HER-2/neu test. The status of a biomarker can signal an abnormal state, analogous to the way the presence of an antibody may signal an infection. Biomarkers are useful tools for cancer detection. They can predict who is likely to develop cancer (BRCA1 mutation status) and/or detect the disease at an early stage (mammographic image). They can indicate a particular stage in the disease (CAT scan image, CA125), guide treatment decisions (circulating tumor cells, pathological response), and, importantly, aid Ventura

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in the identiﬁcation of new targets for drug development (3). Among all the possible forms of biomarkers, genetic biomarkers have received particular attention in cancer research because they guide the discovery of genetic determinants of cancer. A genetic biomarker is credentialed as a genetic determinant if it is not only correlated with a particular type of cancer but is driving the phenotype, causing the disease. This last statement is strongly based in the previous overarching observation that cancer is a genetic disease driven by the overexpression, mutation, and/or deletion of speciﬁc genes. We widely accept in our everyday life the idea that in order to ﬁx something, we ﬁrst need to know what is wrong. By the same reasoning, it is important to ﬁnd not just genetic markers but genetic determinants of each type of cancer, in order to understand where the failure is. Every determinant is also a biomarker, but not every (genetic) marker is a determinant. Several technologies allow the identiﬁcation of aberrant gene expression. Gene expression proﬁling is a powerful parallel approach for viewing the expression of many genes simultaneously in different types of malignant or normal cells. Using computational techniques, differentially expressed genes or informative patterns of expressed genes are mined from large data sets. Gene expression proﬁling has radically transformed many aspects of cancer research (4). No one technique can uncover all genetic markers. For example, gene expression proﬁling cannot identify post-transcriptional changes in protein expression or activity (5). As a particularly well-known example, we can highlight the members of the Ras family, whose mutations cause inappropriate activation of Ras proteins. Ras mutations are very common in human tumors, but because they do not change the level of messenger expression, gene expression proﬁling cannot detect them. However, although measurements of protein levels (and activity) are the ultimate

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Normal State particular biological features

over-expression mutation

specific genes

deletion

Abnormal State (Cancer)

Biomarkers: genetic level

Genetic Marker

…

+

only correlated with the disease (passenger)

alterations that control the biomarker

in vivo/in vitro molecular biology, phenotypic testing, spectroscopic tools

physiological level

Useful for: susceptibility assessment early detection diagnosis evaluating stage treatment decisions

causing the disease (driver)

Genetic Determinant

drug target

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(predict response to specific therapy)

modified biological features

prognosis (survival)

Figure 1 Scheme summarizing the concepts of biomarker, genetic marker, and genetic determinant in cancer. Different levels and types of inquiry are required to credential markers associated with carcinogenesis and cancer progression.

goal for many uses, large numbers of protein levels still cannot be assayed as rapidly or as accurately as RNA. Aberrantly expressed genes pinpointed by gene expression proﬁling can be divided into two categories: genes that are simply correlated with cancer but not functionally related to it, and genes that actually cause the development of cancer—the genetic determinants. There is considerable interest in ﬁnding genetic determinants in cancer, not only to provide a better description of the disease in molecular terms (6) but also to develop targeted therapy, i.e., a new generation of cancer drugs designed to interfere with a speciﬁc molecular target (7). Figure 1 summarizes the connections between genetic markers and determinants in cancer.

Cancer as a Systemic Disease Research into cancer genetics has, so far, primarily taken the form of intensive investigation of individual genes and a small number of interactions between genes. However, like most biological processes, cancer cannot be understood by studying individual com-

ponents only. It is the interactions between these components that deﬁne the properties of the system, suggesting that cancer can only be fully understood, and hence combated, at the systems level. This insight raises two key questions (8). Cancer being such a complex system, how can we predict or understand the effect of a speciﬁc mutation in a particular gene (or overexpression of a gene) on the functionality of the entire system? And even more important, once we have identiﬁed such a gene, how can we predict the effect of a targeted therapy against it? A third question is how to reconcile both approaches to cancer: the search for genetic determinants versus a systems-level understanding. We believe that these perspectives are not in conﬂict, as cancer determinants are components of a system. Integrating parts and processes can enable us to better understand underlying mechanisms and improve our predictive power, and can lead to new therapeutic approaches. We propose this can only be done by combining quantitative experimentation and mathematical tools (see Reference 8 for a review).

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IBC: inﬂammatory breast cancer LABC: locally advanced breast cancer

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Following this line of reasoning, we analyze in detail the case of the most aggressive form of breast cancer, known as inﬂammatory breast cancer (IBC). We ﬁrst present its clinical and pathological features and then describe its molecular biology. We compare the genetic and molecular markers of breast cancer and IBC and subsequently focus on genetic determinants. Finally, we describe therapeutic targets in IBC in connection with the genetic determinants.

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INFLAMMATORY BREAST CANCER: INTRODUCTION IBC is the most aggressive, metastatic, and deadly form of locally advanced breast cancer (LABC). The proportion of IBC is 1%–3% of all breast cancers in the United States (9). It carries a signiﬁcantly worse prognosis than noninﬂammatory breast cancers, with threeyear survival of 32% compared to 60% for other breast cancers (10). The lower survival rate is presumably due to the highly metastatic nature of IBC. Three biological features make IBC unique (11). First, it is rapidly progressive. Second, it is highly angiogenic and angioinvasive. Third, its aggressive behavior and angiogenicity are intrinsic characteristics of the tumor, present from its inception. Clinically, these properties confer on IBC an extremely high metastatic potential: Usually less than 6 months elapse from ﬁrst symptoms to diagnosis of a stage IIIB or IV tumor. At diagnosis, nearly all IBC patients have regional lymph node involvement, and an average of 30% already have gross distant metastasis. Major improvement in survival has resulted from combined-modality treatment, and at present, long-term survival can be achieved in ∼50% of patients treated with neoadjuvant chemotherapy, local/regional treatment, and consolidation chemotherapy (12, 12a, 12b). Despite these advances, IBC is still a huge burden to patients and a challenge to oncologists who treat breast cancer. Therefore, ef202

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forts are focused on understanding its molecular basis.

CLINICAL AND PATHOLOGICAL FEATURES OF IBC IBC was ﬁrst described by Lee & Tannenbaum in 1924 (13). The designation “inﬂammatory” stems from the clinical appearance, which mimics an acute inﬂammation of the breast. The age distribution of IBC is not signiﬁcantly different from that of ordinary inﬁltrating carcinoma, averaging around 55 years, with most patients being postmenopausal (9). In the earliest phase a mass may not be palpable. The typical patient presents with pain and a tender, ﬁrm, and enlarged breast. The skin over the breast is reddened, warm, and thickened, and is termed “peau d’orange” (skin of an orange). These skin changes are due to invasion of the dermal lymphatic vessels by tumor emboli. The lymphatic tumor emboli of malignant tumor cells in primary IBC (de novo IBC) prevent proper drainage of the auxiliary lymph ﬂuid and lead to erythema and edema of the breast. Thus, the rapid increase in breast size due to the obstructed lymph drainage helps in the differentiation of primary IBC from other breast cancer types and secondary IBC (such as breast cancer recurrences that may have some inﬂammatory features) (14). The term inﬂammatory thus refers only to the clinical features and not to the presence of an inﬂammatory inﬁltrate. Primary IBC is often ductal type and has high histologic grade. Pathologically, as mentioned, IBC is highly angiogenic and angioinvasive. Although it is not a speciﬁc histologic subtype of mammary carcinoma, the presence of numerous ectatic and dilated dermal lymphatics clogged by malignant cells constitutes the histologic hallmark that accompanies the symptoms (11).

MOLECULAR BIOLOGY OF IBC Understanding the genetic alterations in IBC is crucial for distinguishing IBC tumors from

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non-IBC tumors and for designing IBCspeciﬁc treatments. Until recently, there were no known biological markers to deﬁne the IBC phenotype. However, rapid advances in molecular biology and cancer models have yielded new information on the molecular genetics of this disease (15, 16). Two IBC cell lines, SUM149 and SUM190, were derived from primary human IBC tumors (17) and enabled in vitro models and useful xenograft in immunocompromised nude mice. These developments were critical in facilitating the genotypic and phenotypic characterization of IBC.

Breast Cancer/IBC Molecular and Genetic Markers The main molecular candidates to be genetic determinants of major breast cancer phenotypes include hormone receptors, growth factor receptor, the p53 tumor suppressor gene, E-cadherin, and various angiogenic factors. Several studies have analyzed these candidates in IBC. Hormone receptors. In general, the absence of estrogen receptor and progesterone receptor expression correlates with shorter disease-free survival and overall poor clinical outcome of breast cancer patients, including those with IBC (18). However, the expression status of these receptors is not sufﬁciently robust to predict outcome in IBC without regard for chemotherapy or in a multivariate analysis. Growth factor receptor (Her-2/neu). Her-2/neu, encoded by ERBB2, is a member of the epidermal growth factor receptor family. The ERBB2 gene is ampliﬁed and overexpressed in ∼15% of human breast cancers. Patients with breast tumors exhibiting the ERBB2 ampliﬁcation have a poorer clinical outcome and shorter survival (19). Although excessive Her-2/neu is often present in the IBC signature, it is nonspeciﬁc, meaning that this marker alone cannot lead to accurate IBC diagnosis or prognosis.

The p53 tumor suppressor gene. The p53 gene is mutated or absent in ∼50% of breast cancers (20). The p53 gene product can act as a negative regulator of cell proliferation through induction of cell cycle arrest or through induction of the apoptotic machinery. The primary well-known function of wild-type p53 is to maintain the integrity of the genome and to prevent the accumulation of harmful mutations. Mutant p53 promotes genome instability and enables the accumulation of gene mutations that are thought to drive breast cancer progression. Mutations in p53 are also associated with a reduced response to chemotherapy (20). A study of p53 in IBC suggests that missense mutation and nuclear exclusion are the two major mechanisms of p53 inactivation in this form of breast cancer (21). Further studies (22, 23) have suggested p53 status can be a prognostic marker and a relatively useful adjunct to stage and receptor status to evaluate different treatment regimens in IBC.

Tumor suppressor gene: gene that normally restrains cell growth; when it is missing or inactivated by mutation, cells can grow uncontrolled

E-cadherin. E-cadherin is a transmembrane glycoprotein that mediates epithelial cell-tocell adhesion (24). It is postulated to be a tumor suppressor protein, as loss of Ecadherin function or expression has been implicated in cancer progression and metastasis. E-cadherin downregulation decreases the strength of cellular adhesion within a tissue, resulting in an increase in cellular motility. In some cases, this in turn may allow cancer cells to cross the basement membrane and invade surrounding tissues. In breast cancer, loss of E-cadherin contributes to increased proliferation, invasion, and metastasis (25). E-cadherin-negative status has been correlated with dedifferentiation, histologic grade (26), metastasis (27), and decreased patient survival. However, in IBC it was shown (25) that tumors were predominately E-cadherin-positive even in the metastatic sites. A 10- to 20-fold overexpression of E-cadherin was observed in an IBC animal model (28). Overexpression of E-cadherin in IBC appears to maintain the

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HME cells: human mammary epithelial cells RhoC GTPase: low-molecularweight guanine-nucleotidebinding protein involved in all aspects of cellular motility and invasion, including polarity, cytoskeletal organization, and signal transduction from the environment Rho-GEF: Rho guanine-nucleotide exchange factor Rho-GAP: RhoGTPase-activating protein Rho-GDI: RhoGDP-dissociation inhibitor

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loose integrity of the tumor emboli as they invade and grow in the dermal lymphatics. This unique feature was identiﬁed as part of the “inﬂammatory signature” of IBC (29). Angiogenic factors. As noted above, the inﬂammation of the breast in IBC is caused by blockage of the dermal lymphatics by tumor inﬁltrate, and not by inﬁltration of inﬂammatory cells. IBC tumors produce negligible levels of most inﬂammatory cytokines, including IFN-γ, IL-1, and IL-12 (11). Nonetheless, IBC tumors are highly angiogenic and angioinvasive. Indeed, IBC tumor cell lines and tumor specimens have been reported to secrete high levels of angiogenic factors, including basic ﬁbroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), IL-6, and IL-8 (11). The VEGF receptor-3 is found on the epithelium of lymphatic vessels and is known to bind the VEGF family members VEGF-C and VEGF-D (30). A variety of breast cancer cell lines were screened for expression levels of the VEGF family members (VEGF-A, -B, -C, and -D) (31). VEGF-A and -B, which are important in regulating tumor angiogenesis, were expressed by all types of breast cancers analyzed. However, VEGF-D was detected only in an IBC cell line and in a tumor cell line that was developed from an inﬂammatory skin metastasis. These results suggest the angiogenic factors and/or their receptors can be approached as novel therapeutic targets to inhibit lymphangiogenesis and angiolymphatic invasion of IBC.

Genetic Markers and Genetic Determinants for IBC The previous section listed several general breast cancer molecular markers, most of which are not speciﬁc for IBC. In search of IBC-speciﬁc genetic markers and genetic determinants, Merajver and coworkers utilized differential display technology to identify genes differentially expressed between the IBC cell line SUM149 (17) and human mam204

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mary epithelial (HME) cells (15). This study was the ﬁrst of its kind for IBC. Seventeen transcripts were identiﬁed as being differentially expressed between SUM149 and HME. Eight were expressed solely by the normal cell lines and the other nine were expressed solely by the tumor cell line. Expression patterns of all 17 transcripts were further conﬁrmed in 20 archival IBC and 30 non-IBC LABC tissue samples by in situ hybridization. Among those genes, ARHC (RhoC GTPase) was predominately overexpressed in the IBC tumors (91% positive in IBC versus 38% positive in non-IBC), while another gene, WISP3/LIBC, was lost in most of the IBC specimens (80% negative in IBC versus 21% negative in non-IBC). RhoC GTPase. The Rho (Ras homology) gene was ﬁrst isolated from Aplysia and has been highly conserved throughout evolution (32). RhoC belongs to the Ras superfamily of low-molecular-weight guaninenucleotide-binding proteins, being highly homologous to two other members of the family, RhoA (92% similarity in protein sequence) and RhoB (33). The Rho GTPases transduce extracellular signals of growth factor receptors and integrins to the cytoplasm (34) through a tightly regulated and highly complex signaling pathway. Like other small GTPases, RhoC cycles between a GDP-bound inactive and a GTP-bound active state, thus acting as a molecular on/off switch (35) involved in all aspects of cellular motility and invasion, including polarity, cytoskeletal organization, and signal transduction from the environment (36). The GDP-GTP cycle is controlled by the opposing activities of Rho guaninenucleotide exchange factors (Rho-GEFs), which catalyze the exchange of GDP for GTP, and Rho-GTPase-activating proteins (RhoGAPs), which increase the rate of GTP hydrolysis into GDP. Another layer of regulation is provided by Rho-GDP–dissociation inhibitors (Rho-GDIs), which sequester Rho away from the GDP-GTP cycle (37).

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In the cytosol of resting cells, Rho is present in the GDP-bound form complexed with Rho-GDI. When cells are stimulated by certain extracellular signals, Rho-GDI is dissociated and speciﬁc GEFs are activated. In a GTP-bound, activated form, the Rho protein acquires a C-terminal lipid modiﬁcation by farnesylation and geranylgeranylation, localizes to the cell membrane, and interacts with diverse effector proteins, including Rho kinase (ROCK), citron kinase, protein kinase N, and Diaphanous (Dia) (34). Because the Rho GTPases are closely related to Ras oncogenes, they have long been suspected of involvement in tumorigenesis. In the case of Ras, a mutation in the gene causes the abnormality. However, it is remarkable that breast cancers almost never harbor Ras mutations. Although no known mutation was found within Rho genes, aberrant Rho expression is found in various types of cancers. RhoA overexpression has been observed in breast cancer, testicular germ cell tumors, and colon cancer (38). Because of the high homology between RhoC and RhoA, RhoC had always been regarded as a duplicated homolog of RhoA (39) and largely ignored. However, crucial studies in human tissues have unequivocally shown that overexpression of RhoC is strongly associated with very aggressive breast cancer with poor clinical outcomes (40– 42). Upregulation of RhoC is independently identiﬁed in many types of malignant cancers, including IBC (15), bladder cancer (43), ovarian cancer (44), pancreatic cancer (45), melanoma (46), and hepatocellular carcinoma (47). In melanoma, overexpression of RhoC enhances the ability of melanoma cells to exit the blood and colonize the lungs. The essential role of RhoC in tumor metastasis has been conﬁrmed in vivo thanks to a RhoC-deﬁcient mouse model. Hakem and colleagues (48) showed that RhoC is dispensable for embryonic and postnatal development. When the RhoC− /− mice were crossed, Hakem et al. demonstrated that loss of RhoC does not affect tumor development but does decrease tu-

mor cell motility and metastatic cell survival, thereby drastically inhibiting metastasis. Given that RhoC GTPase is a speciﬁc genetic marker for IBC, the next question is whether it is a genetic determinant. Only in vitro and in vivo approaches can answer this question (Figure 1). In what follows, we describe experiments that proved RhoC is driving the phenotype in IBC. To study the contribution of RhoC GTPase to an IBC-like phenotype (16), stable transfectants of HME cells overexpressing RhoC were generated. The HME-RhoC transfectants formed large colonies under anchorage-independent growth conditions, were more motile, and were invasive. In conjunction with an increase in motility, overexpression of RhoC enhanced the formation of actin stress ﬁber and focal adhesion contact. Furthermore, orthotopic injection into immunocompromised mice led to tumor formation. These data indicate that RhoC GTPase can lead to a highly invasive phenotype similar to that seen in IBC. Seeking further potential downstream effects of RhoC overexpression, van Golen et al. (49) showed that stable transfectants of human breast cells overexpressing RhoC secreted signiﬁcantly higher amounts of angiogenic factors (such as VEGF, bFGF, IL-6, and IL-8) than the control lines. Production of the angiogenic factors was abolished when the RhoC-overexpressing cells were treated with the pan-Rho-speciﬁc inhibitor C3 exo-transferase. Comparative microarray analysis revealed that stable RhoC overexpression induced in spontaneously immortalized mammary epithelial cells (MCF10A) upregulated genes involved in cell proliferation, invasion/ adhesion, and angiogenesis (50). Messenger RNA levels of cyclin D1, ﬁbronectin, VEGF-C, caveolin-2, and CXCL1 were increased, while the level of IGFBP-2 (insulinlike growth factor binding protein 2) was decreased. The upregulated gene products are involved in cell cycle (cyclin D1), cellmatrix adhesion (ﬁbronectin, caveolin-2), and

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Oncogene: mutant form of a gene that plays a role in normal cell proliferation. The oncogene is associated with the development of cancer through uncontrolled growth IGFBP: insulin-like growth factor binding protein

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angiogenesis (CXCL1 and VEGF-C, mentioned above), where they greatly enhance the invasive and motile phenotype of RhoCoverexpressing breast cells. RhoC-mediated downregulation of IGFBP-2, a negative regulator of the potent mitogen insulin-like growth factor (IGF), supports the notion that RhoC overexpression enhances an IBC-like, invasive phenotype in normal breast cells by modulation of IGF signaling. Taken together, the evidence shown in References 16, 49, and 50 strongly suggests that the overexpression of RhoC GTPase is essential in driving the highly invasive IBC phenotype. WISP3. The paper by van Golen et al. (15) that ﬁrst identiﬁed speciﬁc signature markers of IBC also identiﬁed WISP3 (Wnt-inducible signaling pathway protein 3) as a major determinant. WISP3 (LIBC, CCN6, IGFBPrP3) has been identiﬁed as a member of the CCN family of proteins, a group of extremely cysteine-rich secreted proteins with important biological functions in normal physiology as well as in carcinogenesis (51). CCN proteins contain an N-terminal secretory signal followed by four distinct motifs with homology to IGFBP, von Willebrand factor type C, and thrombospondin 1, and a C-terminal region with heparin-binding motifs and sequence similarities to the C termini of von Willebrand factor and mucins (52–54). WISP3 was also independently identiﬁed from a screen of differentially expressed transcripts in Wnt-1-transformed colon cells (53). In fact, WISP3 was initially thought to be an IGFBP-related protein (IGFBP-rP) on the basis of its protein sequence (15). IGFBPrPs show relatively low afﬁnity for IGF and are involved in cell growth (55). In addition, IGFBP-rP1 (T1A12/mac25) was proposed to be a potential tumor suppressor protein for prostate cancer (56). Downregulation of IGFBP-rP1 expression is associated with progression of breast cancer (57) and prostate cancer (56). Transfection of IGFBP-rP1 in prostate cancer cells signiﬁcantly reduced the metastatic phenotype (56).

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As mentioned, WISP3 was found to be speciﬁcally lost in human IBC tumors compared to stage-matched non-IBC tumors (15). Later on, the full-length WISP3 cDNA was sequenced and cloned into an expression vector (58). The resulting construct was introduced into the SUM149 cell line (which lacks WISP3 expression). In soft agar, stable WISP3 transfectants formed signiﬁcantly fewer colonies than controls. Stable WISP3 transfectants lost their ability to invade and had reduced angiogenic potential. WISP3 transfection was effective in suppressing in vivo tumor growth in nude mice. Mice with WISP3-expressing tumors had signiﬁcantly longer survival than those with vector-control transfectant tumors. This work demonstrated that WISP3 acts as a tumor suppressor gene in the breast; loss of WISP3 expression contributes to the phenotype of IBC by regulating tumor cell growth, invasion, and angiogenesis in vitro and in vivo. Because RhoC and WISP3 appear to be concordantly altered in IBC, studies were performed to understand whether their expression levels are mechanistically linked, and furthermore, whether their concordant expression changes act synergistically (59). In this work, by using an antisense approach, WISP3 expression was blocked in nontransformed HME cells. The WISP3-knockdown cells had signiﬁcantly higher expression of RhoC and increased cellular proliferation, anchorage-independent growth, and secretion of VEGF. Conversely, restoration of WISP3 expression in the highly malignant IBC cell line SUM149 decreased expression of RhoC. This study demonstrated that the genes encoding WISP3 and RhoC are altered mutually in HME cells and in SUM149; it provided further evidence that these two genes act in concert to give rise to the highly aggressive IBC phenotype, leading to the hypothesis that they cooperate in the development of IBC. The function of WISP3 was investigated in relationship to its structure (58). The ﬁndings indicate that WISP3 is secreted into the

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conditioned media and into the lumens of normal breast ducts. Once secreted, WISP3 was able to decrease, directly or through induction of other molecule(s), the IGF-1-induced activation of the type I IGF receptor (IGFIR) and two of its main downstream signaling molecules, IRS1 and ERK-1/2, in SUM149 IBC cells. Furthermore, WISP3 containing conditioned media slowed the growth of SUM149 cells. This work clariﬁed the mechanism of WISP3 function by demonstrating that it is secreted and that, once in the extracellular media, it induces a series of molecular events that leads to modulation of IGFIR signaling pathways and cellular growth in IBC. Finally, it was also shown that inhibition of WISP3 in HME cells results in the loss of a growth regulatory function that protects HME cells from the tumorigenic effects of growth factors, particularly IGF-1 (61). WISP3-deﬁcient HME cells showed increased motility and invasiveness and developed features of epithelial-mesenchymal transition. Coordinate changes in RhoC GTPase and WISP3 expression in IBC are fundamental to the pathogenesis of this disease and provide promising new prognosis tools and therapeutic targets.

NOVEL THERAPEUTIC TARGETS Novel therapeutics that speciﬁcally target IBC but are not connected with the genetic determinants mentioned above were reviewed in Reference 62. Here we focus on therapeutics targeting Rho/RhoC GTPase. It is the coordinate changes in RhoC and WISP3 expression that make IBC unique. We have described extensive experimental evidence that the genes encoding RhoC and WISP3 are genetic determinants for IBC. However, since WISP3 is lost in human IBC, it is not a promising therapeutic target, although important to understand pathogenesis. In contrast, RhoC GTPase is a very attractive prognostic marker and therapeutic target

owing to its pathogenetic role in IBC metastasis and its critical role in the IBC signaling network as a kind of phenotype “molecular switch.” The prognostic value of RhoC expression in breast cancer in general was evaluated using tissue microarray analysis of 801 tissue cores from 280 patients (42). These samples represented a wide range of normal breast tissue and breast disease, including intraductal hyperplasia, ductal carcinoma in situ (DCIS), invasive carcinomas, and distant metastases. The tissue microarrays were immunostained using a polyclonal anti-RhoC antibody (39). All samples of normal breast epithelium had negative to weak staining, whereas staining intensity increased in hyperplasia, DCIS, invasive carcinoma, and metastases. In patients with invasive carcinoma, high RhoC expression was associated with features of aggressive behavior including high histologic grade, positive lymph nodes, and negative hormonal receptor status. Of great interest, RhoC expression status was found to be a good prognostic marker of overall survival of breast cancer patients in multivariate analyses, performing as well as stage. In addition, high RhoC expression was an independent predictor of poor response to doxorubicin-based chemotherapy. In the future, RhoC expression status may help select patients who may beneﬁt from novel RhoC inhibitor therapy. There are several ways (reviewed in Reference 34) in which Rho proteins could be targeted by pharmacological agents. One approach is prevention of correct targeting of the protein. As already mentioned, Rho proteins require lipid modiﬁcations at their C termini, and compounds have been developed to prevent these modiﬁcations (63). Inhibition of Rho-GEFs may also be an effective strategy to inhibit Rho. Experimentally, Rho mutants that titrate Rho-GEFs into nonfunctional complexes away from the endogenous Rho proteins have also proven to interfere with Rho-protein function. Disruption of Rho protein–effector complexes is yet another approach. Overexpression of minimal

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Rho protein binding domains can sequester GTP-bound Rho proteins away from their effectors. A ﬁnal strategy is inhibition of effector protein activity. As mentioned above, active Rho-GTP becomes prenylated (lipid-attached) at the C terminus for membrane binding, in the same manner as closely related Ras proteins (34). RhoA and RhoC are both geranylgeranylated (GG), whereas RhoB exists in both GG and farnesylated (F) forms (64, 65). Protein farnesylation is catalyzed by farnesyltransferase. Farnesyltransferase inhibitors (FTIs) were originally developed for treating Ras-transformed tumors, but their effect on RhoB GTPase was the underlying mechanism for FTI-induced apoptosis of the Ras-transformed tumor cells (66, 67). Van Golen and colleagues hypothesized that FTI causes the accumulation of RhoB-GG and alters overall Rho signaling (68). FTItreated RhoC-overexpressing breast cells and SUM149 cells signiﬁcantly decreased motility, invasion, and anchorage-independent growth on soft agar. Transient transfection of RhoB-GG into the same cells reproduced the effects of FTI, suggesting that FTI-induced reversion of the RhoC phenotype may be mediated by an increase in RhoB-GG levels. This in vitro study showed that FTI was effective in suppressing malignant phenotypes in cultured cells and may be useful in future clinical applications. Studies of RhoA, RhoB, and RhoC as predictive markers of response to FTI treatment are under way (S.D. Merajver, unpublished). In addition to FTIs,

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other small-molecule inhibitors of RhoC GTPase are currently under development in several laboratories. Although signiﬁcant improvement has been made in IBC treatment (12, 14), the overall survival rates of IBC patients are still poor compared to other types of breast cancer. The development of novel therapeutics speciﬁcally for IBC is vital for improving outcome. The combination of traditional and innovative tools may help identify drug targets and predict the effect of drugs in complicated pathways (see Reference 8 for an elegant review).

CONCLUDING REMARKS We have reviewed the notions of biomarker, genetic marker, and genetic determinant in cancer, with particular focus on IBC. We have described the research trajectory that starts with ﬁnding molecular/genetic differences between cancerous and healthy cells and leads to new cancer biomarkers that in principle constitute rational drug targets. The IBC example offers a helpful model for the credentialing of molecular signatures utilizing in vivo and in vitro readouts. Despite signiﬁcant advancements in IBC diagnosis and treatment, further work is needed. RhoC is itself a promising target for treatment, and future RhoC-speciﬁc inhibitors should have the potential to block the RhoCdependent metastatic pathway, prevent IBC cells from spreading, and signiﬁcantly improve outcomes for patients with IBC.

SUMMARY POINTS 1. Cancer is a genetic disease. Its development and spread are caused by the overexpression, mutation, and/or deletion of speciﬁc genes. 2. A biomarker is a ﬂag indicating an abnormal state, such as cancer. 3. A genetic marker is a biomarker at the genetic level, usually found by comparing molecular and genetic properties of cancerous and healthy cells. 4. Gene expression proﬁling is the standard technique used to identify aberrant gene expression.

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5. A genetic determinant is a genetic marker that is not only correlated with but actually causes a disease or major features of a syndrome. 6. In vivo and in vitro molecular biology is necessary to validate a proposed genetic determinant. 7. A genetic determinant has the potential to become a drug target.

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8. Coordinate changes in RhoC GTPase and WISP3 gene expression are essential in driving the highly invasive phenotype of IBC. Therefore, RhoC GTPase and WISP3 are deemed genetic determinants of this disease.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENT This work was supported by a DOD-BCRP multidisciplinary postdoctoral award (A.C.V.), The Burroughs Wellcome Fund (S.D.M.), The Breast Cancer Research Foundation (S.D.M.), and NIH grant RO1-CA66712 (S.D.M.).

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15. First study done to identify IBC-specific genetic markers and genetic determinants.

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34. Review of the role of Rho proteins in cancer and their potential as therapeutic targets.

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:199-212. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan1 and D. Gary Gilliland1,2 1

Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115; 2 Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115; email: [email protected]

Annu. Rev. Med. 2008. 59:213–22

Key Words

First published online as a Review in Advance on October 5, 2007

chronic myelogenous leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), idiopathic myeloﬁbrosis (IMF), hematopoietic stem cell (HSC)

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.061506.154159 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0213$20.00

Abstract Myeloproliferative disorders (MPDs) are characterized by a clonal expansion of myeloid cells. Over the past two years, the identiﬁcation of the JAK2V617F mutation in most cases of polycythemia vera (PV) as well as ∼50% of patients with essential thrombocythemia (ET) and idiopathic myeloﬁbrosis (IMF) has greatly advanced our understanding of MPDs. The JAK2V617F mutation alters the JAK2 tyrosine kinase to confer constitutive activation and affect downstream signaling pathways. Data from mouse models demonstrate that the mutation is sufﬁcient for development of PV, but additional work is needed to better understand how this allele functions in ET and IMF. Regardless of the various pathologies, the JAK2V617F discovery highlights the importance of JAK-STAT signaling in myeloid differentiation and focuses effort on developing a clinically relevant JAK2 inhibitor.

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INTRODUCTION MPD: myeloproliferative disorder CML: chronic myelogenous leukemia PV: polycythemia vera

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ET: essential thrombocythemia IMF: idiopathic myeloﬁbrosis AML: acute myeloid leukemia JH: JAK homology

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Myeloproliferative disorders (MPDs) are a class of stem cell–derived hematological malignancies that result in overexpansion of mature myeloid cells. Two main classes of MPDs are recognized by the World Health Organization: (a) chronic myelogenous leukemia (CML), characterized by the Philadelphia (Ph1 ) translocation, which frequently results in the BCR-ABL fusion gene; and (b) the Ph1 -negative MPDs, polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myeloﬁbrosis (IMF) (1). Less common disorders, such as chronic neutrophilic leukemia and chronic eosinophilic leukemia/hypereosinophilic syndrome, are also classiﬁed as chronic myeloid disorders but are grouped separately as “atypical MPD” (2). Clinical manifestations of these disorders include splenomegaly, thrombosis, bone marrow ﬁbrosis, extramedullary hematopoiesis, and leukemic transformation demonstrated in the bone marrow. The latter manifests as increased and clustered megakaryocytes in ET and PV, increased granulopoiesis in PV and IMF and increased erythropoiesis in PV (3). CML, PV, ET, and IMF were all recognized to have signiﬁcant overlap in both clinical and biological features and thus were thought of as related diseases in 1951 (4). In 1960, cytogenetic characterization of CML discovered the Ph1 chromosome (5). Lacking obvious cytogenetic differences in the remaining three diseases, multiple groups analyzed bone marrow samples from PV and ET patients. Interestingly, the samples exhibited similar cytokine hypersensitivity to erythropoietin (6, 7) and growth of erythroid progenitors in the absence of exogenous erythropoietin (8). In addition, similar disease progression patterns were observed among the disorders, including acute myeloid leukemia (AML) as well as PV, ET, and IMF (9). This observation implied that the molecular mechanism of transformation was the same or similar among the disorders. In the

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late 1970s through the early 1980s, Fialkow and colleagues analyzed X chromosome inactivation patterns in women who had PV, ET, IMF, or CML and who concurrently carried a polymorphic variant of the gene for glucose-6-phosphate dehydrogenase. From the data they established that all four diseases were clonal stem cell disorders (10–12). More recently, several independent groups described a somatic mutation involving a protein tyrosine kinase in a signiﬁcant number of patients with MPD (13–16). Each employing different experimental strategies, they discovered a surprisingly high incidence of a JAK2 point mutation at codon 617, subsequently named JAK2V617F. A single nucleotide change at 1849 of exon 12 results in a valine-to-phenylalanine substitution. The occurrence of the mutation in Ph1 negative MPD was found to be approximately 95% in PV, 50% in ET, and 50% in IMF (17).

JAK2 AND SIGNALING JAK2 belongs to the Janus family of kinases, which also includes JAK1, JAK3, and TYK2. The acronym JAK stands for Just Another Kinase, a reference to the discovery of multiple tyrosine kinase family members through a PCR screen spanning the conserved kinase domains (18). The unique structure of JAK kinases distinguished them from other tyrosine kinases. The most signiﬁcant features are the presence of seven JAK homology domains ( JH1 through JH7) and the absence of Src homology binding domains (SH2 or SH3) (19) (Figure 1a). The two carboxy-terminal JH domains retain high homology to tyrosine kinase domains. Only one retains function; the other, lacking critical amino acids for kinase activity, is called the pseudokinase domain. Mutational analysis has shown that deletion of the JH2 domain negatively regulates JAK2 kinase activity (20). The same study suggested that interaction between JH1 and JH2 was responsible for JAK2 kinase inhibition.

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Figure 1 Role of JAK2. (a) Representation of JAK2 showing JAK homology ( JH) domains 1–7, including the kinase domain ( JH1) and pseudokinase domain ( JH2), SH2-like domains, and FERM domain. Arrow indicates V617F mutation site. (b) Receptor signaling involving JAK2 activation. Ligand binds the cytokine receptor, causing a conformational change that brings JAK2 molecules in contact with one another. Auto- and trans-phosphorylation occurs on JAK2 and the receptor. Signal transducers and activators of transcription (STAT) molecules are recruited and phosphorylated, and dimers translocated to the nucleus. Inhibition molecules regulate JAK2 activation by dephosphorylation (SHP-1) and by competing for STAT binding sites (PIAS or SOCS).

Janus kinases mediate cytokine/growth factor receptor signaling via auto- or transphosphorylation (Figure 1b). Ligand binding induces receptor oligomerization and the recruitment of JAKs to bind via their FERM domain, a region that contains a band 4.1, Ezrin, radixin and moesin domain shared by the focal adhesion kinase (FAK) family (21). The juxtaposition of JAKs results in their phosphorylation by each other and/or other kinase members on key tyrosine residues, which increases overall JAK activity. The activated JAKs then phosphorylate target tyrosine residues on the receptor that serve as docking sites for signaling molecules containing SH2 binding domains, including signal trans-

ducers and activators of transcription (STAT) proteins, as well as other phosphokinases and adaptor molecules. Following their phosphorylation, STATs dimerize and translocate to the nucleus to interact with speciﬁc DNA regulatory elements and induce target gene transcription. Regulation of JAK signaling is tightly controlled by several mechanisms to prevent excessive signaling or overactivation, which lead to autoimmune disorders or malignant transformation (Figure 1b). Mechanisms for control of cytokine signaling include direct dephosphorylation by Src homology tyrosine phosphatases (SHP-1), inhibition by suppressors of cytokine signaling (SOCS) family www.annualreviews.org • JAK2 and Myeloproliferative Diseases

STAT: signal transducers and activators of transcription

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members through direct binding to JAK2 on tyrosine 1007 (22), and competition for STAT binding sites and binding to negative regulators of STAT gene transcription such as protein inhibitors of activated STAT (PIAS) (23).

MUTATIONS IN JAK2

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As illustrated by Ph1 -positive CML, MPDs frequently involve the deregulation of a tyrosine kinase. Molecular characterization of the (9;22)(q34;q11) translocation discovered that the resulting fusion gene, BCR-ABL, produced an activated kinase (24). Subsequent work has demonstrated that BCR-ABL protein expression is both necessary and sufﬁcient for the development of CML. If BCR-ABL kinase activity is inhibited, downstream signaling pathways can be effectively shut down. The work of many researchers has resulted in vastly improved patient diagnosis, treatment, and longevity. Because JAK’s activation is essential for cytokine-mediated signaling, and abnormalities in its tyrosine kinase activity frequently result in disease, its presence in hematological malignancy is not surprising. The JAK2 gene, located on chromosome 9p24, has been identiﬁed as a fusion partner in multiple translocations. In t(9;12)(p24;p13), the resultant TEL-JAK2 translocation is associated with both T and pre-B acute lymphoid leukemia and atypical CML (13). Another translocation involving JAK2, t(8;9)(p22;24), which results in the PCM1-JAK2 fusion, has recently been characterized in a myeloproliferative/myelodysplastic disorder and a CML-like disease (25, 26). In both types of JAK2 translocations, deregulated kinase activity is thought to play a major role in the pathogenesis of disease. The JAK2V617F mutation has been reported in >95% of patients with PV and 50%–60% of patients with ET or IMF (17, 27). The mutation is somatically acquired, and a subset of patients with PV and IMF demonstrate homozygosity as a result of mitotic recombination. Subsequent reports have 216

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observed JAK2V617F in patients with other myeloid malignancies, including chronic myelomonocytic leukemia, myelodysplasia, and, in rare cases, AML (28–30). Patient samples have shown the JAK2V617F protein in progenitors and myeloid cells, including cells that demonstrate a hematopoietic stem cell phenotype as well as more mature progenitors (15, 31) (Figure 2a). It is believed the mutation is myeloid lineage speciﬁc because it is present in erythroid and granulocytemacrophage progenitors as well as CD34derived erythroblasts and platelets (13, 15). However, recent reports show that B, T and NK cells express JAK2V617F. These ﬁndings conﬁrm the stem cell nature of JAK2V617Fpositive MPDs, but it is not clear whether or how this mutation affects lymphoid lineages (32, 33). Since the discovery of the mutation, studies have focused on elucidating the JAK2V617F mechanism for transformation. JAK2V617F occurs within the autoinhibitory JH2 domain or pseudokinase domain. Structurally, the mutation is not well understood because the interaction between the kinase domain and the pseudokinase domain is unknown. However, the working hypothesis is that JAK2V617F relieves inhibition by the pseudokinase domain and results in constitutive activation of kinase activity. Resolution of a cocrystal structure of both domains will be needed to understand how the mutation alters their interactions and their subsequent functions.

MODELS OF JAK2V617F In vitro studies have demonstrated that the JAK2V617F mutation is associated with constitutive phosphorylation of JAK2 (34) and erythropoietin hypersensitivity (13, 14, 34). Initial studies showed that expression of JAK2V617F resulted in the transformation of Ba/F3 cells to IL-3-independent growth, whereas wild-type JAK2 did not (13). Similarly, coexpression of JAK2V617F and erythropoietin receptor, thrombopoietin

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Figure 2 Effect of JAK2V617F on hematopoietic cell populations. (a) Acquisition of JAK2V617F mutation in human cells results in expansion of the hematopoietic stem cell (HSC) compartment and various downstream populations. Expansion of the common myeloid progenitor (CMP), megakaryocyte/erythroid progenitor (MEP), granulocyte/monocyte progenitor (GMP), and their progeny is seen in polycythemia vera (PV) and idiopathic myeloﬁbrosis (IMF). Expansion of the CMP, MEP, and differentiated progeny is seen in essential thrombocythemia (ET). (b) Similar patterns of expanded compartments are seen in bone marrow transplant (BMT) mouse models of Jak2V617F. HSC, CMP, MEP, GMP, and their differentiated progeny all exhibit a phenotype. CLP, common lymphoid progenitor.

receptor, prolactin receptor, or granulocyte macrophage colony stimulating factor (GMCSF) receptor (all homodimeric Type 1 cytokine receptors) also resulted in cellular transformation (14, 35). In contrast to the earlier studies, later work showed that in the absence of the expression of Type 1 cytokine receptors, JAK2V617F acts as a monomeric cytosolic protein and remains inactive (35). Regardless of the differing data, both reports

suggest that either IL-3 receptors or Type 1 receptors are required to provide a scaffold for JAK2V617F proteins to bind to, where they become physically close to one another so that trans-phosphorylation can occur even in the absence of cytokines. Whereas in vitro experimentation is needed to understand the JAK2V617F mechanism for pathogenesis, mouse modeling is crucial to delineate in vivo effects of the www.annualreviews.org • JAK2 and Myeloproliferative Diseases

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mutation. Early reports showed that transplantation of JAK2V617F retrovirally transduced bone marrow cells resulted in increased erythrocytosis in the recipient mice at 4 weeks after transplant. No increase in erythrocytosis was seen in recipients of wildtype JAK2 or empty vector transduced bone marrow cells (13, 14). Subsequent transplantation experiments have shown the development of polycythemia in all recipient mice, with differences dependent on background strain (36, 37) (Figure 2b). Disease latency was observed as early as 7 weeks after transplant, with mice surviving up to 32 weeks (36). Mice developed many key features associated with PV, including leukocytosis peaking at 90 days (37), splenomegaly due to extramedullary hematopoiesis, cytokineindependent colony formation, and low serum erythropoietin. Also observed were regression of polycythemia and the development of ﬁbrosis with neutrophilia, mimicking human PV and its evolution to myeloﬁbrosis (37).

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PATHOLOGIES ASSOCIATED WITH JAK2V617F Although the retroviral transplant mouse models have provided valuable evidence that the acquisition of Jak2V617F is sufﬁcient for development of PV, additional research is needed to understand its effects on ET and IMF. One explanation is gene dosage. It has been shown that 25% of patients with PV are homozygous for the JAK2V617F mutation, whereas most patients with ET are heterozygous or wild-type (14). Homozygosity increases the expression of the mutated protein and eliminates the competition with wild-type protein. These data suggest that higher levels of JAK2V617F expression support an erythroid phenotype and lower levels support a megakaryocytic phenotype (Figure 3a). The dosage hypothesis is compatible with the mouse models, where overexpression of Jak2V617F induces a PV-like disease that can progress to an IMF-like disease. 218

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However, this hypothesis can only be veriﬁed using a knock-in mouse model in which the endogenous promoter regulates gene expression levels and allows the comparison of heterozygous versus homozygous Jak2V617F mutations. Another explanation for the various pathologies associated with JAK2V617F is that other, unidentiﬁed mutations are required for the different phenotypes (Figure 3b). JAK2V617F has a preference for myeloid lineages or immature myeloid lineage progenitors. This would imply that the acquisition of JAK2V617F at the stem cell level dictates the lineages that are affected. Given the high incidence of the JAK2V617F mutation in PV, its presence is likely required for this disorder to develop, and possibly precedes other secondary lesions. In addition, the reduced occurrence of the mutation in patients with ET implies that other mutations can be responsible for the development of ET. However, for JAK2V617F-positive ET, other events would most likely be primary to the JAK2V617F mutation, since a secondary mutation would imply that ET occurs after a polycythemic stage. For both PV and ET, progression to IMF may occur after the accumulation of more mutations in the stem cell (Figure 3b). This hypothesis is attractive because most hematopoietic malignancies involve more than one transforming event. Finally, receptor interaction may play an important role in lineage restriction. JAK2 is a crucial molecule for signal transduction, along with many Type 1 receptors including erythropoietin receptor and thrombopoietin receptor, as well as cytokine class 1 receptor family members such as IL-3 and GM-CSF receptors. These receptors are expressed speciﬁcally during myeloid and erythroid differentiation. It has been proposed that the level of interaction between the receptor and JAK2, speciﬁcally JAK2V617F, may inﬂuence the expression of the activated phenotype (21). Thus, with cellspeciﬁc receptor expression and the potential

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Figure 3 Causes of phenotype variability exhibited by JAK2V617F. (a) Gene dosage. Heterozygous contribution of JAK2V617F may lead to essential thrombocythemia (ET). Homozygous contribution and a lack of wild-type (WT) JAK2 may lead to more severe phenotypes, such as polycythemia vera (PV) and idiopathic myeloﬁbrosis (IMF). (b) Unidentiﬁed mutations. JAK2V617F or other mutations can lead to ET. However, JAK2V617F is required for phenotypes such as PV, with secondary mutations needed for the progression to IMF. (c) Speciﬁc receptors are expressed on the surface of myeloid cells. The type of receptor may result in higher levels of JAK2V617F activation that inﬂuence the phenotype of disease.

for stronger interactions with JAK2V617 (Figure 3c), the affected cell populations may be activated to varying degrees.

THERAPIES AND FUTURE RESEARCH Although questions still remain concerning the role and functional consequences of the JAK2V617F mutation, its discovery has led to the reevaluation of treatments for myeloproliferative diseases. Clearly, with the recognition of this mutation, MPDs can now be viewed as stem cell disorders rather than reactive conditions, although its presence alone cannot distinguish PV from ET or IMF. Understanding the molecular pathogenesis of

diseases—for example, knowing the role of BCR-ABL in CML and now JAK2V617F in PV and some cases of ET and IMF—has laid the foundation for investigating smallmolecule inhibitors for JAK2. Current JAK2 inhibitors have shown promise by reducing growth of JAK2V617F-positive cell lines and primary cells (14, 31, 38); thus, efforts are ongoing to develop compounds for clinical use. However, it is not known whether an inhibitor can show speciﬁc activity against the mutant without disrupting wild-type JAK2 and causing signiﬁcant hematological toxicity. Cost management may become an even larger issue, since most conventional therapies are achieved at relatively modest cost compared to a potentially expensive long-term therapy. www.annualreviews.org • JAK2 and Myeloproliferative Diseases

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Despite the unanswered questions, with the success of imatinib for CML as inspiration, researchers and physicians are hopeful about the

development of therapeutic JAK2 inhibitors for treating patients in accelerated phases of disease.

SUMMARY POINTS 1. MPD includes CML, PV, ET, and IMF. 2. ∼95% of PV and >50% of ET and IMF patients harbor JAK2V617F. 3. JAK2V617F mutation induces stem cell disease with myeloid lineage-speciﬁc phenotype. 4. JAK2V617F mutation confers constitutive tyrosine kinase activation. Annu. Rev. Med. 2008.59:213-222. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

5. Mutant expression converts Ba/F3 cells to cytokine-independent growth. 6. Mouse models demonstrate JAK2V617F is sufﬁcient for PV-like disease.

FUTURE ISSUES 1. Structural consequences of JAK2V617F mutation on pseudokinase interaction with kinase domain. 2. Requirement for speciﬁc receptors (Type 1 versus cytokine class 1 receptors). 3. Various pathologies observed in JAK2V617F-positive disease, PV versus ET versus IMF. 4. Development of JAK2 small-molecule inhibitors.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENT D.G.G. is an Investigator of the Howard Hughes Medical Institute, and a Doris Duke Charitable Foundation Distinguished Clinical Scientist Award Recipient.

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4. Dameshek W. 1951. Some speculations on the myeloproliferative syndromes. Blood 6:372– 75 5. Nowell PC, Hungerford DA. 1960. Chromosome studies on normal and leukemic human leukocytes. J. Natl. Cancer Inst. 25:85–109 6. Krantz SB. 1968. Response of polycythemia vera marrow to erythropoietin in vitro. J. Lab. Clin. Med. 71:999–1012 7. Ward HP, Vautrin R, Kurnick J, et al. 1974. Presence of a myeloproliferative factor in patients with polycythemia vera and agnogenic myeloid metaplasia. I. Expansion of the erythropoietin-responsive stem cell compartment. Proc. Soc. Exp. Biol. Med. 147:305–8 8. Prchal JF, Axelrad AA. 1974. Letter: Bone-marrow responses in polycythemia vera. N. Engl. J. Med. 290:1382 9. Tefferi A, Silverstein MN. 1998. Treatment of polycythaemia vera and essential thrombocythaemia. Baillieres Clin. Haematol. 11:769–85 10. Adamson JW, Fialkow PJ, Murphy S, et al. 1976. Polycythemia vera: stem-cell and probable clonal origin of the disease. N. Engl. J. Med. 295:913–16 11. Fialkow PJ, Jacobson RJ, Papayannopoulou T. 1977. Chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet and monocyte/macrophage. Am. J. Med. 63:125–30 12. Fialkow PJ, Faguet GB, Jacobson RJ, et al. 1981. Evidence that essential thrombocythemia is a clonal disorder with origin in a multipotent stem cell. Blood 58:916–19 13. James C, Ugo V, Le Couedic JP, et al. 2005. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 434:1144–48 14. Levine RL, Wadleigh M, Cools J, et al. 2005. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myeloﬁbrosis. Cancer Cell 7:387–97 15. Baxter EJ, Scott LM, Campbell PJ, et al. 2005. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 365:1054–61 16. Kralovics R, Passamonti F, Buser AS, et al. 2005. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N. Engl. J. Med. 352:1779–90 17. Tefferi A, Gilliland DG. 2005. JAK2 in myeloproliferative disorders is not just another kinase. Cell Cycle 4:1053–56 18. Wilks AF. 1989. Two putative protein-tyrosine kinases identiﬁed by application of the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86:1603–7 19. Wilks AF, Harpur AG, Kurban RR, et al. 1991. Two novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, deﬁne a new class of protein kinase. Mol. Cell. Biol. 11:2057–65 20. Saharinen P, Takaluoma K, Silvennoinen O. 2000. Regulation of the Jak2 tyrosine kinase by its pseudokinase domain. Mol. Cell Biol. 20:3387–95 21. Funakoshi-Tago M, Pelletier S, Matsuda T, et al. 2006. Receptor speciﬁc downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2. EMBO J. 25:4763–72 22. Feng J, Witthuhn BA, Matsuda T, et al. 1997. Activation of Jak2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop. Mol. Cell Biol. 17:2497–501 23. Khwaja A. 2006. The role of Janus kinases in haemopoiesis and haematological malignancy. Br. J. Haematol. 134:366–84 24. Heisterkamp N, Stam K, Groffen J, et al. 1985. Structural organization of the bcr gene and its role in the Ph translocation. Nature 315:758–61 www.annualreviews.org • JAK2 and Myeloproliferative Diseases

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25. Heiss S, Erdel M, Gunsilius E, et al. 2005. Myelodysplastic/myeloproliferative disease with erythropoietic hyperplasia (erythroid preleukemia) and the unique translocation (8;9)(p23;p24): ﬁrst description of a case. Hum. Pathol. 36:1148–51 26. Reiter A, Walz C, Watmore A, et al. 2005. The t(8;9)(p22;p24) is a recurrent abnormality in chronic and acute leukemia that fuses PCM1 to JAK2. Cancer Res. 65:2662–67 27. Levine RL, Gilliland DG. 2007. JAK-2 mutations and their relevance to myeloproliferative disease. Curr. Opin. Hematol. 14:43–47 28. Levine RL, Loriaux M, Huntly BJ, et al. 2005. The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia. Blood 106:3377–79 29. Steensma DP, Dewald GW, Lasho TL, et al. 2005. The JAK2 V617F activating tyrosine kinase mutation is an infrequent event in both “atypical” myeloproliferative disorders and myelodysplastic syndromes. Blood 106:1207–9 30. Jelinek J, Oki Y, Gharibyan V, et al. 2005. JAK2 mutation 1849G>T is rare in acute leukemias but can be found in CMML, Philadelphia chromosome-negative CML, and megakaryocytic leukemia. Blood 106:3370–73 31. Jamieson CH, Gotlib J, Durocher JA, et al. 2006. The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation. Proc. Natl. Acad. Sci. USA 103:6224–29 32. Bogani C, Guglielmelli P, Antonioli E, et al. 2007. B-, T-, and NK-cell lineage involvement in JAK2V617F-positive patients with idiopathic myeloﬁbrosis. Haematologica 92:258–59 33. Larsen TS, Christensen JH, Hasselbalch HC, et al. 2007. The JAK2 V617F mutation involves B- and T-lymphocyte lineages in a subgroup of patients with Philadelphiachromosome negative chronic myeloproliferative disorders. Br. J. Haematol. 136:745–51 34. Zhao R, Xing S, Li Z, et al. 2005. Identiﬁcation of an acquired JAK2 mutation in polycythemia vera. J. Biol. Chem. 280:22788–92 35. Lu X, Levine R, Tong W, et al. 2005. Expression of a homodimeric type I cytokine receptor is required for JAK2V617F-mediated transformation. Proc. Natl. Acad. Sci. USA 102:18962–67 36. Wernig G, Mercher T, Okabe R, et al. 2006. Expression of Jak2V617F causes a polycythemia vera-like disease with associated myeloﬁbrosis in a murine bone marrow transplant model. Blood 107:4274–81 37. Lacout C, Pisani DF, Tulliez M, et al. 2006. JAK2V617F expression in murine hematopoietic cells leads to MPD mimicking human PV with secondary myeloﬁbrosis. Blood 108:1652–60 38. Li Z, Xu M, Xing S, et al. 2007. Erlotinib effectively inhibits JAK2V617F activity and polycythemia vera cell growth. J. Biol. Chem. 282:3428–32

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:213-222. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet,1 Marcia L. Shew,1 and Jessica A. Kahn2 1

Department of Pediatrics, Section of Adolescent Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202; email: [email protected]; [email protected]

2

Division of Adolescent Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229; email: [email protected]

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Key Words

The Annual Review of Medicine is online at http://med.annualreviews.org

human papillomavirus, HPV, vaccination, immunization, adolescent

This article’s doi: 10.1146/annurev.med.59.092806.131644 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0223$20.00

Abstract Human papillomavirus (HPV) is a necessary, though not sufﬁcient, cause of cervical cancer. Two vaccines have been developed that prevent two HPV types associated with 70% of cervical cancers. One of the vaccines (a quadrivalent vaccine) also prevents two HPV types associated with 90% of genital warts. Both HPV vaccines have shown very good efﬁcacy and safety. This review summarizes the guidelines for use of the quadrivalent vaccine published by the Advisory Committee on Immunization Practices, presents data on vaccine efﬁcacy and safety, and gives an overview of the ﬁndings of costeffectiveness studies. In addition, we summarize the research on the attitudes of parents and health care providers toward HPV vaccine and critically evaluate controversial and challenging issues surrounding HPV vaccination, including concerns about sexual disinhibition and potential obstacles to vaccine distribution and uptake.

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INTRODUCTION HPV: human papillomavirus STI: sexually transmitted infection

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More than 100 types of human papillomavirus (HPV) have been identiﬁed, with >40 specifically infecting the anogenital areas (1). Genital HPV infections are transmitted primarily through sexual contact; prevalence rates peak among young persons after the initiation of sexual activity and decline after age 30 (2, 3). HPV infection is a highly prevalent sexually transmitted infection (STI) (4–6). In a large cross-sectional nationally representative sample of women in the United States, HPV prevalence rates among women who had engaged in sexual intercourse were 39.6% for those aged 14–19 years and 49.3% for those aged 20–24 years (6). Cumulative prevalence rates as high as 82% have been found in adolescent populations (7). Low-risk HPV types, such as types 6 and 11, cause genital warts, low-grade cervical dysplastic lesions, and respiratory papillomatosis. High-risk HPV types, such as types 16, 18, 31, and 45, are responsible for cervical and other genital tract cancers and for both low- and high-grade dysplastic (precursor) lesions (8). It is estimated that ∼90% of genital warts are caused by HPV types 6 and 11 (9), and that 70% of cervical cancers are caused by HPV types 16 and 18 (8). Most genital HPV infections are asymptomatic and transient (10). In some women, HPV infection may cause abnormal cervical cytology on Papanicolaou (Pap) tests, including atypical squamous cells of undetermined signiﬁcance, low-grade squamous intraepithelial lesion, or high-grade squamous intraepithelial lesion. Abnormal cytology may indicate cervical dysplasia, a histologic diagnosis. Mild cervical dysplasia often regresses, but severe cervical dysplasia usually progresses to cervical cancer. Longitudinal studies have established that persistence of high-risk, or cancer-associated, HPV types is necessary for progression to severe cervical dysplasia and cervical cancer. Persistence of high-risk HPV is a necessary, but not sufﬁcient, cause of cervi-

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cal cancer. Other factors that contribute to cervical carcinogenesis are viral characteristics, sexual behaviors (e.g., condom nonuse and multiple sexual partners), chlamydial infections, immunosuppression, and smoking (11). Cervical cancer is the second most commonly diagnosed cancer among women worldwide, accounting for >250,000 deaths per year. Approximately 80% of deaths due to cervical cancer occur in less developed regions of the world because Pap screening programs do not exist in those regions (12). Because of the well-organized Pap screening program in the United States, cervical cancer incidence and mortality are relatively low compared to those of other cancers; however, cervical cancer still causes ∼3700 deaths per year in the United States (13). In 2000, it was estimated that the lifetime ﬁnancial burden of HPV disease in the United States was $3.9 billion and that 90% of this amount was spent on followup of abnormal Pap tests and treatment of precancerous lesions (14). Two vaccines have been developed to prevent HPV infection. One vaccine, approved by the U.S. Food and Drug Administration (FDA) in June 2006, prevents HPV types 6, 11, 16, and 18, protecting against the types most frequently associated with both cervical cancers and genital warts (15). A second vaccine, not yet licensed but currently under review by the FDA, prevents HPV types 16 and 18 (16). Both vaccines show sustained efﬁcacy of at least ﬁve years with good safety proﬁles (17–19). Here we review the guidelines for HPV vaccine administration published by the Advisory Committee on Immunization Practices (ACIP), address vaccine efﬁcacy and safety data, discuss the ﬁndings of cost-effectiveness studies, summarize the literature on parental and physicians’ knowledge about HPV and attitudes about HPV vaccination, and discuss different immunization strategies and issues.

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ACIP GUIDELINES FOR QUADRIVALENT VACCINE ADMINISTRATION The ACIP recommends routine vaccination with the quadrivalent HPV vaccine of all 11- to 12-year-old girls, catch-up vaccination of all 13- to 26-year-old young women not previously vaccinated, and vaccination of 9- to 10-year-old girls at the provider’s discretion (20). The vaccine is targeted toward 11- to 12-year-old girls because vaccination is most effective in preventing type-speciﬁc HPV infection if given prior to sexual initiation. However, the ACIP notes that vaccination may still provide protection among sexually active young women, as demonstrated in clinical trials (17, 21). Pap testing and HPV DNA testing are not recommended prior to vaccination. The dosing schedule that was evaluated in clinical trials and is recommended by the ACIP is 0, 2, and 6 months. However, recognizing that this ideal schedule may not always be followed, the ACIP offers the following additional recommendations: 1. The ﬁrst and second doses must be separated by at least 4 weeks. 2. The second and third doses must be separated by at least 12 weeks. 3. If the dosing schedule has been interrupted at any point, the vaccine series should not be restarted, but the required dose should be administered as soon as possible. The HPV vaccine can be administered during the same visit as other age-appropriate vaccines, such as the meningococcal conjugate vaccine (MCV4) and the tetanus, diphtheria, and pertussis booster (Tdap). According to the ACIP, lactating women can receive the vaccine, as can young women who are immunocompromised. Although the quadrivalent vaccine has not been associated with any pregnancy-related adverse events, currently there are insufﬁcient data on the potential effects of the vaccine on pregnant women to advance the recommendation.

Finally, the ACIP emphasizes the importance of continued cervical cancer screening in vaccinated women, because (a) 30% of cervical cancers are caused by types not contained in the vaccine, (b) the vaccine may not be 100% effective, and (c) the vaccine has no therapeutic value for those already infected (20). Additional details regarding the ACIP guidelines can be found in Reference 20.

VACCINE EFFICACY AND SAFETY Clinical trials have demonstrated that HPV vaccines are effective and safe, but for both ethical and scientiﬁc reasons, surrogate end points were viral persistence, genital dysplasia, and genital warts as opposed to cervical cancer. The quadrivalent vaccine has demonstrated efﬁcacy not only in participants who followed exact clinical protocols, but also in trial populations with less strenuously deﬁned criteria. In the per-protocol population, the vaccine demonstrated 100% efﬁcacy in preventing warts and/or vulvar or vaginal dysplastic lesions and 98% efﬁcacy in preventing high-grade cervical lesions (18–22). In the intention-to-treat population analysis (deﬁned as including those with infection or disease associated with vaccine types before vaccination and/or protocol violations), the quadrivalent vaccine was 44% effective in preventing high-grade cervical lesions and 73% percent effective in reducing warts as well as vulvar and vaginal lesions (21, 22). Similarly, the bivalent vaccine demonstrated 100% efﬁcacy in preventing persistent infection in cervical samples and cervical intraepithelial neoplasia (CIN) lesions associated with vaccine types in the accordingto-protocol analysis. In the intention-to-treat analysis (deﬁned as receiving at least one dose of the vaccine and testing negative for high-risk HPV types or no abnormalities on Pap testing at a screening visit that was within 90 days of enrollment), the vaccine was 94% efﬁcacious in preventing persistent www.annualreviews.org • Cervical Cancer Vaccine

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infection at 12 months and 96% efﬁcacious in preventing abnormal cytology (17, 19). Additional studies continue to demonstrate high efﬁcacy, particularly among women not previously infected with types 16 and 18 (21, 22). Both vaccines have been well tolerated without serious vaccine-related side effects. No therapeutic effects have been observed in women who have existing HPV infection or HPV-related disease caused by vaccine-speciﬁc types (20). Research on the quadrivalent vaccine in young men indicates that it stimulates a strong immunogenic response and has no signiﬁcant short-term safety issues (23). Vaccine efﬁcacy in men has not yet been demonstrated, but studies are under way to evaluate the efﬁcacy of the vaccine in preventing warts as well as penile, perineal, and perianal cancers. Studies of HPV vaccine safety, immunogenicity, and efﬁcacy in certain populations are ongoing. Follow-up studies of women vaccinated with bivalent or quadrivalent vaccine report sustained efﬁcacy of both vaccines for at least ﬁve years (17, 18). In addition, a modeling study suggests that antibody levels may remain above those stimulated by natural infection for 12 years or more (24). Finally, a study of women 60 months postvaccination found that an additional dose of the vaccine induced a strong anamnestic response typical of vaccines with long-lasting efﬁcacy (25). The immunogenicity and clinical efﬁcacy of HPV vaccination in individuals with chronic disease, particularly those who are immunosuppressed, are still unclear. However, previous work has shown that other viral vaccines (e.g., hepatitis A and B) generate an effective, though attenuated, immune response in those who are immunocompromised (26, 27). In addition, individuals infected with human immunodeﬁciency virus (HIV) generate an immune response to natural HPV infection (28, 29). Future clinical trials will generate more deﬁnitive data concerning the immunogenicity and clinical efﬁcacy of HPV vaccines in immunocompromised individuals. The quadrivalent vaccine

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also appears to be safe in pregnant women: clinical trials have demonstrated no increase in spontaneous loss rate or fetal malformations as compared to placebo groups, and the vaccine has been classiﬁed as Category B in terms of risk in pregnancy (20). Longer-term and larger studies that examine the safety of HPV vaccines in pregnancy are in progress. Given the current status of clinical trial data, the ACIP does not recommend vaccination in women known to be pregnant.

COST-EFFECTIVENESS Several cost-effectiveness studies on HPV vaccination have been carried out, using different methodologies and assumptions (30–35). All of these studies suggest that vaccinating 12-year-old girls against HPV can be cost-effective. One of these studies found that the most effective strategy for reduction of cancer morbidity and mortality was vaccination of girls, followed by annual cervical screening starting at age 18 (30). Another study determined that the ideal approach would be vaccination of girls at age 12, followed by cytologic screening every three years starting at age 25 (31). The potential effects of vaccinating males, however, were not considered in either of these papers. Taira et al. (32) found that vaccination of both females and males would not be cost-effective, but Elbasha et al. (33) found that it would. The divergent ﬁndings are likely attributable to differences in modeling assumptions; for example, Elbasha et al. included HPV types 6, 11, 16, and 18 in their model, whereas Taira et al. considered only the two oncogenic types. All of these studies, which used very different approaches to analysis, found the vaccination of 12-year-old girls against HPV to be a cost-effective strategy. These studies did not consider costs such as lost productivity due to cancer-related illness and death. Inclusion of such factors would only improve the cost-effectiveness of HPV vaccination.

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KNOWLEDGE AND ATTITUDES OF PARENTS AND HEALTH CARE PROVIDERS The research published to date on knowledge about HPV and attitudes about HPV vaccination relies on data collected prior to the licensure and availability of HPV vaccine. These studies, therefore, were carried out before information about HPV and HPV vaccine was widely disseminated to the public and professionals by pharmaceutical companies, the media, and professional organizations. It is likely that knowledge about HPV has increased and that attitudes about vaccination have changed somewhat since these studies were implemented.

Parents In 2006, Zimet et al. (36) reviewed ten published reports on parental attitudes about HPV/STI vaccination. These studies, which included both qualitative and quantitative methodologies, all found that parents expressed moderate to strong interest in vaccinating their preadolescent and adolescent children against STI. All but two of the studies were carried out in the United States, and Zimet et al. recommended that future research include parents from other regions of the world, particularly developing countries. The same review examined research on knowledge about HPV and HPV-related conditions (36). Across multiple studies, women demonstrated a poor understanding of HPV, often reporting no knowledge of the virus or unawareness of the associations of HPV with Pap testing and with cervical cancer. At the same time, other research has indicated that women are very interested in learning more about HPV infection; many state that information about HPV should be provided to young women prior to the initiation of sexual activity (37, 38). Results of studies published since the review by Zimet et al. have been largely consistent with previous ﬁndings (see Table 1)

(39–44). With one exception (39), these studies found the majority of parent participants willing to have their children vaccinated against HPV. Brabin et al. (41) focused on British parents’ attitudes about adolescent self-consent for vaccination and found that nearly half of parents agreed that HPV vaccine should be provided to adolescents at sexual health clinics without parental consent. Across all studies, opposition to HPV vaccine was associated with concern about vaccination leading to sexual disinhibition, viewing one’s child as being at low risk for infection, and worries about vaccine safety. Although these recent studies were carried out in several countries (e.g., United Kingdom, Netherlands, Finland), only one study examined attitudes of parents in a developing country, Vietnam (44). Results from the Vietnam study are comparable to those reported in other research, with parents strongly supportive of HPV vaccination for their children. Further research on HPV vaccine acceptability in developing countries is needed.

Health Care Providers Providers’ attitudes and knowledge about HPV and HPV vaccination are likely to inﬂuence their immunization practices. Zimet et al. (36) reviewed four studies that examined attitudes of nurse practitioners, obstetrician/ gynecologists, family practice physicians, and pediatricians toward recommending HPV/STI vaccines for their adolescent patients. Across health care provider types, these studies found relatively strong endorsement of HPV vaccination. However, all health care provider groups expressed greater reluctance to vaccinate younger adolescents compared to older ones. Studies that assessed knowledge about HPV found that both pediatricians and family practice physicians lacked awareness of several aspects of HPV infections (45–47). A more recent large-scale study of pediatricians found strong support for HPV vaccination of older adolescents but substantially less support for recommending the www.annualreviews.org • Cervical Cancer Vaccine

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HPV vaccine acceptability among parents: studies from 2006 and 2007

Year (Reference)

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Outcome measures

Region

Participants

Method

2006 (39)

United Kingdom

Parents of 8–10-year-old children participated in 6 focus groups of 4–5 parents each

Focus groups

Parental knowledge about HPV and attitudes about HPV vaccination for their children

Parents had little awareness of HPV. Parents were confused about the degree of protection against cervical cancer offered by a 16/18 bivalent vaccine. Parents expressed some concerns about possible side effects of vaccination and about a vaccine designed to prevent a sexually transmitted infection. Overall, parents were more comfortable with vaccinating adolescent than preadolescent children.

2007 (40)

California, United States

522 parents with at least one daughter 18 years old or younger

Random-digitdial phone survey

Parental attitudes about vaccinating a daughter against HPV prior to her thirteenth birthday and sixteenth birthday, respectively

75% of parents said they would likely vaccinate a daughter prior to age 13. Attitudes about HPV vaccination varied with ethnicity, race, education, religious service attendance, and political leaning. Parents in favor of vaccination emphasized the importance of protecting children from harm due to HPV infection. Parents opposed to vaccination expressed concerns about vaccine safety and sexual disinhibition.

2007 (41)

Manchester, United Kingdom

305 parents of 11–12-year-old children

Semi-qualitative study

Parental attitudes about provision of HPV vaccine without parental consent

13.8% of parents strongly agreed and 33.8% agreed that HPV vaccine should be provided to adolescents without parental consent. Parents in favor of adolescent self-consent emphasized issues of autonomy and competence. Parents opposed referred to parental rights and emphasized the importance of parental involvement and guidance.

2007 (42)

The Netherlands

356 parents of 10–12-year-old children

Phone interview

Parental knowledge about HPV and attitudes about HPV vaccination for their children

29.5% of parents had heard of HPV and only 14.3% knew of its relationship to cervical cancer. 87.9% said they would have their child vaccinated. 23% thought that their child should be involved in the HPV vaccination decision-making process.

2007 (43)

Finland

727 parents of 15-year-old children

Mailed questionnaire

Parental knowledge about HPV and attitudes about HPV vaccination for their children

79% of parents had heard of HPV. 86% were interested in vaccinating their child against HPV. Parental opposition to vaccination was associated with lower knowledge about HPV, perception that their adolescent was at low risk for infection, and concerns about vaccine safety and sexual disinhibition.

2007 (44)

Da Nang, Vietnam

181 mothers of 10–18-year-old daughters

Questionnaire

Parental attitudes about HPV vaccination for their children

91% of parents said they were likely or very likely to vaccinate their daughters. 90% did not believe that HPV vaccination would lead to earlier initiation of intercourse.

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vaccine for 10- to 12-year-old girls (48). In this same study, pediatricians demonstrated great variability in their knowledge about HPV. Nearly all respondents knew that HPV caused genital warts, but >30% did not know that HPV causes virtually all cervical cancers. A qualitative interview study with 31 pediatricians working in diverse clinical settings found strong interest in HPV vaccination and support for universal age-based recommendations (49). However, respondents noted that cultural and religious factors may inﬂuence parental attitudes about HPV vaccine and some felt that these issues may be difﬁcult for them to address with parents. Participants were mixed in their views about school-entry mandates for HPV vaccine, with about one third expressing support for mandates, one third opposing them, and one third unsure. The studies of providers, like the parent studies, were carried out prior to licensure of the quadrivalent vaccine. Since the vaccine was licensed, a great deal of information has been provided to health care professionals regarding HPV infection and the ACIP recommendation for universal vaccination of all 11- to 12-year-old girls. Multiple studies indicate that providers look to professional organizations for guidance with regard to new vaccines (46, 50, 51). It is likely, therefore, that attitudes about HPV vaccination have shifted toward greater acceptance of vaccinating younger adolescents. Now that HPV vaccine is available, it will be important to study the actual HPV vaccination practices of health care providers.

CONTROVERSIES Even before the quadrivalent HPV vaccine was licensed, some media reports focused on presumed controversies surrounding HPV vaccination (52). However, the scientiﬁc research described in the previous section suggests that for most parents, this vaccine is not, in fact, controversial. In most studies, the large majority of parents have expressed an interest in preventing cervical cancer in their daugh-

ters through vaccination. Research indicates that for the relatively small group of parents with doubts about HPV vaccination, opposition arises from concerns about sexual disinhibition and about implied consent for sexual initiation.

Sexual Disinhibition The issue of sexual disinhibition as it relates to HPV vaccination has been addressed in several articles (36, 53–55). The question is whether HPV vaccination will give 11- to 12-year-old girls a false sense of protection against STIs, leading them to initiate sexual behavior at an earlier age or not use condoms when sexually active. Now that HPV vaccine is licensed and available, carefully designed studies may be able to answer this question directly. To date, it has been addressed only indirectly. The notion of risk compensation or risk homeostasis has supporters and detractors (56, 57), but it is not clear to what extent it applies to HPV vaccination. A basic assumption underlying the compensation or disinhibition argument is that worries about HPV infection have motivated young women to delay initiation of coitus. However, research has repeatedly demonstrated that most young women do not know about HPV, so concerns about HPV could not have had an inhibitory inﬂuence on them. In addition, to the extent that fear of STI does delay the initiation of sexual intercourse, fear of non-vaccine-preventable infections such as HIV, chlamydia, gonorrhea, and genital herpes would remain inhibitory. Several other areas of research provide further indirect evidence that HPV vaccination is not likely to result in disinhibition. Studies on school-based sex education and condom distribution programs and on provision of emergency contraception have found no evidence that these programs have led to earlier initiation of sexual intercourse or engagement in riskier sexual behaviors (58, 59). Another study reviewed research on sexual risk reduction interventions and employed a variety of www.annualreviews.org • Cervical Cancer Vaccine

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mathematical modeling strategies in order to assess the likelihood of sexual risk compensation and the potential effects on HIV/STI risk (60). A small degree of risk compensation was found, but not nearly enough to negate the salutary effects of risk-reduction behaviors (e.g., increased condom use). Issues of risk compensation or disinhibition have also been studied with respect to the introduction of highly active antiretroviral treatment (HAART) for persons with AIDS. Two related questions have been raised: Annu. Rev. Med. 2008.59:223-236. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

1. Do HAART-related reductions in viral load lead HIV-infected individuals to engage in more unprotected sexual behavior? 2. By altering the perception of AIDS from a fatal disease to a chronic, manageable illness, has HAART led HIV-negative but at-risk populations to engage in riskier sexual behavior? Some of the research examining these issues has produced evidence of risk compensation related to HAART or low viral load (61), but most research indicates no association of HAART with increases in risky sexual behaviors (62, 63). In addition, even when the possibility of disinhibition is discussed, there is no suggestion that HAART should be withheld owing to these concerns. Rather, interventions are proposed that would emphasize the importance of maintaining safer sexual behaviors to minimize transmission of HIV, to decrease the probability of exposure to resistant viral strains, and to protect against other STI (64). Implementing this same approach makes sense with respect to HPV vaccination. Recipients of the vaccine and/or their parents should receive anticipatory guidance emphasizing the importance of safe sexual behavior and the ongoing need for regular cervical screening.

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or preadolescent daughter vaccinated against HPV is conveying approval of sexual behavior. This concern has received some media coverage (65), but several arguments suggest that worries about implied permission to have sex are misguided (55). From a practical viewpoint, like any preventive vaccine, HPV vaccine will be maximally effective if administered prior to exposure to the virus. The targeting of 11- to 12-year-olds for vaccination, therefore, makes a great deal of sense because very few girls in this age range have engaged in sexual behaviors (66). In addition, age-based immunization programs have proven to be more effective at maximizing vaccine coverage than risk-based approaches, partly because it is difﬁcult to determine who is at risk. An additional justiﬁcation for targeting younger adolescent girls for HPV vaccination is that studies indicate a stronger immunogenic response in younger compared to older adolescents (23). Finally, it may not be necessary to describe HPV vaccine as an STI vaccine to an 11- or 12-year-old. The modes of transmission of infections targeted by other vaccines are not typically discussed with children and young adolescents. HPV vaccination is a health protective measure designed to reduce the risk of an adverse outcome, cervical cancer. It is similar in this respect to many other widely advocated measures, including use of bicycle helmets, brushing and ﬂossing of teeth, tetanus vaccination, and use of sunscreen. Research suggests that rather than encouraging riskier behaviors, health-protective behaviors tend to co-occur (67). That is, for instance, persons who use seatbelts are also more likely to have a healthier diet. Furthermore, as pointed out by Haber et al. (55), when we have children vaccinated against tetanus, we are not implying that they should play with dirty, rusty nails. Similarly, it should not be assumed that in vaccinating adolescents against HPV we are endorsing earlier involvement in sexual activity. Rather, by vaccinating girls and young women against HPV, we are communicating our desire to protect them from

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the potentially devastating effects of cervical cancer.

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VACCINE DELIVERY CHALLENGES Now that one HPV vaccine is available and a second is likely to become available shortly, we face the logistical and policy challenges of delivering the vaccine to preadolescents and early adolescents. One potentially significant obstacle to delivery is the cost of HPV vaccine. Although most insurance companies have already decided to cover the vaccine, the extent of reimbursement is variable. The Vaccine for Children Program is designed to cover the cost of vaccination for uninsured and underinsured children and certainly will help to decrease disparities in access to HPV vaccine. Financial barriers are likely to pose an even greater problem in developing countries, where the burden of cervical cancer is much greater than in the United States but personal income and national wealth are limited (68). In addition to ﬁnancing issues, the logistical challenges of any vaccine delivery and possible cultural issues related to an STI vaccine may hinder the introduction of HPV vaccine in the developing world (69). A number of authors have discussed some of the unique political, cultural, and organizational challenges associated with the introduction of any new immunization program in developing countries (70–72). The majority of vaccines are given to younger children and are timed around a well-established schedule of health care visits. Vaccinating adolescents is a relatively new phenomenon and one that poses unique challenges (73). For instance, a regular health care visit at age 11 or 12 is not well-established and many providers may have limited experience providing health care services to young adolescents. In order to achieve optimal coverage, alternative approaches to vaccination may need to be explored, including school-based and pharmacy-based programs (73).

One approach to minimizing health care disparities and ensuring that the greatest number of young women are vaccinated would be for states to require HPV vaccination at middle school entry. A number of states have considered implementing mandates, with some states passing and others defeating mandate legislation (74). The issue of compulsory HPV vaccination also has received a great deal of attention in the professional literature, with arguments for and against school entry requirements (55, 75– 77). The controversies surrounding mandatory HPV vaccination and the apparent rush to pass legislation have created a degree of backlash and shifted attention away from the beneﬁts of HPV vaccination to issues of governmental coercion (55, 77). What has been missing, but is beginning to emerge, is an open, reasoned discussion of the pros and cons of school-entry requirements for HPV vaccination.

SUMMARY Multiple large-scale research studies have demonstrated that the bivalent and quadrivalent HPV vaccines are efﬁcacious for at least ﬁve years and safe. Ongoing surveillance studies will evaluate long-term safety, but there is nothing inherent in either HPV vaccine to suggest any long-term safety issues. There is indirect evidence that the protection afforded by HPV vaccine may last as long as 12 years or more. In addition, all modeling studies indicate that vaccination of 11- and 12-year-old girls is a cost-effective strategy. The ACIP recommends vaccination of all 11- to 12-year-old girls, catch-up vaccination of 13- to 26-yearold young women, and vaccination of 9- to 10-year-old girls at the health care provider’s discretion. Studies carried out prior to vaccine licensure indicated that most parents and health care providers had very positive attitudes about HPV vaccination. However, both parents and providers had misconceptions about HPV and HPV vaccine. Although many of www.annualreviews.org • Cervical Cancer Vaccine

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these misunderstandings may have been addressed by educational efforts undertaken since the quadrivalent vaccine was licensed, it will be important for ongoing research to examine parental decisions about HPV vaccination and health care providers’ immunization practices over time. There is no evidence that adolescents will respond to HPV vaccination with sexual disinhibition; however, direct study of this issue can only begin now that the vaccine is available. Although the quadrivalent vaccine has only been licensed since June of 2006, there are al-

ready concerns and controversies regarding vaccine cost and delivery issues. The rapid pursuit of legislation in a number of states to mandate HPV vaccine for girls at middle school entry led to a great deal of criticism and served to distract from the potential enormous beneﬁts of this vaccine. Delivery of HPV vaccine in developing countries, where the need for the vaccine is particularly acute, also is likely to be very difﬁcult. In addition to the cost of the vaccine, unique cultural, political, and logistical challenges will need to be studied and addressed.

FUTURE ISSUES 1. Evaluation of the long-term safety and efﬁcacy of HPV vaccines, including among pregnant women, males, and individuals who are immunosuppressed. 2. Identiﬁcation of predictors of HPV vaccine acceptance and evaluation of brief behavioral interventions designed to optimize HPV vaccine uptake. 3. Assessment of post-HPV-vaccination changes in attitudes and behavior that may be attributable to receipt of vaccine. 4. Development and evaluation of biomedical, political, logistic, and ﬁnancial strategies for world-wide delivery of HPV vaccine, particularly in developing countries.

DISCLOSURE STATEMENT G.D.Z. has received speaking fees from Merck and Co., Inc. and has served as a consultant to M2 Communications and SciMed, two medical education companies. M.L.S. is a clinical investigator for research grants and serves as a consultant for Merck and Co., Inc.

LITERATURE CITED 1. Bryan JT. 2007. Developing an HPV vaccine to prevent cervical cancer and genital warts. Vaccine 25:3001–6 2. Winer RL, Lee S-K, Hughes JP, et al. 2003. Genital human papillomavirus: infection incidence and risk factors in a cohort of female university students. Am. J. Epidemiol. 157:218–26 3. Tarkowski TA, Koumans EH, Sawyer M, et al. 2004. Epidemiology of human papillomavirus infection and abnormal cytologic test results in an urban adolescent population. J. Infect. Dis. 189:46–50 4. Kahn JA, Lan D, Kahn RS. 2007. Sociodemographic factors associated with high-risk HPV infection in a national sample of U.S. women. Obstet. Gynecol. 110:87–95 5. Giuliano AR, Harris R, Sedjo RL, et al. 2002. Incidence, prevalence, and clearance of type speciﬁc human papillomavirus infections: the Young Women’s Health Study. J. Infect. Dis. 186:462–69 232

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6. Dunne EF, Unger ER, Sternberg M, et al. 2007. Prevalence of HPV infection among females in the United States. JAMA 297:813–19 7. Brown DR, Shew ML, Qadadri B, et al. 2005. A longitudinal study of genital human papillomavirus infection in a cohort of closely followed adolescent women. J. Infect. Dis. 191:182–92 8. Bosch FX, Lorincz A, Munoz N, et al. 2002. The causal relation between human papillomavirus and cervical cancer. J. Clin. Pathol. 55:244–65 9. Greer CE, Wheeler CM, Ladner MB, et al. 1995. Human papillomavirus (HPV) type distribution and serological response to HPV type 6 virus-like particles in patients with genital warts. J. Clin. Microbiol. 33:2058–63 10. Moscicki A-B, Schiffman M, Kjaer S, et al. 2006. Chapter 5: Updating the natural history of HPV and anogenital cancer. Vaccine 24(Suppl. 3):42–51 11. Munoz N, Castellsague X, de Gonzalez AB, et al. 2006. Chapter 1: HPV in the etiology of human cancer. Vaccine 24(Suppl. 3):1–10 12. Parkin DM, Bray F, Ferlay J, et al. 2005. Global cancer statistics, 2002. CA Cancer J. Clin. 55:74–108 13. Jemal A, Siegel R, Ward E, et al. 2007. Cancer statistics, 2007. CA Cancer J. Clin. 57:43–66 14. Chesson HW, Blandford JM, Gift TL, et al. 2004. The estimated direct medical cost of sexually transmitted diseases among American youth, 2000. Perspect. Sex. Reprod. Health 36:11–19 15. Villa LL, Costa RL, Petta CA, et al. 2005. Prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 18) L1 virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre phase II efﬁcacy trial. Lancet Oncol. 6:271–78 16. Harper DM, Franco EL, Wheeler C, et al. 2004. Efﬁcacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. Lancet 364:1757–65 17. Harper DM, Franco EL, Wheeler CM, et al. 2006. Sustained efﬁcacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomised control trial. Lancet 367:1247–55 18. Villa LL, Costa RLR, Petta CA, et al. 2006. High sustained efﬁcacy of a prophylactic quadrivalent human papillomavirus types 6/11/16/18 L1 virus-like particle vaccine through 5 years of follow-up. Br. J. Cancer 95:1450–66 19. Koutsky LA, Harper DM. 2006. Chapter 13: Current ﬁndings from prophylactic HPV vaccine trials. Vaccine 24(Suppl. 3):114–21 20. Centers for Disease Control and Prevention. 2007. Quadrivalent human papillomavirus vaccine: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 56(No. RR-2):1–26 21. FUTURE II Study Group. 2007. Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions. N. Engl. J. Med. 356:1915–27 22. Garland SM, Hernandez-Avila M, Wheeler CM, et al. 2007. Quadrivalent vaccine against human papillomavirus to prevent anogenital diseases. N. Engl. J. Med. 356:1928–43 23. Block SL, Nolan T, Sattler C, et al. 2006. Comparison of the immunogenicity and reactogenicity of a prophylactic quadrivalent human papillomavirus (Types 6, 11, 16, and 18) L1 virus-like particle vaccine in male and female adolescents and young adult women. Pediatrics 118:2135–45 24. Fraser C, Tomassini JE, Xi L, et al. 2007. Modeling the long-term antibody response of a human papillomavirus (HPV) virus-like particle (VLP) type 16 prophylactic vaccine. Vaccine 25:4324–33 www.annualreviews.org • Cervical Cancer Vaccine

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25. Olsson S-E, Villa LL, Costa RLR, et al. 2007. Induction of immune memory following administration of a prophylactic quadrivalent human papillomavirus (HPV) types 6/11/16/18 L1 virus-like particle (VLP) vaccine. Vaccine 25:4931–39 26. Ballout A, Gofﬁn E, Yombi JC, et al. 2005. Vaccinations for adult solid organ transplant recipient: current recommendations. Transplant. Proc. 37:2826–27 27. Laurence JC. 2005. Hepatitis A and B immunizations of individuals infected with human immunodeﬁciency virus. Am. J. Med. 118(10A):75S–83S 28. Viscidi RP, Snyder B, Cu-Uvin S, et al. 2005. Human papillomavirus capsid antibody response to natural infection and risk of subsequent HPV infection in HIV-positive and HIV-negative women. Cancer Epidemiol. Biomarkers Prev. 14:283–88 29. Silverberg MJ, Schneider MF, Silver B, et al. 2006. Serological detection of human papillomavirus type 16 infection in human immunodeﬁciency virus (HIV)-positive and high-risk HIV-negative women. Clin. Vaccine Immunol. 13:511–19 30. Kulasingam SL, Myers ER. 2003. Potential health and economic impact of adding a human papillomavirus vaccine to screening programs. JAMA 290:781–89 31. Goldie SJ, Kohli M, Grima D, et al. 2004. Projected clinical beneﬁts and cost-effectiveness of a human papillomavirus 16/18 vaccine. J. Natl. Cancer Inst. 96:604–15 32. Taira AV, Neukermans CP, Sanders GD. 2004. Evaluating human papillomavirus vaccination programs. Emerg. Infect. Dis. 10:1915–23 33. Elbasha EH, Dasbach EJ, Insinga RP. 2007. Model for assessing human papillomavirus vaccination strategies. Emerg. Infect. Dis. 13:28–41 34. Garnett GP, Kim JJ, French K, et al. 2006. Chapter 21: Modelling the impact of HPV vaccines on cervical cancer and screening programmes. Vaccine 24(Suppl. 3):178–86 35. Sanders GD, Taira AV. 2003. Cost effectiveness of a potential vaccine for human papillomavirus. Emerg. Infect. Dis. 9:37–48 36. Zimet GD, Liddon N, Rosenthal SL, et al. 2006. Chapter 24: Psychosocial aspects of vaccine acceptability. Vaccine 24(Suppl. 3):201–9 37. Holcomb B, Bailey JM, Crawford K, et al. 2004. Adults’ knowledge and behaviors related to human papillomavirus infection. J. Am. Board Fam. Pract. 17:26–31 38. Anhang R, Wright TC Jr, Smock L, et al. 2004. Women’s desired information about human papillomavirus. Cancer 100:315–20 39. Noakes K, Yarwood J, Salisbury D. 2006. Parental response to the introduction of a vaccine against human papilloma virus. Hum. Vaccin. 2:243–48 40. Constantine NA, Jerman P. 2007. Acceptance of human papillomavirus vaccination among Californian parents of daughters: a representative statewide analysis. J. Adolesc. Health 40:108–15 41. Brabin L, Roberts SA, Kitchener HC. 2007. A semiqualitative study of attitudes to vaccinating adolescents against human papillomavirus without parental consent. BMC Pub. Health 7:20 42. Lenselink CH, Gerrits MMJG, Melchers WJG, et al. 2008. Parental acceptance of human papillomavirus vaccines. Eur. J. Obstet. Gynecol. Reprod. Biol. In press 43. Woodhall SC, Lehtinen M, Verho T, et al. 2007. Anticipated acceptance of HPV vaccination at the baseline of implementation: a survey of parental and adolescent knowledge and attitudes in Finland. J. Adolesc. Health 40:466-69 44. Dinh TA, Rosenthal SL, Doan ED, et al. 2007. Attitudes of mothers in Da Nang, Vietnam toward a human papillomavirus vaccine. J. Adolesc. Health 40:559–63 45. Kahn JA, Zimet GD, Bernstein DI, et al. 2005. Pediatricians’ intention to administer human papillomavirus vaccine: the role of practice characteristics, knowledge, and attitudes. J. Adolesc. Health 37:502–10

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46. Riedesel JM, Rosenthal SL, Zimet GD, et al. 2005. Attitudes about human papillomavirus vaccine among family physicians. J. Pediatr. Adolesc. Gynecol. 18:391–98 47. Jain N, Irwin KL, Montano D, et al. 2006. Family physicians’ knowledge of genital human papillomavirus (HPV) infection and HPV-related conditions, United States, 2004. Fam. Med. 38:483–89 48. Daley MF, Liddon N, Crane LA, et al. 2006. A national survey of pediatrician knowledge and attitudes regarding human papillomavirus vaccination. Pediatrics 118:2280–89 49. Tissot AM, Zimet GD, Rosenthal SL, et al. 2007. Effective strategies for HPV vaccine delivery: the views of pediatricians. J. Adolesc. Health 41:119–25 50. Mays RM, Zimet GD. 2004. Recommending STI vaccination to parents of adolescents: the attitudes of nurse practitioners. Sex. Trans. Dis. 31:428–32 51. Raley JC, Followwill KA, Zimet GD, et al. 2004. Gynecologists’ attitudes regarding human papilloma virus vaccination: a survey of fellows of the American College of Obstetricians and Gynecologists. Infect. Dis. Obstet. Gynocol. 12:127–33 52. Silverman E. 2005. Cancer vaccine will be a hard sell. Newark Star Ledger, Apr. 3, Business Sec., p. 1 53. Liddon N. 2006. Record of the meeting of the Advisory Committee on Immunization Practices: behavioral issues related to HPV vaccination. http://www.cdc.gov/nip/ACIP/ minutes/acip min feb06.pdf 54. Lo B. 2006. HPV vaccine and adolescents’ sexual activity. BMJ 332:1106–7 55. Haber G, Malow RM, Zimet GD. 2007. The HPV vaccine mandate controversy. J. Pediatr. Adolesc. Gynecol. In press 56. Wilde GJS. 1998. Risk homeostasis theory: an overview. Inj. Prev. 4:89–91 57. O’Neill B, Williams A. 1998. Risk homeostasis hypothesis: a rebuttal. Inj. Prev. 4:92–93 58. Kirby D. 2002. The impact of schools and school programs upon adolescent sexual behavior. J. Sex Res. 39:27–33 59. Raine TR, Harper CC, Rocca CH, et al. 2005. Direct access to emergency contraception through pharmacies and effect on unintended pregnancy and STIs: a randomized controlled trial. JAMA 293:54–62 60. Pinkerton SD. 2001. Sexual risk compensation and HIV/STD transmission: empirical evidence and theoretical considerations. Risk Anal. 21:727–36 61. Van de Ven P, Mao L, Fogarty A, et al. 2005. Undetectable viral load is associated with sexual risk taking in HIV serodiscordant gay couples in Sydney. AIDS 19:179–84 62. Crepaz N, Hart TA, Marks G. 2004. Highly active antiretroviral therapy and sexual risk behavior. JAMA 292:224–36 63. Elford J. 2006. Changing patterns of sexual behaviour in the era of highly active antiretroviral therapy. Curr. Opin. Infect. Dis. 19:26–32 64. Elford J, Hart G. 2005. HAART, viral load and sexual risk behaviour. AIDS 19:205–7 65. Rubin R. 2005. Injected into a controversy. USA Today Oct. 19, p. 8D 66. Abma JC, Martinez GM, Mosher WD, et al. 2004. Teenagers in the United States: sexual activity, contraceptive use, and childbearing, 2002. Vital Health Stat. 23:1–87 67. Fortenberry JD, Costa FM, Jessor R, et al. 1997. Contraceptive behavior and adolescent lifestyles: a structural modeling approach. J. Res. Adolesc. 7:307–29 68. Agosti JM, Goldie SJ. 2007. Introducing HPV vaccine in developing countries—key challenges and issues. N. Engl. J. Med. 356:1908–10 69. Kane MA, Sherris J, Coursaget P, et al. 2006. Chapter 15: HPV vaccine use in the developing world. Vaccine 24(Suppl. 3):132–39 70. Stanton BF. 2004. Assessment of relevant cultural considerations is essential for the success of a vaccine. J. Health Popul. Nutr. 22:286–92 www.annualreviews.org • Cervical Cancer Vaccine

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71. Streeﬂand PH. 2003. Introduction of a HIV vaccine in developing countries: social and cultural dimensions. Vaccine 21:1304–9 72. Gauri V, Khaleghian P. 2002. Immunization in developing countries: its political and organizational determinants. World Dev. 30:2109–32 73. Middleman AB. 2007. Adolescent immunizations: policies to provide a shot in the arm for adolescents. J. Adolesc. Health 41:109–18 74. Charo RA. 2007. Politics, parents, and prophylaxis—mandating HPV vaccination in the United States. N. Engl. J. Med. 356:1905–8 75. Colgrove J. 2006. The ethics and politics of compulsory HPV vaccination. N. Engl. J. Med. 355:2389–91 76. Zimmerman RK. 2006. Ethical analysis of HPV vaccine policy options. Vaccine 24:4812–20 77. Gostin LO, DeAngelis CD. 2007. Mandatory HPV vaccination: public health vs private wealth. JAMA 297:1921–23

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:223-236. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina Cougar Biotechnology, Los Angeles, California 90024; email: [email protected]

Annu. Rev. Med. 2008. 59:237–50

Key Words

First published online as a Review in Advance on July 16, 2007

monoclonal antibody, immunotherapy, CD20 antigen, follicular lymphoma, diffuse large B cell lymphoma

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.060906.220345 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0237$20.00

Abstract The anti-CD20 monoclonal antibody rituximab, ﬁrst approved for clinical use in 1997, has changed the standard of care for many patients with non-Hodgkin’s lymphoma (NHL). Recent data from large randomized clinical trials conﬁrm that the addition of rituximab to standard chemotherapy regimens (chemoimmunotherapy) improves both response rates and survival outcomes in patients with follicular NHL and diffuse large B cell lymphoma (DLBCL), the two most common subtypes of NHL. Population-based analyses have found substantial improvements in NHL survival over the past decade; studies indicate that rituximab has favorably altered the longterm prognosis of follicular NHL and DLBCL patients. This review discusses the clinical development of rituximab-based therapies for patients with low-grade or follicular NHL and newly diagnosed DLBCL, highlighting recent key randomized trials with a focus on survival outcomes.

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INTRODUCTION NHL: non-Hodgkin’s lymphoma

CD20 antigen: transmembrane protein expressed speciﬁcally on B cells, but not found on pre B cells or stem cells Chemoimmunotherapy: use of chemotherapy combined with an immune therapy, such as a monoclonal antibody

Cisplatin (1978)

Rate per 100,000*

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DLBCL: diffuse large B cell lymphoma, a subtype of NHL

Non-Hodgkin’s lymphoma (NHL) is the ﬁfth most frequently diagnosed cancer in the United States. NHL accounts for 4% of all new cancer cases and 3% of all cancer-related deaths in both men and women (1). It is estimated that in 2007 there will be 63,191 new NHL diagnoses and 18,660 NHL-related deaths. Approximately 85% to 90% of all NHL cases in the United States and Western Europe are of B cell origin. Diffuse large B cell lymphoma (DLBCL) and follicular lymphoma are the most common NHL subtypes, accounting for about 31% and 22%, respectively, of new NHL cases (2). Data from the Surveillance, Epidemiology and End Results (SEER) program show that although the incidence of NHL continues to rise in the United States, there has been a signiﬁcant decline in NHL-related mortality over the past decade. From 1991 through 2003, the most recent period for which data are available, the incidence of NHL rose at a rate of 0.4% per year according to jointpoint regression analysis (3). From 1991 through 1997, mortality also rose at an annualized rate of 1.6%. However, from 1997 through 2003, mortality rates declined by 2.8% per year. Although throughout this time NHL

Figure 1 Mortality rates of non-Hodgkin’s lymphoma (NHL) and new drugs approved in the United States for the treatment of NHL.

238

Etoposide (1983)

Fludarabine (1991)

Ibritumomab tiuxetan (2002) Tositumomab Rituximab (2003) (1997)

10

NHL Mortality 5

0 1975

1980

1985

*Age adjusted to 2000 US standard population

Molina

therapies continued to be evaluated and reﬁned, it is interesting that the downward shift in NHL mortality coincided with the introduction of rituximab (Rituxan®, MabThera®) (Figure 1). Rituximab is a chimeric monoclonal IgG1 κ antibody directed against the CD20 antigen, which is expressed on the surface of nearly all malignant and normal B cells (4, 5). Binding of rituximab to the CD20 antigen induces cell lysis and apoptosis, and it also sensitizes cells to the cytotoxic effects of chemotherapy (5–7). In 1997, clinical efﬁcacy with rituximab was ﬁrst reported in patients with relapsed lowgrade B cell NHL (8). These ﬁndings heralded a new era of immunotherapy for the treatment of B cell lymphomas. Since then, the role of rituximab in the treatment of NHL has expanded across both indolent and aggressive subtypes (9). In low-grade or follicular lymphoma, rituximab has been shown to be effective as a single agent and in combination with chemotherapy (chemoimmunotherapy) in patients with newly diagnosed disease and those with relapsed or refractory disease. Findings from recent randomized clinical trials conﬁrm that in newly diagnosed follicular NHL, the addition of rituximab to frontline

1990

1995

2000

2005

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chemotherapy improves response rates as well as progression-free and survival endpoints (10–16). Rituximab maintenance therapy can effectively prolong progression-free survival, although additional clinical trials and longer follow-up are needed to ascertain the impact of maintenance therapy on overall survival (17–21). In DLBCL, data from three randomized trials conﬁrm that the addition of rituximab to CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) or CHOP-like chemotherapy improves both response and survival outcomes in previously untreated patients (22–25). The role of maintenance rituximab in DLBCL is less well deﬁned. In patients who have received rituximab and chemotherapy in the frontline setting, it appears unlikely that maintenance rituximab provides any additional clinical beneﬁt (24). This review focuses on the development of rituximab-based therapies and novel chemoimmunotherapy regimens in patients with low-grade or follicular NHL and newly Table 1

diagnosed DLBCL, highlighting key randomized studies and survival outcomes.

RITUXIMAB IN LOW-GRADE OR FOLLICULAR NHL Single-Agent Rituximab The U.S. Food and Drug Administration’s initial approval of rituximab in November 1997 was for the treatment of relapsed or refractory low-grade or follicular NHL. Overall response rates across phase II studies with a standard course of rituximab, 375 mg/m2 weekly for 4 to 8 doses, typically ranged from 40% to 50% (Table 1), including studies that enrolled patients with bulky or advanced disease (26–31). Median response duration was ∼12 months in most patients, though response duration tended to be shorter in patients with bulky or advanced disease. In studies that enrolled newly diagnosed patients, overall response rates of 70%–80% with median response durations of 18–26 months were seen (32–34).

CHOP: chemotherapy regimen consisting of cyclophosphamide, doxorubicin, vincristine, and prednisone

Selected studies of single-agent rituximab in low-grade or follicular NHLa Patients

References

Response (%)

Population

Rituximab treatment

(n)

CR

OR

Time-to-event outcome

32, 33

follicular NHL, low tumor burden

375 mg/m2 weekly × 4

49

49

80

median PFS = 18 months 5-year RFS = 28%

34

follicular NHL, Grade 1

375 mg/m2 weekly × 4

36

36

72

median TTP = 26 months

6

48

median DR = 11.2 months

Newly diagnosed

Relapsed/refractory 26, 27

low-grade or follicular NHL

375 mg/m2 weekly × 4

166

28

follicular NHL

375 mg/m2 weekly × 4

70

3

46

median DR = 11 months

29

follicular NHL, advanced stage

375 mg/m2 weekly × 4

30

17

47

median DR = 5.8 months

30

low-grade or follicular NHL, bulky disease

375 mg/m2 weekly × 4

31

3

39

median DR = 5.9 months

31

low-grade or follicular NHL

375 mg/m2 weekly × 8

37

14

43

median DR = 13.4+ months

a Abbreviations: NHL, non-Hodgkin’s lymphoma; CR, complete response; OR, overall response, PFS, progression-free survival; TTP, time to progression; DR duration of response.

www.annualreviews.org • Rituximab in Non-Hodgkin’s Lymphoma

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Table 2 Randomized trials of chemotherapy with or without rituximab in newly diagnosed follicular NHLa Response (%) References

Treatment

Patients (n)

CR/CRub

OR

Overall survival

10, 11

R-CVP CVP

162 159

41 10

81 57

4-year

83% 77%

0.029

12

R-CHOP CHOP

223 205

20a 17a

96 90

2-year

95% 90%

0.016

14

R-MCP MCP

105 96

50 25

92 75

4-year

87% 74%

0.0096

15, 16

R-CHVP-αIFN CHVP-αIFN

175 183

79 63

84 73

3.5-year

91% 84%

0.029

p

a

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Abbreviations: R = rituximab; CVP = cyclophosphamide, vincristine, prednisone; CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone; MCP = mitoxantrone, chlorambucil, prednisolone; CVHP-αIFN = cyclophosphamide, doxorubicin, etoposide, prednisone, plus α-interferon; CR/CRu = complete response/complete response unconﬁrmed; OR = overall response. b Patients meeting CR criteria without conﬁrmed negative bone marrow biopsy were deﬁned as partial responders instead of CRu.

Chemoimmunotherapy

CR/CRu: complete response/complete response unconﬁrmed

240

Long-term follow-up has found a signiﬁcant improvement in overall survival with the addition of rituximab to chemotherapy across four randomized trials enrolling patients with newly diagnosed advanced follicular lymphoma (Table 2) (10–16). Marcus et al. (10, 11) recently reported that adding rituximab to CVP (cyclophosphamide, vincristine, prednisone) chemotherapy increased both complete and overall response rates and more than doubled the median time to disease progression (34 months versus 15 months) compared to CVP alone ( p < 0.0001). The four-year disease-free survival rate for patients with a complete response (CR or CRu) was 54% with R-CVP compared to 17% with CVP ( p = 0.0001). The addition of rituximab also signiﬁcantly improved overall survival. With a median follow-up time of 53 months, four-year estimated overall survival rates were 83% with R-CVP versus 77% with CVP ( p = 0.029). Hiddemann et al. (12) found that the addition of rituximab to CHOP resulted in a signiﬁcantly higher overall response rate— 96% versus 90% with CHOP alone ( p = 0.011). Time to treatment failure (TTF) was also longer with R-CHOP. Although the meMolina

dian follow-up time of 18 months was relatively short, there was a statistical difference in TTF, with the median not yet reached with R-CHOP compared to 2.6 years for CHOP alone ( p < 0.001). Only 28 treatment failures occurred with R-CHOP compared to 61 with CHOP. Estimated 2-year overall survival rates were 95% with R-CHOP and 90% with CHOP ( p = 0.016), with 6 deaths in the R-CHOP group compared to 17 deaths in the CHOP group within the ﬁrst 3 years. The addition of rituximab to MCP (mitoxantrone, chlorambucil, prednisolone) chemotherapy also improved response rates and survival times (13, 14). In patients with advanced follicular lymphoma, the overall response rate with R-MCP was 92% compared to 75% with MCP ( p = 0.0004), with complete response rates of 50% and 25%, respectively ( p = 0.0009). With a median followup time of 47 months, R-MCP resulted in longer progression-free survival ( p < 0.0001), with an estimated four-year progression-free survival rate of 71% versus 41% with MCP. Overall survival also favored R-MCP, with estimated four-year overall survival rates of 87% versus 74% ( p = 0.0096). Foussard and colleagues (15, 16) found that the addition of rituximab to

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a combination regimen of CHVP-αIFN (cyclophosphamide, doxorubicin, etoposide, prednisone, followed by α2-interferon) also improved response and survival outcomes. For R-CHVP-αIFN the overall response rate was 84% versus 73% for CHVP-αIFN ( p = 0.004). With a median follow-up time of 3.5 years, event-free survival and overall survival outcomes also signiﬁcantly favored the addition of rituximab. Comparative estimated event-free survival rates were 81% versus 62% ( p = 0.002), and overall survival rates were 91% versus 84% ( p = 0.029). An ongoing randomized trial is comparing rituximab alone to chemotherapy with CNOP (cyclophosphamide, mitoxantrone, vincristine, and prednisone) and chemoimmunotherapy with CNOP plus rituximab in patients with newly diagnosed indolent lymphoma. Preliminary results from 195 patients indicate no statistical difference in either overall response rates (85% for rituximab, 83% for CNOP, and 90% for R-CNOP) or two-year overall survival rates (87%, 84%, and 78%, respectively) (35). Further details of this study and additional follow-up time are needed to more fully evaluate these results. An overall survival beneﬁt from the addition of rituximab to chemotherapy in patients with indolent NHL was also identiﬁed in a meta-analysis conducted by the Cochrane Hematological Malignancies Group (36). Data from six randomized clinical trials of chemotherapy with or without rituximab in patients with newly diagnosed or relapsed/refractory indolent or mantle cell lymphoma were included (10, 12, 13, 35, 37, 38). Overall survival, the primary study parameter, was evaluated using data from 994 patients enrolled across ﬁve trials. This analysis found that adding rituximab to chemotherapy signiﬁcantly improved survival compared to chemotherapy alone, with a hazard ratio of 0.61 (95% CI: 0.47–0.80).

Rituximab Maintenance The use of maintenance rituximab can improve disease control in patients with indolent

or follicular NHL. Results of randomized studies suggest clinical beneﬁt from several different rituximab extended-dosing and maintenance strategies following singleagent rituximab therapy, chemotherapy, or chemoimmunotherapy. In a phase III trial, follicular NHL patients with responding or stable disease following a standard course of rituximab were randomized either to maintenance rituximab, given as a single dose at week 12 and months 5, 7, and 9, or to observation (17). Among 151 patients, maintenance rituximab produced signiﬁcant improvements in both response duration, 36 months versus 16 months ( p = 0.0039), and event-free survival time, 23 months versus 12 months ( p = 0.02), compared to observation. Hainsworth et al. (18) evaluated maintenance rituximab in 90 patients with indolent NHL who had initially responsive or stable disease. A standard course of rituximab, repeated at six-month intervals, was compared to retreatment with rituximab at the time of disease progression. The use of maintenance rituximab increased the overall response rate from 39% following initial therapy to 52%. Time to disease progression was 31 months with maintenance rituximab versus 7 months without ( p = 0.007). Hochster et al. evaluated maintenance rituximab following chemotherapy in patients with newly diagnosed advanced follicular NHL (19). This phase III trial enrolled 237 patients with responding or stable disease following CVP chemotherapy, who were randomized either to maintenance rituximab, given as four weekly doses every six months for two years, or to observation. Both time to disease progression and overall survival were signiﬁcantly prolonged with maintenance rituximab (Table 3). Comparative four-year progression-free survival rates were 56% versus 33% ( p < 0.0001), and four-year overall survival rates were 88% versus 72% ( p = 0.03). Other investigators reported that maintenance rituximab improved response and survival outcomes in patients with relapsed

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Table 3 Randomized trials of maintenance rituximab following chemotherapy or chemoimmunotherapy in low-grade or follicular NHLa Treatment References

Population

Initial

Maintenance

Patients (n)

Overall survival

p

19

newly diagnosed advanced stage follicular NHL

CVP

R Obs.

237

4-year

88% 72%

0.03

20

relapsed/refractory follicular NHL

R-CHOP CHOP

R Obs.

334

3-year

85% 77%

0.011

21

relapsed/refractory follicular and mantle cell lymphoma

R-FCM FCM

R Obs.

176

3-year

77% 57%

0.10

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a Abbreviations: R = rituximab; CVP = cyclophosphamide, vincristine, prednisone; CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone; FCM = ﬂudarabine, cyclophosphamide, mitoxantrone; Obs. = observation.

or refractory follicular NHL initially treated with CHOP alone or CHOP with rituximab (Table 3) (20). Following an initial randomization to either CHOP or R-CHOP, responding patients underwent a second randomization either to maintenance rituximab, given as a single dose every three months for two years, or to observation. With initial therapy, the addition of rituximab to CHOP signiﬁcantly improved both the overall response rate, 85% versus 72% ( p < 0.001), and the median progression-free survival time, 33 months versus 20 months ( p < 0.001). With a median follow-up time of 33 months from the second randomization, median progression-free survival was more than three times longer with rituximab maintenance— 52 months compared to 15 months with observation ( p < 0.0001). Progression-free survival improved with maintenance rituximab following both CHOP and R-CHOP. Overall survival also improved with maintenance rituximab; three-year overall survival rates (from second randomization) were 85% versus 77% ( p = 0.011). Forstpointner et al. evaluated maintenance rituximab following FCM (ﬂudarabine, cyclophosphamide, mitoxantrone) chemotherapy with or without rituximab in patients with relapsed or refractory follicular or mantle cell lymphoma (21). The initial treatment randomization of R-FCM versus FCM was stopped after the ﬁrst 147 patients upon recognition of a signiﬁcantly higher overall 242

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response rate, 79% versus 58% ( p = 0.01), and longer progression-free survival ( p = 0.014) with R-FCM (37). All patients enrolled thereafter received R-FCM as initial therapy. Responding patients underwent a second randomization either to maintenance rituximab, given as four weekly doses at three months and nine months, or to observation. Duration of response was signiﬁcantly longer with maintenance rituximab. With a median follow-up time of 26 months, median time to disease progression had not been reached in the maintenance group compared to 17 months for observation ( p < 0.001). Overall survival rates at three years were 77% with maintenance rituximab and 57% without, although this difference did not reach statistical signiﬁcance. Emerging evidence suggests a potential role for maintenance rituximab in patients with low-grade or follicular NHL. However, in the above studies, very different maintenance rituximab strategies were used, and an optimal rituximab maintenance schedule and duration of treatment remain to be deﬁned. Different frontline modalities were also used, and results of these studies need to be interpreted in light of evolving standards of care.

RITUXIMAB IN AGGRESSIVE NHL In patients with newly diagnosed DLBCL, the addition of rituximab to CHOP or CHOPlike chemotherapy has been shown to improve

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Randomized trials of chemotherapy with or without rituximab in newly diagnosed DLBCLa Response (%)

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References

Population

Treatment

Patients (n)

CR/CRu

OR

Overall survival

75 63

82 69

5-year

58% 45%

0.0073

77 76

3-yearb

67% 58%

0.05

3-year

93% 84%

0.0001

22, 23

newly diagnosed advanced DLBCL, age 60–80 years

R-CHOP CHOP

399

24

newly diagnosed DLBCL, age ≥ 60 years

R-CHOP CHOP → R versus Obs.

546

25

newly diagnosed, good prognosis DLBCL, age 18–60 years

R-CHOP-likec CHOP-likec

823

86 68

p

a DLBCL = diffuse large B cell lymphoma; R = rituximab; Obs. = observation; CR/CRu = complete response/complete response unconﬁrmed; OR = overall response. b Secondary analysis of induction therapy without maintenance rituximab. c CHOP-like chemotherapy included CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), CHOEP (cyclophosphamide, doxorubicin, vincristine, etoposide, prednisone), MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin), and PMitCEMO (mitoxantrone, cyclophosphamide, etoposide, vincristine, bleomycin, prednisone).

response and survival outcomes across three randomized trials (Table 4). These data were used to support an extension of rituximab’s approved indications by the U.S. Food and Drug Administration in 2006. In the seminal study coordinated by the Groupe d’Etudes des Lymphomes de l’Adulte (GELA), the addition of rituximab to CHOP signiﬁcantly improved response rate and survival times in newly diagnosed DLBCL patients aged 60–80 years (22). The overall response rate with R-CHOP was 82% compared to 69% with CHOP alone ( p = 0.005). With longterm follow-up, progression-free survival outcomes continued to signiﬁcantly favor R-CHOP ( p < 00001), with comparative ﬁve-year progression-free survival rates of 54% versus 30% (23). Overall survival was also longer with R-CHOP, with comparative 5-year overall survival rates of 58% versus 45% ( p = 0.0073). Another trial has also compared R-CHOP to CHOP in newly diagnosed DLBCL patients aged ≥60 years (24). This phase III US Intergroup trial differed in design from the GELA trial in that patients who responded to initial therapy underwent a sec-

ond randomization to maintenance rituximab or observation. In contrast to the GELA trial, there was no difference in overall response following initial therapy, with rates of 77% and 76% with R-CHOP and CHOP, respectively. The lack of difference in response rates between the two study arms of the US Intergroup trial may be related to the requirement for the use of fewer cycles of chemotherapy, fewer rituximab treatments, and a different schedule of rituximab administration, as well as enrollment of a higher perecntage of high-risk patients compard to the GELA trial. Another potential contibuting factor was the requirement for conﬁrmation of responses at four weeks or later and the timing of second randomization within that period. Survival analyses from this study were relatively complex. However, with a median follow-up time of 3.5 years, the estimated three-year progression-free survival rate was longer for R-CHOP compared to CHOP induction, 53% versus 46% ( p = 0.04). Because of the potential confounding effects of maintenance rituximab on the induction comparison, an inverse probability-weighted statistical analysis was performed to compare

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induction regimens in the absence of maintenance rituximab. In this secondary analysis, overall survival was signiﬁcantly longer with R-CHOP; the estimated three-year overall survival rate was 67% with R-CHOP compared to 58% with CHOP alone ( p = 0.05). This trial also evaluated maintenance rituximab following either CHOP or R-CHOP. There was a signiﬁcant difference in the effect of maintenance therapy according to which induction therapy was given; maintenance rituximab prolonged failure-free survival following CHOP but not R-CHOP ( p = 0.05). After the second randomization, estimated two-year failure-free survival rates were as follows: 79% for R-CHOP plus maintenance, 77% for R-CHOP alone, 74% for CHOP plus maintenance, and 45% for CHOP alone. Overall, these ﬁndings indicate that the use of rituximab either as part of initial therapy or as maintenance therapy in responding patients signiﬁcantly prolongs failure-free survival ( p < 0.001). These ﬁndings further indicate that use of maintenance rituximab following initial chemoimmunotherapy that includes rituximab is unlikely to provide additional beneﬁt. In younger patients with good-prognosis DLBCL, response and survival beneﬁts from the addition of rituximab to CHOP or CHOP-like chemotherapy have recently been reported (25). This phase III study enrolled newly diagnosed DLBCL patients aged 18– 60 with zero or one risk factor according to the age-adjusted International Prognostic Index (IPI) (39). Patients were randomized to receive frontline chemotherapy (one of four regimens selected by the participating institution) with or without rituximab. Most patients (92%) received CHOP or CHOEP (CHOP plus etoposide). Chemotherapy plus rituximab produced a higher rate of CR/CRu than chemotherapy alone, 86% versus 68% ( p < 0.0001). With a median follow-up time of 34 months, patients who received rituximab with chemotherapy had signiﬁcantly longer progression-free and overall survival times. Comparative three-year progression-free sur-

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CLL: chronic lymphocytic leukemia

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vival estimates were 85% versus 68% ( p < 0.0001), and three-year overall survival estimates were 93% versus 84% ( p = 0.0001). Improved clinical outcomes with the addition of rituximab were seen with both CHOP and CHOEP regimens.

DISCUSSION Rituximab has become an integral component in the management of many patients with NHL. Treatment guidelines established by the National Comprehensive Cancer Network (NCCN) suggest rituximab-based therapies for patients with follicular NHL and DLBCL, and they indicate that rituximab is an optional component of therapy for other forms of NHL, including chronic lymphocytic leukemia (CLL), marginal zone lymphoma, and mantle cell lymphoma (40). Newer treatment options, and in particular the introduction of rituximab, appear to have improved the long-term prognosis of patients with follicular NHL and DLBCL. Across a series of studies conducted in patients with follicular NHL over the past three decades by the Southwest Oncology Group (SWOG), four-year overall survival estimates have improved from 69% with CHOP alone to 91% with CHOP plus monoclonal antibody therapy ( p < 0.001) (41). Substantial improvements in survival outcomes in patients with follicular NHL have also been reported by the Gruppo Italiano Studio Linfomi (GISL) and these are thought to be related primarily to the use of rituximab in combination with chemotherapy (42). The results of recent phase III randomized trials further support an improving prognosis. In newly diagnosed follicular NHL patients, the combination of rituximab with CVP, CHOP, MCP, and CHVPαIFN increases response rates and prolongs progression-free and overall survival times (11, 12, 14, 16). In patients with relapsed or refractory disease, combinations of rituximab with CHOP or FCM have improved response rate and time to disease progression (20, 37).

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Figure 2

PRIMA

Rituximab x 8 in combination with

Maintenance Rituximab x 1 every 8 weeks for 24 months

CVP x 8 cycles CR/PR

CHOP x 6 cycles FCM x 6 cycles

3-year follow-up

Observation for 24 months

N = 1200 patients

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Enrollment: previously untreated stage III/IV follicular NHL (Grade 1, 2, or 3 by World Health Organization (WHO) criteria)

Maintenance rituximab can extend response duration and time to disease progression following rituximab monotherapy, chemotherapy, or chemoimmunotherapy (17–21). Overall survival beneﬁts with maintenance rituximab have been seen in some randomized trials (19, 20), although additional trials and follow-up are needed to conﬁrm a longterm clinical beneﬁt. Additional information on the beneﬁt of maintenance rituximab following chemoimmunotherapy for patients with previously untreated follicular NHL will be provided by the Primary Rituximab and Maintenance (PRIMA) trial (Figure 2). The trial is coordinated by GELA and is being conducted at sites primarily in Europe and Australia. The primary study endpoint is event-free survival, with response, progression-free survival, and overall survival evaluated as secondary endpoints. Accrual of 1200 patients was recently completed. In patients with DLBCL, the addition of rituximab to CHOP or CHOP-like chemotherapy has improved response and survival outcomes in newly diagnosed patients older than 60 years as well as younger patients with good prognostic features in phase III trials (22–25). A population-based analysis conducted in British Columbia supports these ﬁndings, noting a substantial survival beneﬁt with the addition of rituximab to CHOP across all age groups (43). The role of maintenance rituximab in patients with DLBCL

Study design of the PRIMA trial, evaluating maintenance rituximab versus observation following frontline therapy with rituximab combined with one of three chemotherapy regimens in patients with newly diagnosed follicular NHL.

is less clear, but it seems unlikely that it will provide any further beneﬁt to those who have received rituximab as part of their initial therapy. The improved survival outcomes in DLBCL resulting from the combination of rituximab with CHOP or CHOP-like regimens have prompted an adjustment to the IPI classiﬁcation to provide a clinically more useful tool (44). The revised International Prognostic Index (R-IPI) maintains the same baseline criteria but identiﬁes three prognostic groups: very good (0 risk factors), good (1 or 2 risk factors), and poor (3 to 5 risk factors). Estimated survival rates at four years using RIPI are 94%, 79%, and 53%, respectively. It is interesting that the IPI no longer identiﬁes a group with a risk rate below 50%, again suggesting that the prognosis for these patients has improved. An increase in adverse events is always a concern with the addition of any new agent to an established regimen. However, across these phase III trials, the addition of rituximab did not substantially increase adverse event rates in either follicular NHL or DLBCL patients. Although one study reported an increase in severe granulocytopenia with R-CHOP compared to CHOP (12), severe infections were rare and not different between study arms. Most of the trials found the addition of rituximab to chemotherapy to be generally well tolerated, and no clinically meaningful differences in toxicities were seen.

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REACH Enrollment: • Active CLL • Binet A, B, C • Fludarabine sensitive

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Figure 3 Study design of the REACH trial, evaluating chemotherapy with or without rituximab for patients with CLL.

N = 620 patients The use of rituximab in other B cell NHL subtypes, including CLL, mantle cell lymphoma, and Waldenstrom macroglobulinemia, as well as Hodgkin lymphoma and posttransplant lymphoproliferative disorders, is currently under investigation. Indications of response and potential survival improvements have been seen in phase II studies of rituximab in combination with chemotherapy for patients with newly diagnosed or relapsed/refractory CLL (45–48). These ﬁndings are the basis of two ongoing randomized

Fludarabine Cyclophosphamide Rituximab x 6 courses Fludarabine Cyclophosphamide x 6 courses clinical trials, one in newly diagnosed CLL patients and the other in patients with relapsed or refractory disease, which will evaluate chemotherapy with ﬂudarabine and cyclophosphamide with or without rituximab. Accrual goals have been met for the former study, with completion of accrual for the latter expected by the end of 2007. Both studies use the same design (Figure 3), with progression-free survival as the primary endpoint. Preliminary results are anticipated in 2008.

SUMMARY POINTS 1. In newly diagnosed low-grade or follicular NHL, the addition of rituximab to chemotherapy signiﬁcantly improves overall response rates and disease control. It may also improve overall survival. 2. In relapsed or refractory low-grade or follicular NHL, the addition of rituximab to chemotherapy signiﬁcantly improves overall response rates and disease control. 3. The use of maintenance rituximab in patients with follicular NHL extends progression-free survival, and additional clinical studies are ongoing to assess impact on overall survival. 4. In newly diagnosed DLBCL, the addition of rituximab to CHOP or CHOP-like chemotherapy signiﬁcantly improves overall response rates, disease control, and overall survival. 5. In DLBCL patients who have received rituximab as part of frontline chemoimmunotherapy, it is unlikely that maintenance rituximab offers any additional beneﬁt.

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FUTURE ISSUES 1. The clinical beneﬁt of maintenance rituximab therapy following initial treatment with rituximab chemoimmunotherapy combinations in patients with previously untreated follicular lymphoma is not yet well deﬁned. 2. The optimum schedule and duration of maintenance rituximab has yet to be deﬁned. 3. It is not yet known if chronic rituximab therapy may lead to selection of rituximabresistant cells.

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4. Will second- and third-generation anti-CD20 antibodies, currently under development, further improve clinical outcomes?

DISCLOSURE STATEMENT The author was employed at Biogen Idec when this manuscript was submitted for publication. Rituximab was discovered at Idec prior to the Biogen Idec merger. It is currently comarketed as Rituxan® by Genentech, Inc. and Biogen Idec in the United States, and as MabThera® by Roche outside the United States.

ACKNOWLEDGMENTS The author thanks Christine Gutheil for editorial assistance. The author also expresses great appreciation to all of the patients who participated in clinical trials of rituximab.

LITERATURE CITED 1. American Cancer Society. 2007. Cancer Facts & Figures 2007. Atlanta, GA: Am. Cancer Soc. 2. The Non-Hodgkin’s Lymphoma Classiﬁcation Project. 1997. A clinical evaluation of the International Lymphoma Study Group classiﬁcation of non-Hodgkin’s lymphomas. Blood 89:3909–18 3. Ries LAG, Harkins D, Krapcho M, et al. 2006. SEER Cancer Statistics Review, 1975– 2003. Based on Nov. 2005 SEER data submission. Bethesda, MD: Nat. Cancer Inst., http://seer.cancer.gov/csr/1975 2003/ 4. Anderson KC, Bates MP, Slaughenhoupt BL, et al. 1984. Expression of human B cellassociated antigens on leukemias and lymphomas: a model of human B cell differentiation. Blood 63:1424–33 5. Reff ME, Carner K, Chambers KS, et al. 1994. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood 83:435–45 6. Flieger D, Renoth S, Beier I, et al. 2000. Mechanism of cytotoxicity induced by chimeric mouse human monoclonal antibody IDEC-C2B8 in CD20-expressing lymphoma cell lines. Cell Immunol. 204:55–63 7. Demidem A, Lam T, Alas S, et al. 1997. Chimeric anti-CD20 (IDEC-C2B8) monoclonal antibody sensitizes a B cell lymphoma cell line to cell killing by cytotoxic drugs. Cancer Biother. Radiopharm. 12:177–85 ´ 8. Maloney DG, Grillo-Lopez AJ, White CA, et al. 1997. IDEC-C2B8 (rituximab) antiCD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin’s lymphoma. Blood 90:2188–95 www.annualreviews.org • Rituximab in Non-Hodgkin’s Lymphoma

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9. Cvetkovic RS, Perry CM. 2006. Rituximab: a review of its use in non-Hodgkin’s lymphoma and chronic lymphocytic leukaemia. Drugs 66:791–820 10. Marcus R, Imrie K, Belch A, et al. 2005. CVP chemotherapy plus rituximab compared with CVP as ﬁrst-line treatment for advanced follicular lymphoma. Blood 105:1417–23 11. Marcus RE, Solal-Celigny P, Imrie K, et al. 2006. MabThera (rituximab) plus cyclophosphamide, vincristine and prednisone (CVP) chemotherapy improves survival in previously untreated patients with advanced follicular non-Hodgkin’s lymphoma. Blood ASH Annu. Meet. Abstr. 108:481 12. Hiddemann W, Kneba M, Dreyling M, et al. 2005. Frontline therapy with rituximab added to the combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) signiﬁcantly improves the outcome for patients with advanced-stage follicular lymphoma compared with therapy with CHOP alone: results of a prospective randomized study of the German Low-Grade Lymphoma Study Group. Blood 106:3725–32 13. Herold M, Dolken G, Fiedler F, et al. 2003. Randomized phase III study for the treatment of advanced indolent non-Hodgkin’s lymphomas (NHL) and mantle cell lymphoma: chemotherapy versus chemotherapy plus rituximab. Ann. Hematol. 82:77–79 14. Herold M, Haas A, Srock S, et al. 2007. Rituximab added to ﬁrst-line mitoxantrone, chlorambucil, and prednisolone chemotherapy followed by interferon maintenance prolongs survival in patients with advanced follicular lymphoma: an East German Study Group Hematology and Oncology study. J. Clin. Oncol. 251986–92 15. Salles GA, Foussard C, Mounier N, et al. 2004. Rituximab added to αIFN+CHVP improves the outcome of follicular lymphoma patients with a high tumor burden: ﬁrst analysis of the GELA-GOELAMS FL-2000 randomized trial in 359 patients. Blood ASH Annu. Meet. Abstr. 104:160 (Abstr.) 16. Foussard C, Mounier N, Van Hoof A, et al. 2006. Update of the FL2000 randomized trial combining rituximab to CHVP-interferon in follicular lymphoma (FL) patients (pts). J. Clin. Oncol. ASCO Annu. Meet. Proc. 24:7508 (Abstr.) 17. Ghielmini M, Schmitz SF, Cogliatti SB, et al. 2004. Prolonged treatment with rituximab in patients with follicular lymphoma signiﬁcantly increases event-free survival and response duration compared with the standard weekly × 4 schedule. Blood 103:4416–23 18. Hainsworth JD, Litchy S, Shaffer DW, et al. 2005. Maximizing therapeutic beneﬁt of rituximab: maintenance therapy versus retreatment at progression in patients with indolent non-Hodgkin’s lymphoma—a randomized phase II trial of the Minnie Pearl Cancer Research Network. J. Clin. Oncol. 23:1088–95 19. Hochster HS, Weller E, Gascoyne RD, et al. 2005. Maintenance rituximab after CVP results in superior clinical outcome in advanced follicular lymphoma (FL): results of the E1496 phase III trial from the Eastern Cooperative Oncology Group and the Cancer and Leukemia Group B. Blood ASH Annu. Meet. Abstr. 106:349 (Abstr.) 20. van Oers MH, Klasa R, Marcus RE, et al. 2006. Rituximab maintenance improves clinical outcome of relapsed/resistant follicular non-Hodgkin lymphoma in patients both with and without rituximab during induction: results of a prospective randomized phase 3 intergroup trial. Blood 108:3295–301 21. Forstpointner R, Unterhalt M, Dreyling M, et al. 2006. Maintenance therapy with rituximab leads to a signiﬁcant prolongation of response duration after salvage therapy with a combination of rituximab, ﬂudarabine, cyclophosphamide, and mitoxantrone (R-FCM) in patients with recurring and refractory follicular and mantle cell lymphomas: results of a prospective randomized study of the German Low Grade Lymphoma Study Group (GLSG). Blood 108:4003–8

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22. Coifﬁer B, LePage E, Briere J, et al. 2002. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N. Engl. J. Med. 346:235–42 23. Feugier P, Van Hoof A, Sebban C, et al. 2005. Long-term results of the R-CHOP study in the treatment of elderly patients with diffuse large B-cell lymphoma: a study by the Groupe d’Etudes des Lymphomes de l’Adulte. J. Clin. Oncol. 23:4117–26 24. Habermann TM, Weller EA, Morrison VA, et al. 2006. Rituximab-CHOP versus CHOP alone or with maintenance rituximab in older patients with diffuse large B-cell lymphoma. J. Clin. Oncol. 24:3121–27 25. Pfreundschuh M, Trumper L, Osterborg A, et al. 2006. CHOP-like chemotherapy plus rituximab versus CHOP-like chemotherapy alone in young patients with good-prognosis diffuse large-B-cell lymphoma: a randomised controlled trial by the MabThera International Trial (MInT) Group. Lancet Oncol. 7:379–91 ´ 26. McLaughlin P, Grillo-Lopez AJ, Link BK, et al. 1998. Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program. J. Clin. Oncol. 16:2825–33 ´ 27. McLaughlin P, Hagemeister FB, Grillo-Lopez AJ. 1999. Rituximab in indolent lymphoma: the single-agent pivotal trial. Semin. Oncol. 26(5 Suppl. 14):79–87 28. Foran JM, Gupta RK, Cunningham D, et al. 2000. A UK multicentre phase II study of rituximab (chimaeric anti-CD20 monoclonal antibody) in patients with follicular lymphoma, with PCR monitoring of molecular response. Br. J. Haematol. 109:81–88 29. Feuring-Buske M, Kneba M, Unterhalt M, et al. 2000. IDEC-C2B8 (rituximab) anti-CD20 antibody treatment in relapsed advanced-stage follicular lymphomas: results of a phase-II study of the German Low-Grade Lymphoma Study Group. Ann. Hematol. 79:493–500 ´ 30. Davis TA, White CA, Grillo-Lopez AJ, et al. 1999. Single-agent monoclonal antibody efﬁcacy in bulky non-Hodgkin’s lymphoma: results of a phase II trial of rituximab. J. Clin. Oncol. 17:1851–57 ´ 31. Piro LD, White CA, Grillo-Lopez AJ, et al. 1999. Extended rituximab (anti-CD20 monoclonal antibody) therapy for relapsed or refractory low-grade or follicular non-Hodgkin’s lymphoma. Ann. Oncol. 10:655–61 32. Colombat P, Salles G, Brousse N, et al. 2001. Rituximab (anti-CD20 monoclonal antibody) as single ﬁrst-line therapy for patients with follicular lymphoma with a low tumor burden: clinical and molecular evaluation. Blood 97:101–6 33. Solal-Celigny P, Salles GA, Brousse N, et al. 2004. Single 4-dose rituximab treatment for low-tumor burden follicular lymphoma: survival analyses with a follow-up of at least 5 years. Blood ASH Annu. Meet. Abstr. 104:585 (Abstr.) 34. Witzig TE, Vukov AM, Habermann TM, et al. 2005. Rituximab therapy for patients with newly diagnosed, advanced-stage, follicular grade I non-Hodgkin’s lymphoma: a phase II trial in the North Central Cancer Treatment Group. J. Clin. Oncol. 23:1103–8 35. Rivas-Vera S, Baez E, Sobrevilla-Calvo P, et al. 2005. Is ﬁrst line single agent rituximab the best treatment for indolent non-Hodgkin’s lymphoma? Update of a multicentric study comparing rituximab vs CNOP vs rituximab plus CNOP. Blood ASH Annu. Meet. Abstr. 106:2431 (Abstr.) 36. Schulz H, Skoetz N, Bohlius J, et al. 2005. Does combined immunochemotherapy with the monoclonal antibody rituximab improve overall survival in the treatment of patients with indolent non-Hodgkin lymphoma? Preliminary results of a comprehensive meta-analysis. Blood ASH Annu. Meet. Abstr. 106:351 (Abstr.) www.annualreviews.org • Rituximab in Non-Hodgkin’s Lymphoma

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37. Forstpointner R, Dreyling M, Repp R, et al. 2004. The addition of rituximab to a combination of ﬂudarabine, cyclophosphamide, mitoxantrone (FCM) signiﬁcantly increases the response rate and prolongs survival as compared with FCM alone in patients with relapsed and refractory follicular and mantle cell lymphomas: results of a prospective randomized study of the German Low-Grade Lymphoma Study Group. Blood 104:3064–71 38. Lenz G, Dreyling M, Hoster E, et al. 2005. Immunochemotherapy with rituximab and cyclophosphamide, doxorubicin, vincristine, and prednisone signiﬁcantly improves response and time to treatment failure, but not long-term outcome in patients with previously untreated mantle cell lymphoma: results of a prospective randomized trial of the German Low Grade Lymphoma Study Group (GLSG). J. Clin. Oncol. 23:1984–92 39. The International Non-Hodgkin’s Lymphoma Prognostic Factors Project. 1993. A predictive model for aggressive non-Hodgkin’s lymphoma. N. Engl. J. Med. 329:987–94 40. National Comprehensive Cancer Network, Inc. 2007. NCCN clinical practice guidelines in oncology—Version 1.2007. Non-Hodgkin’s lymphomas. http://www.nccn.org/ professionals/physician gls/PDF/nhl.pdf 41. Fisher RI, LeBlanc M, Press OW, et al. 2005. New treatment options have changed the survival of patients with follicular lymphoma. J. Clin. Oncol. 23:8447–52 42. Sacchi S, Pozzi S, Marcheselli L, et al. 2007. Introduction of rituximab in front-line and salvage therapies has improved outcome of advanced-stage follicular lymphoma patients. Cancer 109:2077–82 43. Sehn LH, Donaldson J, Chhanabhai M, et al. 2005. Introduction of combined CHOP plus rituximab therapy dramatically improved outcome of diffuse large B-cell lymphoma in British Columbia. J. Clin. Oncol. 23:5027–33 44. Sehn LH, Berry B, Chhanabhai M, et al. 2007. The revised International Prognostic Index (R-IPI) is a better predictor of outcome than the standard IPI for patients with diffuse large B-cell lymphoma treated with R-CHOP. Blood 109:1857–61 45. Byrd JC, Rai K, Peterson BL, et al. 2005. Addition of rituximab to ﬂudarabine may prolong progression-free survival and overall survival in patients with previously untreated chronic lymphocytic leukemia: an updated retrospective comparative analysis of CALGB 9712 and CALGB 9011. Blood 105:49–53 46. Keating MJ, O’Brien S, Albitar M, et al. 2005. Early results of a chemoimmunotherapy regimen of ﬂudarabine, cyclophosphamide, and rituximab as initial therapy for chronic lymphocytic leukemia. J. Clin. Oncol. 23:4079–88 47. Wierda W, O’Brien S, Wen S, et al. 2005. Chemoimmunotherapy with ﬂudarabine, cyclophosphamide, and rituximab for relapsed and refractory chronic lymphocytic leukemia. J. Clin. Oncol. 23:4070–78 48. Wierda W, O’Brien S, Faderl S, et al. 2006. A retrospective comparison of three sequential groups of patients with recurrent/refractory chronic lymphocytic leukemia treated with ﬂudarabine-based regimens. Cancer 106:337–45

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:237-250. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Nanotechnology and Cancer James R. Heath and Mark E. Davis

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Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125; email: [email protected]

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Key Words

First published online as a Review in Advance on October 15, 2007

in vitro diagnostics, cancer therapeutics, nanoparticles, quantum dots, nanowires, microﬂuidics

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.061506.185523 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0251$20.00

Abstract The biological picture of cancer is rapidly advancing from models built from phenomenological descriptions to network models derived from systems biology, which can capture the evolving pathophysiology of the disease at the molecular level. The translation of this (still academic) picture into a clinically relevant framework can be enabling for the war on cancer, but it is a scientiﬁc and technological challenge. In this review, we discuss emerging in vitro diagnostic technologies and therapeutic approaches that are being developed to handle this challenge. Our discussion of in vitro diagnostics is guided by the theme of making large numbers of measurements accurately, sensitively, and at very low cost. We discuss diagnostic approaches based on microﬂuidics and nanotechnology. We then review the current state of the art of nanoparticle-based therapeutics that have reached the clinic. The goal of the presentation is to identify nanotherapeutic strategies that are designed to increase efﬁcacy while simultaneously minimizing the toxic side effects commonly associated with cancer chemotherapies.

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INTRODUCTION

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More than 1500 Americans will die from cancer each day this year. Projections of the numbers of expected cancer diagnoses, and the numbers of expected deaths from cancer, are publicly available.1 While the rate of cancers being diagnosed has steadily increased, the normalized numbers of cancer-related deaths has remained virtually unchanged. Against this static background is emerging a new picture of cancer, which is inspiring hopes that the war on cancer may be winnable. In this review, we introduce this picture and discuss how it is driving the development of new diagnostic and therapeutic technologies. In particular, we focus on nanotechnologies and microﬂuidics for in vitro diagnostics and nanotechnologies for drug delivery. These technologies constitute only a few pieces, albeit critical ones, that are being brought together to win the war on cancer. Recent advances, both conceptual and technological, are making it possible to imagine a future in which cancer is a manageable chronic ailment. Consider how cancer was viewed just a few years ago. Most pathology practices were based upon a few phenomenological measurements to assess disease (Figure 1a). An increased understanding of cancer has demonstrated that a given type of cancer can be triggered by different genetic mutations, each of which can lead to a different outcome (e.g., aggressive versus nonaggressive cancer). This understanding led to the model of cancer pathways (2) (Figure 1b). In this model, there are multiple pathways of interacting proteins, each constituting a cascade of molecular events. A given pathway, if genetically altered in speciﬁc ways, is effectively short-circuited and thus constantly activated, even in the absence of signaling molecules. Emerging cancer

1

Statistics related to cancer deaths, cases diagnosed, etc., for years 1997–2007 are available from the American Cancer Society at http://www.cancer.org/docroot/stt/ stt 0.asp.

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molecular therapeutics (3, 4) are designed against speciﬁc pathways, often targeting the genetically altered proteins. Molecular measurements, such as the identiﬁcation of mRNAs (5) or pathway-associated proteins, are increasingly used to identify the altered pathway or the response of the cancer to therapy (6). Such an analysis can potentially indicate an appropriate therapy (7, 8), the progression of the cancer (9), the potential for recurrence after therapy (10), or the potential for drug resistance (8). In vivo molecular imaging is also increasingly employed as a diagnostic of drug efﬁcacy (11). Pathway models are useful but limited. A pathway-based diagnosis typically requires prior knowledge that cancer is present, so it constitutes a more accurate pathology report but not an early detection strategy. Another drawback is that pathway models do not account for the dynamic evolution of cancer, and they underestimate the degree of interconnectivity among the various genes and proteins. Finally, pathway models assume that a given cancer is homogeneous, which is almost certainly incorrect. Network models of disease and disease progression (Figure 1c) are emerging out of systems biology procedures (12), which generally involve deep transcriptome analyses (13), occasionally coupled with focused proteomic investigations (14), all integrated together using computational methods (15). Network models can illustrate how the onset and progression of disease are reﬂected in the form of differentially expressed genes and their associated protein networks. Current network models, though unwieldy, are beginning to lend molecular-level insight into the pathophysiology of disease progression. This, in turn, has implications for cancer clinical care, including the potential for achieving a more informative diagnosis. This increased information content arises from at least three concepts. First, the proteomic and genomic databases may be comparatively mined to generate a list of candidate biomarkers that are detectable in body ﬂuids. Blood, for example,

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b

a

Drug A EGFR/EGFRviii Akt

Ras Sos Grb

P13K FKHR mTOR

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Drug B Erk

c

P70 s6K

s6

Nutrients, ATP, etc.

Healthy Presymptomatic Late-stage Figure 1 These images represent the evolving picture of medicine that is driving the development of nanotechnologies for the investigation, diagnosis, and treatment of cancer. (a) A mammogram exempliﬁes traditional, phenomenological, single-parameter cancer diagnostic techniques. (b) The “cancer pathways” model for understanding the differential response of certain cancers to molecular therapies. Each pathway is a cascade of interacting proteins (circled in yellow). An analysis of multiple mRNAs and proteins from cancerous tissues can lead to the appropriate prescription of drugs. (c) A dynamic network model of disease. This model, compared to the cancer pathways model, more accurately reﬂects the complex interrelationships between various proteins within a biological system. It is thus a truer reﬂection of the molecular nature of the disease, but it also can be mined for biomarkers that can be diagnostic for the progression of the disease. Such biomarkers can potentially be harnessed for detecting disease prior to the emergence of clinical symptoms. (Dynamic network model images courtesy of Leroy Hood.)

is a powerful window into health and disease, but it is a noisy environment, containing >104 proteins that span a concentration range of >109 . The ability to identify organspeciﬁc, secreted proteins in blood is an exam-

ple of a powerful strategy for extracting signal from such an environment (16). Second, if the regulatory networks associated with the relevant proteins are identiﬁed, then measurements of those proteins can be directly www.annualreviews.org • Nanotechnology and Cancer

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correlated with the developing pathophysiology of the disease. Third, the best network models will soon be dynamic models, and thus the extracted molecular signatures of disease are identiﬁed against a time-averaged background. Ultimately, dynamic network models may allow the detection of disease prior to the development of clinical symptoms, thus paving the way for prophylactic therapies. This evolving picture of cancer holds promise for changing both diagnostics and therapeutics. For diagnostics, this scenario permits the asking of many more clinically relevant questions, but it places new demands on both measurement and computational technologies. Information will eventually become the commodity of value, implying that quantitative, sensitive, and multiparameter diagnostic measurements must be accomplished inexpensively, and the results must be rapidly integrated to produce a simple and yet accurate diagnostic conclusion. “Multiparameter” measurements consider genes, proteins, and cells. “Inexpensive” measurements are rapid and routine to execute, requiring small amounts of tissue and minimal sample handling. It is here that nanotechnologies, new chemical methods, and microﬂuidics are emerging as powerful tools. The ability to detect cancer early almost always correlates with the ability to cure the disease, typically with combinations of surgery, radiation therapy, and chemotherapy. Emerging molecular therapeutics have shown promise against very speciﬁc classes of tumors (17, 18), but typically the cancer is kept at bay for only 1–2 years before returning in a drugresistant form (19, 20). The more traditional chemotherapies are, as a rule, more effective against broad patient populations, but they are also accompanied by side effects ranging from hair loss to cardiac arrest. New nanotechnologies for drug encapsulation and delivery are being developed to increase the accuracy of drug delivery to target, while also minimizing the exposure of noncancerous tissues and reducing toxicity.

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EMERGING IN VITRO DIAGNOSTIC TECHNOLOGIES In vitro cancer diagnostics will increasingly mean the measurement of large panels of biomolecules (mRNAs and proteins) from ever-smaller samples. Samples may consist of body ﬂuids, tissues, cells sorted from resected or biopsied tumors, circulating tumor cells (21), etc. To focus our discussion on the rapidly expanding ﬁelds of microﬂuidics (22) and nanotechnologies for in vitro cancer diagnostics, we take a lesson from the semiconductor industry. Integrated circuit manufacture has advanced so that the cost of producing a transistor is a fraction of a penny. This has required high-throughput manufacturing protocols that integrate hundreds of processing steps and many different materials. The analogy to the transistor is the biological measurement, which must cost a few pennies or less for clinical diagnostics to keep pace with the evolving picture of human disease. Our discussion highlights both the progress and the challenges associated with integrating chemistry, biology, device fabrication, etc., into a seamless and highly parallel manufacturing process for enabling inexpensive biological measurements. Antibody reagents constitute a severe roadblock standing in the way of making biological measurements inexpensive. Antibodies are expensive and unstable, and they don’t always have a high afﬁnity or a high selectivity for their cognate proteins. A description of alternative protein capture agents (e.g., peptides, aptamers, biligands) is not presented here, but we would be remiss to ignore this issue.

Protein Assays A common diagnostic technique for measuring proteins is the enzyme-linked immunosorbent assay (ELISA) (23). ELISA variations are often referred to as sandwich assays (24)—the biomolecule to be detected is sandwiched between a surface- bound primary (1◦ )

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antibody and a ﬂuorophore-labeled secondary (2◦ ) antibody. The binding event of the 2◦ antibody is optically detected. ELISA assays are typically carried out in multiwell plates, with one type of protein detected per well. The well is incubated ﬁrst with the 1◦ antibody and then with the sample and the 2◦ antibody. This protocol takes a few hours, but this long timescale is not intrinsic (see below). With a 1◦ antibody that has 10−9 M afﬁnity, an ELISA assay can detect proteins present in the few-picoM concentration range. The limitations of ELISA are multifold. First, ELISA is a single-protein detection method (although extendable to multiple wells). Second, the concentration range over which a given biomolecule may be quantitated is ∼102 , limited by the minimal detectable signal over background and the tendency of ﬂuorophores to photobleach. Third, the need for two antibodies per biomolecule is nontrivial. Signiﬁcant work has gone into improving sandwich assays (25–27). Improvements have included the introduction of ampliﬁcation using electroless Ag deposition on Au nanoparticle-labeled 2◦ antibodies (28), or using nanoparticle-loaded DNA or Raman bio-barcodes (29). These nanotechnologybased ampliﬁcation strategies can (when high-afﬁnity antibodies are available) push the detection threshold into the 100-attoM (10−16 M) range. Such high sensitivity will almost certainly have diagnostic value.

Microfluidics Chips Glass (30), elastomeric (31), or multilayer integrated elastomeric (32, 33) microﬂuidics platforms provide the framework for most of the emerging technologies, and they enable cost reductions both in the consumption of reagents and, under certain conditions, in the time required to perform an assay. The binding kinetics of microﬂuidics-entrained surface immunoassays has been modeled (34, 35). Zimmerman et al. found two kinetic limits. Under low ﬂow velocity, the surface-bound antigen is able to exploit a large fraction of the

analyte, but with slow, diffusion-limited capture kinetics. Higher ﬂow velocities (of order 1 mm·s−1 ) and small active areas (150 nm2 ) provide a limit at which the immunoassay binding kinetics reﬂects the analyte/antigen kon and koff binding constants: d θt = kon C(θmax − θt ) − koff θt , dt

1.

with a characteristic binding time, τ , can be expressed as τ ≈ (kon C + koff )−1 .

2.

Under the (common) conditions in which kon C koff , τ ≈ 1/koff . Here, θ t is the surface density of bound analyte at time t, θ max is the maximum surface density of molecules possible, and C is the target protein concentration. Over fairly broad protein concentration ranges (10−15 –10−10 M), the time to detection can be fast (minutes) for an analyte/antigen binding afﬁnity in the nanomolar range. Thus, the slow rate associated with developing an ELISA immunoassay can be improved through microﬂuidics design. This potential time savings represents a key reduction in the cost of measurements.

Chips for Blood and Tissue Handling Serum proteomics involves separating the cells from the plasma using centrifugation, followed by detection of proteins using mass spectrometry, Western blot, or sandwich assays. Centrifugation requires signiﬁcant handling and sample volumes, and so is expensive. Various methods for the on-chip separation of biological materials have been advanced, including dielectrophoresis (36), microﬁltration (37), acoustic forces (38), and lateral displacement (39), to name a few. Yang et al. (40) recently reported on a microﬂuidics design for the separation of plasma from whole blood. They took advantage of the Zweifach-Fung effect, in which a microﬂuidic channel is split into low-resistance and high-resistance channels. In an optimized design, the blood cells pass into the channel that has the higher ﬂow www.annualreviews.org • Nanotechnology and Cancer

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rate, with ∼20% of the plasma ﬂowing into the low-ﬂow-rate channel. They reported a plasma selectivity with respect to blood hematocrit level of almost 100% regardless of the inlet hematocrit. This technology is remarkably efﬁcient, has no moving parts, is composed only of plastic and glass, and can handle very small amounts of blood. The culturing and handling of tissue and the sorting of cells are important procedures for cancer research, drug screening, cancer immunotherapy (41), and in vitro diagnostics. On-chip techniques for cell culture (42) and sorting are being developed by a number of groups (43). Cell-sorting chips rely largely on microﬂuidics variations of ﬂuorescenceactivated cell sorting (FACS) (44, 45), dielectrophoresis (4), or the antibody array–based technique of panning (47, 48). As of this writing, we have not identiﬁed chip-based automated tissue processors, such as would be necessary to separate and sort speciﬁc cancer cells, immune cells, etc., starting from a solid tumor. A major challenge associated with these blood/tissue-handling chips is to ﬁnd methods to integrate them with measurement assays. Integration of disparate technologies is often considered an applied engineering problem, and is ignored in the academic labs where many of the individual components are invented. However, for the applications discussed herein, integration issues constitute relatively uncharted science.

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Label-Free Measurement Techniques Surface plasmon resonance (SPR) (49), nanowire (50), nanotube (51, 52), and nanocantilever (53, 54) biomolecular sensors are all classiﬁed as label-free measurement technologies, meaning that the binding of the target biomolecule to its surface-bound capture agent is directly detected. Furthermore, under ﬂowing sample conditions, kon , koff , and/or analyte concentration (see Equations 1 and 2) can be directly measured (55, 56) from the dynamic sensing response. SPR is a 256

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commercial product that is rapidly being improved (57),2 but the nanotechnologies have distinct advantages. These include direct electrical readout of the signal [which requires piezoresistive nanocantilevers (59)], increased sensitivity (60) and dynamic range, the ability to detect small molecule-binding events, and a large degree of substrate independence (61). Although nanowire and nanotube sensors have demonstrated the ability to sense single (62) or small panels (63) of cancer serum biomarkers, they are limited for protein sensing in biological media (e.g., an electrolyte) by Debye screening (64). Nanocantilevers and nanowire technologies are immature technologies and, despite promising demonstrations, it is not clear if they will emerge as useful cancer diagnostic tools. Nevertheless, the impressive sensitivity, dynamic range, and batch processability of these devices, coupled with emerging and novel applications (59), imply that they will become signiﬁcant measurement tools.

Tissue Analysis Immunohistochemical staining and related methods (65) represent in situ protein assays that are important for obtaining a molecular diagnosis of cancer from resected tumors. Emerging nanotechnology variants of this method have included the use of semiconductor quantum dots as the antibody ﬂuorescent labels (66) for the staining of breast cancer tissues. The relevant advantages (67) arise from the robust ﬂuorescence properties and the sharp and size-tunable emission spectra of quantum dots, which permit increased multiplexing.

Multiparameter Measurements Cancer diagnostic platforms will ultimately integrate multiplexed assays of cells, mRNAs,

2

For example, Lumera sells an SPR product for evaluating ∼103 protein-protein interactions simultaneously (see http :/ /www.lumera.com/Bioscience/Products/ ProteomicProcessor.php).

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and proteins. However, different and incompatible surface chemistries are required for different classes of biomolecules, and not all are compatible with device fabrication. Antibodies are immobilized onto aldehyde, epoxy, maleimide, or hydrophobic solid supports (68–71). Variables such as pH, ionic strength, hydration, etc., must be controlled to prevent protein denaturation. The best surface for reducing nonspeciﬁc binding of cells while maintaining full antibody functionality is acrylamide (72), which is incompatible with DNA. DNA microarrays are electrostatically absorbed (via spotting) onto amine surfaces. One option for detecting DNA, proteins, and cells on the same platform is to utilize differentially functionalized surfaces (or beads, such as are used by certain Illumina® systems for the codetection of DNA oligomers and proteins). For microﬂuidics-based assays, this adds signiﬁcant manufacturing complexity and cost. In addition, not all of the abovedescribed surface chemistries are stable to the processing steps associated with microﬂuidics fabrication. A second alternative recently demonstrated (48) is to utilize the technique of DNA Encoded Antibody Libraries (DEAL) for multiplexed gene and protein detection and cell sorting. This provides one common surface chemistry (spotted DNA arrays) that is fully compatible with microﬂuidics manufacture. Furthermore, an optimized DEAL assay can be signiﬁcantly faster and more sensitive than conventional immunoassays; it can cover a broader dynamic range; and it is far superior to panning as a multiplexed cell-sorting technique.

EMERGING NANOPARTICLE THERAPEUTICS Informative diagnoses of the future will exploit new advances in nanotechnology in order to provide in vitro molecular measurements of pathophysiology from body ﬂuids such as blood. These advanced diagnostic methods will provide information that will allow the design of new intervention strategies,

provided appropriate therapeutics are available. Nanotechnology is playing a role in providing new types of therapeutics for cancer. These nanotherapeutics have the potential to provide effective therapies with minimal side effects. Most cancer patients die from drugresistant, metastatic disease. Thus, the ultimate goal for cancer therapies would be the ability to treat this stage as well as any of those leading up to it. It is hoped that as diagnostic methods improve, treament can be initiated at earlier and earlier stages of disease progression. However, in the most general sense, it would be advantageous to develop therapies that could be employed at all stages of cancer because of the enormous resources that are required to bring a new therapeutic to market. Targeted nanoparticles have the potential to provide therapies not achievable with any other drug modalities. By tuning the size and surface properties of the nanoparticle, manipulation of the pharmacokinetics (PK) from a systemic administration is achievable. Nanoparticles should be larger than ∼10 nm to avoid single-pass renal clearance and not be positively charged to any great extent (minimizing nonspeciﬁc interactions with proteins and cells) in order to allow these PK manipulations. The particles can be tuned to provide long or short circulation times, and with careful control of size and surface properties, they can be directed to speciﬁc cell types within target organs (e.g., hepatocytes versus Kupffer cells in the liver). Other types of therapeutics, such as molecular conjugates (e.g., antibody-drug conjugates), can also meet these minimum speciﬁcations, but targeted nanoparticles are distinguished from all other therapeutic entities by at least four features: 1. The nanoparticle can carry a very large “payload.” For example, a 70-nm nanoparticle can contain ∼2000 siRNA drug molecules (73) whereas antibody conjugates have <10 (74). The nanoparticle payloads are located within the particle and do not participate in the control over PK and biodistribution. In www.annualreviews.org • Nanotechnology and Cancer

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Nano-scaled systems for systemic therapy

Platform

Latest stage of development FDA approved

DaunoXome, Doxil®

albumin-based particles

FDA approved

Abraxane

nanocrystals

FDA approved

Rapamune (oral), Emend (oral)

polymeric micelles

clinical trials

Genexol-PM, SP1049C, NK911, NK012

polymer-based particles

clinical trials

XYOTAX, IT-101, CT-2106, AP5346

dendrimers

preclinical

polyamidoamine (PAMAM)

inorganic or other solid particles

preclinical

carbon nanotubes, silica particles, gold particles

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molecular conjugates, by contrast, the type and number of therapeutic entities conjugated to the targeting ligand (e.g., an antibody) signiﬁcantly modify the overall properties of the conjugate. 2. Nanoparticles are sufﬁciently large to contain multiple targeting ligands that can provide multivalent binding to cell surface receptors (75). Nanoparticles have two parameters for tuning the binding to target cells: (a) the afﬁnity of the targeting moiety and (b) the densities of the targeting moiety. The multivalency effects can lead to very high “effective” afﬁnities when arrangements of low-afﬁnity ligands are used (75–77). Thus, the repertoire of molecules that can be used as targeting agents is greatly expanded, since many low-afﬁnity ligands can be installed on nanoparticles to create higher afﬁnity via multivalent binding to cell surface receptors. 3. Nanoparticles are sufﬁciently large to accommodate multiple types of drug molecules. Numerous therapeutic interventions can be simultaneously applied with a nanoparticle in a controlled manner. 4. Nanoparticles bypass multidrug resistance mechanisms that involve cell surface protein pumps, e.g., glycoprotein P, because they enter cells via endocytosis. These properties provide the opportunity to create therapeutic strategies not possible with non-nanoparticle drugs. A controlled combi258

Examples

liposomes

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nation of these features can minimize side effects while enhancing drug efﬁcacy, and offers the potential to treat drug-resistant disease if the resistance is from cell surface pumps. Clinical results are emerging that suggest nanoparticle therapeutics will lead to new methods of treatment for cancer. Therapeutics that are now classiﬁed as nanoparticles have existed for some time. Table 1 lists nano-scaled systems for systemic therapy and their latest stage of development. Liposomes carrying chemotherapeutic smallmolecule drugs have been approved since the mid-1990s. Liposomes (∼100 nm and larger) can give extended circulation times if they are stabilized (see Doxil® in Table 2) but do not provide intracellular delivery of drug molecules (78). Thus, they are not effective against disease that is resistant to cell surface pumps. Additionally, they provide no control for the time of drug release. Their use is mainly in solubilizing drugs and extending circulation times to favor higher tumor uptake of drugs. Albumin-based nanoparticles were approved by the US Food and Drug Administration in 2005 (79), but are not nanoparticle therapeutics, in that they dissolve upon administration into the circulatory system (note PK parameters for Abraxane versus Taxol® in Table 2). Nanocrystals of drug molecules are also approved for oral administration; however, these nanoparticles never reach the bloodstream. These ﬁrst approved nanoparticle formulations prove that nanoparticle-based therapeutics can safely be

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Table 2

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Comparison of pharmacokinetics (human) of small-molecule drugs to nanoparticle therapeutics

Name

Formulationa

Diameter (nm)

CL (ml/min·kg)

Reference

DOX

0.9% NaCl

—

0.8

14.4

80

SP1049C

pluronic micelle/DOX

22–27

2.4

12.6

80

NK911

PEG-Asp micelle/DOX

40

2.8

6.7

80

Doxil®

PEG-liposome/DOX

80–90

84.0

0.02

80

Taxol®

Cremophor® EL

—

21.8 (20.5)

3.9 (9.2)

79, 80

Genexol

PEG-PLA micelle/paclitaxel

20–50

11.0

4.8

80

Abraxane

albumin/paclitaxel

120b

21.6

6.5

79

XYOTAX

PG/paclitaxel

?

70–120

0.07–0.12

82

LE-SN38

liposome/SN-38

?

7–58

3.5–13.6

83

CT-2106

PG/CPT

?

65–99

0.44

84

IT-101

CD polymer/CPT

30–40

38

0.03

85

t 1/2 (h)

a Abbreviations: DOX, doxorubucin; PEG-PLA, block copolymer of polyethylene glycol-poly(L-lactic acid); PG, polyglutamic acid; CPT, camptothecin; CD polymer, cyclodextrin-containing polymer. b Dissolves upon exposure to blood.

administered to patients and can enhance the safety and efﬁcacy of other drug molecules. However, newer nanoparticle systems have great advantages over these early nanoparticle products. Table 2 compares some nanoparticlebased therapeutics to the drug molecules that they are carrying. The types of particles include liposomes, polymer micelles, and polymer-based nanoparticles. For each case, e.g., DOX versus SP1049C, NK911 and Doxil®, the nanoparticle alters the PK properties of the drug molecule. The listed circulation half-lives are difﬁcult to compare because different models are used for their determination. Clearance rate (the volume of blood/plasma cleared of the drug per time; lower clearance rates indicate higher circulation times) is a common and available PK parameter, and it is a better indicator of circulation differences among these therapeutics. Dramatically reduced clearance rates have been obtained with nanoparticles, e.g., Doxil®, XYOTAX, CT-2106, and IT-101. These nanoparticles can provide longer circulation times that allow them to adequately interrogate the body for the presence of tumors if in fact they extravasate into tumors. Small particles like polymeric micelles (<100 nm) have been shown to accumulate more readily

in tumors than the larger liposomes (80). Additionally, movement of a particle throughout a tumor is also size-dependent. It is speculated that nanoparticles between 10 and 100 nm in diameter will be optimal for tumor penetration. Therefore, careful control of size will be important to the PK, biodistribution, tumor accumulation, and tumor penetration. Some of the nanoparticles that are now in clinical testing also have mechanisms to control the release of the drug. These mechanisms rely on cleavage of a chemical bond between the particle and the drug by (a) hydrolysis, (b) enzymes that are located within and outside of cells (e.g., esterases), or (c) enzymes that are located only within cells (e.g., cathepsin B). This feature is designed into the structures of polymer micelle-based (NK911 and NK012) and polymer-based (XYOTAX, CT-2106, and IT101) nanoparticles. Finally, some of the newer nanoparticles (e.g., IT-101) enter tumor cells and are thus effective against tumors that are resistant to the drug via surface pump mechanisms. Results from clinical trials with the newer nanoparticle-based experimental therapeutics are conﬁrming that common side effects of the drug molecules used can be altered or reduced. Vastly improved side-effect proﬁles are emerging from these nanoparticle treatments. www.annualreviews.org • Nanotechnology and Cancer

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Table 3

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Ligand-targeted therapeutic agents

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Name

Targeting agent

Therapeutic agent

Status

Reference

Mylotarg (antibody-drug)

humanized antibody anti-CD33

calicheamicin

FDA approved

86

Ontak (fusion protein)

IL-2

diphtheria toxin fragment

FDA approved

87

Zevalin (radioimmunotherapy)

mouse antibody anti-CD20

90 Y

FDA approved

88

Bexxar (radioimmunotherapy)

mouse antibody anti-CD20

131 I

FDA approved

88

PK-2 (polymer-drug)

galactose

DOX

Phase I (stopped)

89

MBP-426 (liposome-drug)

transferrin

oxaliplatin

Phase I

90

SGT-53 liposome-plasmid DNA

antibody fragment to transferrin receptor

plasmid DNA with p53 gene

Phase I

91

CALAA-01 (polymer-siRNA)

transferrin

siRNA

Phase I (planned for 2007)

81

The nanoparticles listed in Table 2 reach tumors by passive targeting, meaning they accumulate in tumors because the leaky vasculature of tumors allows the nanoparticles to extravasate while normal vasculature does not (this property partially accounts for the difference in biodistribution between nanoparticles and drug molecules). Active targeting via the inclusion of a targeting ligand on the nanoparticles is envisioned to provide the most effective therapy. Table 3 lists the very few ligandtargeted therapeutics that are either approved or in the clinic. PK-2 can be considered the ﬁrst ligand-targeted nanoparticle to reach the clinic. The galactose ligand was used to target the asialoglycoprotein receptor (ASGPR) that is expressed on hepatocytes in hopes that it was still highly expressed on primary liver cancer cells. However, because the ASGPR is expressed on healthy hepatocytes, the targeted nanoparticles accumulated in normal liver as well as in the tumor. As of mid-2007, MBP-426 and SGT-53 are the only targeted nanoparticle in the clinic. Clinical trials of CALAA-01 are planned to begin in late 2007. All three of these nanoparticles (liposomal delivery of a small-molecule chemotherapeutic, liposomal delivery of plasmid DNA, and polymer delivery of siRNA) use the human protein transferrin to target the transferrin receptors on cancer cells. Transferrin receptor 260

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is known to be upregulated in many types of cancer. A targeted nanoparticle can be very multifunctional [e.g., CALAA-01 is a targeted nanoparticle that has high drug (siRNA) payload per targeting ligand, proven multivalent binding to cancer cell surfaces, and an active drug (siRNA) release mechanism that is triggered upon intracellular localization (73, 81)], and it is expected that these new nanoparticles should perform in superior ways to the older, less functional nanoparticles. Increasingly sophisticated nanoparticles are reaching the clinic, with trial results already inspiring enthusiasm for this type of therapeutic modality. This is only the beginning. The nanoparticle provides the ability to design and tune properties in ways not possible with other types of therapeutics. Thus, as more clinical data become available, the nanoparticle approach will become better and better as the optimal properties will be elucidated from previous experiences in humans. The better side-effect proﬁles enabled by the newer nanoparticles are already improving the quality of life for patients, and there is hope for even more improvement in the future. It is not unreasonable to predict the design of nanoparticle therapeutics sufﬁciently nontoxic for prophylactic use. Such nanotherapeutics would provide a powerful companion to very early in vitro diagnostic detection.

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DISCLOSURE STATEMENT Dr. Davis is a consultant and has stock in Calando Pharmaceuticals and Insert Therapeutics. Dr. Heath is on the board of Homestead Clinical Corp., which has obtained licensing rights to the DEAL technology.

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88. Allen TM. 2002. Ligand-targeted therapeutics in anticancer therapy. Nat. Rev. Cancer 2:750–63 89. Duncan R. 2006. Polymer conjugates as anticancer nanomedicines. Nat. Rev. Cancer 6:688– 701 90. 2006. Safety study of MBP-426 (liposomal oxaliplatin suspension for injection) to treat advanced or metastatic solid tumors. ClinicalTrials.gov Identiﬁer NCT00355888. http://www. clinicaltrials.gov/ct/show/NCT00355888 91. 2007. Safety study of infusion of SGT-53 to treat solid tumors. ClinicalTrials.gov Identiﬁer NCT0040613. http://www.clinicaltrials.gov/ct/show/NCT00355888

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:251-265. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones Department of Urology, Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California 90089; email: jones [email protected]

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Key Words

First published online as a Review in Advance on October 15, 2007

DNA methylation, histone modiﬁcation, CpG islands

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.061606.095816 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0267$20.00

Abstract Deregulation of gene expression is a hallmark of cancer. Although genetic lesions have been the focus of cancer research for many years, it has become increasingly recognized that aberrant epigenetic modiﬁcations also play major roles in the tumorigenic process. These modiﬁcations are imposed on chromatin, do not change the nucleotide sequence of DNA, and are manifested by speciﬁc patterns of gene expression that are heritable through many cell divisions. We review these modiﬁcations in normal and cancer cells and the evolving approaches used to study them. Additionally, we outline advances in their potential use for cancer diagnostics and targeted epigenetic therapy.

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INTRODUCTION

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DNMT: DNA methyltransferase Nucleosome: the core unit of chromatin, composed of 147 base pairs of DNA wrapped around a histone octamer. It was initially viewed as a structural component of chromatin, but now its composition and position are considered important in the control of gene expression HDAC: histone deacetylase

During normal development, somatic cells that are descended from a single progenitor, and contain a similar genotype, differentiate to acquire diverse functions and features by expressing and repressing different sets of genes. This process is brought about by modiﬁcations that affect how the genetic material is packaged and utilized without changing its nucleotide sequence. Importantly, these “epigenetic” modiﬁcations are maintained through cell division. The involvement of these modiﬁcations in cancer states has been increasingly recognized and is the subject of this review.

THE INTERPLAY OF EPIGENETIC MODIFICATIONS Epigenetic modiﬁcations can be generally divided into three interacting processes: DNA methylation, histone modiﬁcation, and chromatin remodeling. DNA methylation is catalyzed by at least three DNA methyltransferases (DNMTs) that add methyl groups to the 5 portion of the cytosine ring to form 5 methyl-cytosine. During S-phase, DNMTs, found at the replication fork, copy the methylation pattern of the parent strand onto the daughter strand, making methylation patterns heritable over many generations of cell divisions. In mammalian genomes, this modiﬁcation occurs almost exclusively on cytosine residues that precede guanine—i.e., CpG dinucleotides. The term CpG applies to both methylated and unmethylated dinucleotides; the “p” refers to the phosphate moiety that connects deoxycytidine and deoxyguanosine. CpGs occur in the genome at a lower frequency than would be statistically predicted because methylated cytosines can spontaneously deaminate to form thymine. This is not efﬁciently recognized by the DNA repair machinery, so C-T mutations accumulate during evolution. As a result, ∼99% of the genome is CpG depleted. The other ∼1% is composed of discrete regions

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that have a high (G+C) and CpG content. These regions are called CpG islands (1, 2). CpG islands are mostly found at the 5 regulatory regions of genes, and ∼60% of human gene promoters are embedded in CpG islands. Although most of the CpG dinucleotides are methylated, the persistence of CpG islands suggests that they are not methylated in the germ line and so did not undergo CpG depletion during evolution (3). Around 90% of CpG islands are estimated to be unmethylated in somatic tissues (4), and the expression of genes that contain CpG islands is not generally regulated by their methylation. However, under some circumstances CpG islands do get methylated, resulting in long-term gene silencing. DNA methylation is essential for normal development, as mice lacking any one of the enzymes responsible for placing the mark die in the embryonic stages or shortly after birth (5, 6). As a silencing mechanism, it plays a role in the normal transcriptional repression of repetitive and centromeric regions, X chromosome inactivation in females, and genomic imprinting (7). The silencing mediated by DNA methylation occurs in conjunction with histone modiﬁcations and nucleosome remodeling, which together establish a repressive chromatin structure (Figure 1A). The functional link between DNA methylation and histone modiﬁcations was initially established by studies showing that histone deacetylases (HDACs) are recruited to methylated DNA by methyl-CpG binding proteins (8, 9). Histones, which are the building blocks of nucleosomes, undergo numerous post-translational modiﬁcations that regulate chromatin structure, gene expression, and DNA repair (10). The most studied histone modiﬁcations are methylation and acetylation of lysine residues. Until recently, histone methylation was considered a permanent mark placed on chromatin. However, several histone demethylating enzymes have been discovered in recent years (10), and both acetylation and methylation are now

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A Normal

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Figure 1 Epigenetic patterns in normal and cancer cells. (A) DNA methylation. In normal cells, nearly all of the CpG dinucleotides are methylated whereas CpG islands, mostly residing in 5 regulatory regions of genes, are unmethylated. In cancer cells, many CpG islands become hypermethylated, in conjunction with silencing of their cognate genes, while global hypomethylation, mostly at repetitive elements, occurs. (B) Chromatin and histone modiﬁcation. Active genes are associated with acetylation of histone tails, methylation of lysine 4 on histone H3 (H3K4), and nucleosome depletion at their promoters. The promoters of silenced genes (drawn here in conjunction with DNA hypermethylation) become associated with nucleosomes, lose acetylation and H3K4 methylation marks, and gain repressive methylation marks such as lysine 9 or 27 on histone H3, which recruit repressive complexes. Methylated DNA binding proteins link methylated DNA with the histone modiﬁcation and nucleosome remodeling machineries (not shown).

considered reversible modiﬁcations catalyzed by enzymes having opposing activities. In general, regions silenced by DNA methylation also show hypoacetylation and hypermethylation of speciﬁc histone lysine residues, such as lysine 9 or 27 in histone H3 (10). In contrast, promoters of actively transcribed genes show hyperacetylation of histones H3 and H4, and methylation of lysine 4 of histone H3 (H3K4) (11, 12). DNA methylation and histone modiﬁcations function in close interplay with nucleosome remodeling and positioning complexes that bind speciﬁc histone modiﬁcations, such as trimethylated H3K4 (13, 14) and methyl CpG binding proteins (15), and move nucle-

osomes on DNA by ATP-dependent mechanisms. Nonmethylated CpG island promoters are usually hypersensitive to nucleases and are relatively depleted of nucleosomes, whereas methylated promoters have nucleosomes on them and are nuclease resistant (16, 17, 17a) (Figure 1B).

CANCER: A MODIFIED EPIGENOME When a general role for DNA methylation in gene silencing was established more than 25 years ago (18), it was proposed that aberrant patterns of DNA methylation might play a role in tumorigenesis (19). Initial studies www.annualreviews.org • Cancer Epigenetics

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MicroRNAs: small, noncoding RNA molecules, approximately 22 nucleotides long that bind to the mRNA of target genes to negatively control their expression. MicroRNAs have essential roles in normal development and their expression patterns are linked to cancer development Methylomes: Distinct DNA methylation proﬁles in tumors, tissues, or different cell types CpG island methylator phenotype (CIMP): a trait exhibited by a subset of tumors that show an exceptionally high frequency of methylation of distinct CpG islands

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found evidence for a decrease in the total 5-methylcytosine content in tumor cells (20), and the occurrence of global hypomethylation in cancer was ﬁrmly established in subsequent studies. Hypomethylation occurs mainly at DNA repetitive elements and might contribute to the genomic instability frequently seen in cancer (20). Hypomethylation might also contribute to overexpression of oncogenic proteins and was shown to be associated with loss of imprinting of IGF2 (insulin growth factor 2), leading to aberrant activation of the normally silent maternally inherited allele. This was found to be associated with an increased risk for colon cancer (21). The mechanisms underlying global hypomethylation patterns are currently unknown. Aberrant hypermethylation at normally unmethylated CpG islands occurs parallel to global hypomethylation (Figure 1A). The CpG island promoter of the Rb (Retinoblastoma) gene, found to be hypermethylated in retinoblastoma, was the ﬁrst tumor suppressor shown to harbor such a modiﬁcation (22). This discovery was soon followed by studies showing promoter hypermethylation and silencing of other tumor suppressor genes such as VHL (von Hippel–Lindau) in renal cancer (23), the cell cycle regulator CDKN2 A/p16 in bladder cancer (24), the mismatch repair gene hMLH1 in colon cancer (25), and many others. On the basis of these ﬁndings, it was proposed that epigenetic silencing of tumor suppressor genes by DNA methylation can serve as an alternative “hit” to mutation and/or deletion in Knudson’s two-hit carcinogenesis model (26). This led to the notion that ﬁnding hypermethylated genes would result in the discovery of new tumor suppressors. An example is ID4, a proposed tumor suppressor, which was found to be hypermethylated in hematological malignancies but for which no mutations were detected in tumors (27). The development of large-scale unbiased methods for detecting methylation, such as restriction landmark genomic scanning (RLGS) and array-based techniques (see below), led Gal-Yam et al.

to a ﬂurry of studies reporting numerous hypermethylated genes in cancer (see Reference 28 for a partial list). It is now established that aberrant hypermethylation at CpG island promoters is a hallmark of cancer. Notably, not only protein-coding genes undergo these modiﬁcations; CpG island promoters of noncoding microRNAs were shown to be hypermethylated in tumors, possibly contributing to their proposed roles in carcinogenesis (29, 30). What is the origin for the deregulated methylation patterns in cancer? Initially it was suggested that like genetic mutations, de novo hypermethylation events are stochastically generated, and that the ﬁnal patterns observed are a result of growth advantage and selection (30a). However, several observations made in recent years should be noted: First, hypermethylation events are already apparent at precancerous stages, such as in benign tumors and in tumor-predisposing inﬂammatory lesions (31, 32). Second, there seem to be deﬁned sets of hypermethylated genes in certain tumors. These differential methylation signatures, or “methylomes,” may even differentiate between tumors of the same type, as was recently shown for the CpG island methylator phenotype (CIMP) in colon cancer (33). Third, although many hypermethylated genes have tumor-suppressing functions, not all are involved in cell growth or tumorigenesis. Furthermore, some of these genes are not expressed in the corresponding normal tissue, so their methylation does not result in their de novo silencing in the cancer cells (34; E. Nili Gal-Yam, G. Egger, A. Tanay, P. A. Jones, unpublished data). Thus, although the hypothesis of stochastic methylation and selection is probably true for some cases, the observations detailed above suggest that these patterns may be generated by upstream-acting “programs” that have gone wrong. Evidence for such a program involving the Polycomb group complexes (PcGs) is emerging. PcGs are protein complexes responsible for maintenance of long-term silencing of genes, which is

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mediated by methylation of lysine 27 of histone H3 at the repressed regions. The enzyme that catalyzes this modiﬁcation is EZH2, which is known to be upregulated in tumors and involved in tumor progression (35). In embryonic stem cells, repression of a large set of developmental genes mediated by PcGs is thought to maintain these cells in a pluripotent state (36, 37). Several studies have recently shown that these genes are prone to be hypermethylated in cancer, suggesting a functional link between the two repressing systems and lending support to the idea of an epigenetic stem cell signature in cancer (38–40). Future studies that analyze global methylation patterns after manipulation of PcG components are needed to provide further insights into the role of this system in aberrant DNA methylation. As discussed above, silenced hypermethylated promoters are generally associated with hypoacetylation of lysine residues on histones H3 and H4 and hypermethylation of lysine 9 or lysine 27 on histone H3, which mediate the formation of a repressive chromatin structure (Figure 1B). Global histone modiﬁcations are also altered in cancer: Leukemias, colon cancers, and cell lines derived from them exhibit

loss of acetylation at lysine 16 and trimethylation at lysine 20 of histone H4. These changes seem to occur at hypomethylated repetitive elements (41). The mechanisms responsible for alterations of these global patterns are mostly unknown but may involve the disruption of the enzymes responsible for these modiﬁcations (28).

DETECTION OF EPIGENETIC MODIFICATIONS DNA Methylation Various approaches exist to study DNA methylation at speciﬁc loci (Figure 2). The oldest approach relies on the use of methylation-sensitive restriction enzymes (MSREs), which distinguish between methylated and nonmethylated sites. These were initially used in conjunction with Southern blotting to analyze methylation status at candidate genes. This technique is labor-intensive, requires large quantities of high-quality DNA not readily obtained from tumors, and depends on the existence of the enzymes’ speciﬁc recognition sites. Nevertheless, MSRE-based techniques are also being

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Figure 2 Approaches for detection of epigenetic marks. DNA methylation can be detected by three main approaches: one based on bisulﬁte conversion, which changes the nucleotide sequence depending on the methylation state of cytosines; another based on methylation-sensitive restriction enzymes, which differentially digest methylated and unmethylated DNA; and a third based on pulldown of methylated DNA by 5 -methylcytosine binding proteins. Alternatively, speciﬁc activation of genes after treatment with the demethylating agent 5 -aza-2 deoxycytidine identiﬁes potentially methylated genes that need to be conﬁrmed by direct analyses. Histone modiﬁcations are usually detected by chromatin immunoprecipitation. These approaches, initially used to detect modiﬁcations at candidate regions, have also been adopted for genome-wide studies (see text for details). www.annualreviews.org • Cancer Epigenetics

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Oligonucleotide tiling arrays: microarrays on which overlapping oligonucleotides, usually 25–50 base pairs long, are printed, covering contiguous regions of the genome. Used to interrogate enrichment of genomic regions that are bound by speciﬁc factors or modiﬁcations

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adopted for large-scale analyses, as detailed below. Methods based on bisulﬁte conversion provide the most accurate methylation detection at the genomic-sequence level. Bisulﬁte treatment of DNA results in deamination of nonmethylated cytosines to uracils while methylated cytosines are not altered (42). This change in the nucleotide sequence, reﬂecting the initial methylation pattern, can be interrogated by various methods. Genomic bisulﬁte sequencing, performed after PCR ampliﬁcation and cloning of the region of interest, is considered the gold standard for methylated cytosine detection; this method gives the exact methylation status for each CpG site. However, because of the large amount of locus-speciﬁc ampliﬁcation and sequencing involved, this is currently not the preferred method for high-throughput methylation analyses. Methylation-speciﬁc PCR (MSP) or its quantitative derivatives, such as Methyl-light (42a), amplify converted DNA using primer sets that are speciﬁc either for the methylated or unmethylated DNA (43). These sensitive techniques have become the most common methylation detection tools using a candidate gene approach, and they allow for the analysis of small quantities of DNA derived from archived tissue. However, as only totally methylated or totally unmethylated molecules are ampliﬁed in these techniques, the exact pattern of methylation is not reﬂected in the result. Additionally, owing to their high sensitivity, rigorous negative (unmethylated) and positive (totally methylated) controls should be used. Other methods based on bisulﬁte-converted DNA, such as MS-SNuPE or pyrosequencing, have been adapted from the ﬁeld of single nucleotide polymorphism (SNP) detection; these enable the accurate quantiﬁcation of methylation at discrete CpG sites within a given region (44, 45). With the realization that aberrant methylation patterns are common in cancer and the advent of genomic technologies to detect them, the ﬁeld has moved from candiGal-Yam et al.

date gene approaches to methods that detect methylation on a large scale in an unbiased manner. In restriction landmark genomic scanning (RLGS), the DNA from tumor and healthy tissue is cleaved by methylationsensitive enzymes, radiolabeled, separated by two-dimensional gel electrophoresis with further enzyme digestion, and autoradiographed. Comparison between the normal and tumor gels reveals spots with differential intensity, representing differential methylation and/or copy number at speciﬁc loci. Although only ∼1000 CpG islands can be interrogated in this manner, this was one of the ﬁrst techniques that compared global methylation proﬁles in a large number of tumor samples, and a nonrandom and type-speciﬁc pattern of promoter hypermethylation was found in tumors (46). Methods relying on microarray technologies have further advanced the study of genomic methylation. An early example was the differential methylation hybridization method (DMH), in which DNA is cleaved by MSREs, labeled, and hybridized to a CpG island array. A differential hybridization signal between normal and tumor DNA reﬂects differential methylation at a speciﬁc CpG island (47). More recently developed techniques rely on the ability of proteins or antibodies to bind speciﬁcally to methylated DNA (48, 49). The methylated DNA immunoprecipitaion (MeDIP) technique, for example, utilizes antibodies that speciﬁcally recognize 5-methylcytosine to immunoprecipitate methylated DNA, resulting in its enrichment in the sample. Coupling this method with oligonucleotide tiling arrays covering the majority of human promoters (50) or the complete Arabidopsis thaliana genome (51) resulted in the ﬁrst high-resolution methylomes to date and promises to be a powerful tool for genome-wide methylation detection in various applications. An alternative approach to detect aberrantly methylated regions relies on the treatment of cells with demethylating compounds such as 5-aza-2 deoxycytidine, which results in the demethylation and transcriptional

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upregulation of speciﬁc genes (52). The use of these compounds in conjunction with expression microarrays enables large-scale screening for differentially expressed genes in treated compared to nontreated cells. An advantage of this approach is that it detects functionally relevant changes in methylation, which are assumed to affect the tumorigenic process, rather than simply the hypermethylation itself. However, as elevated expression of a gene after drug treatment could be due to indirect effects of the drug, the actual methylation status of the identiﬁed genes needs to be conﬁrmed by other methods such as those described above. Another drawback is that the actual experiments can only be performed on cultured cell lines, which do not necessarily reﬂect the situation in the tumors themselves.

HISTONE MODIFICATIONS The detection of histone modiﬁcations largely relies on the existence of high-quality antibodies that recognize speciﬁc modiﬁcation on various amino acid residues of histones. Western blots and immunostaining can be used to detect global levels or localization patterns for these modiﬁcations in the nucleus. The now commonly used chromatin immunoprecipitation (ChIP) technique enables researchers to measure the enrichment of speciﬁc histone modiﬁcations at deﬁned genomic regions. This technique can be scaled to global studies, mainly by combining it with microarray technology (ChIP-chip). ChIPchip can be used to study modiﬁcations at deﬁned genomic entities such as promoters or CpG islands, or in contiguous genomic regions or even whole chromosomes using recently developed oligonucleotide tiling arrays. A drawback to ChIP-chip is the inability to study repetitive elements, as their inclusion in the arrays will interfere with hybridizations and skew the results. Additionally, a bias may be introduced by the ampliﬁcation performed to obtain the large amounts of DNA needed for hybridizations. ChIP-derived DNA can also be sequenced, with the number of se-

quence reads aligning to a speciﬁc genomic locus deﬁned as enrichment at this locus (53). Advantages of this approach are relative ease of analysis, unbiased results, and the fact that the nucleotide sequence of the pulled down fragments is precisely known. Furthermore, rapid developments in sequencing techniques may eventually render ChIP sequencing cheaper and more timely than conventional ChIP-chip (54).

EPIGENETIC DIAGNOSTICS Early detection and risk assessment remain high priorities in oncology. Ideal tumor markers would have high sensitivity and speciﬁcity and be present in sufﬁcient amounts to reveal minimal disease in peripheral samples. Detection of hypermethylated DNA is considered a promising diagnostic tool in cancer because aberrant methylation events are abundant in tumors, occur early in the tumorigenic process, and different cancers exhibit speciﬁc hypermethylation patterns. Because they are DNA markers, they are more stable than RNA or proteins. Furthermore, whereas detection of other DNA aberrations such as point mutations often requires examination of different sites within a gene in various patients, promoter hypermethylation usually occurs over the same region of a given gene, simplifying the design of a detection assay. During the past decade, many studies have detected tumor-derived free circulating hypermethylated DNA in plasma or serum of patients with cancer. Additionally, hypermethylated DNA was obtained from various body ﬂuids of cancer patients, such as urine, stool, saliva, bronchoalveolar lavage (BAL), sputum, mammary aspiration ﬂuid, pancreatic juice, peritoneal ﬂuid, and vaginal secretions (55). Many of these samples can be obtained with minimal invasiveness and thus are suitable for large population screening. Most of these studies were performed using the highly sensitive bisulﬁte-based MSP methods and provide a basis for future clinical trials using DNA methylation markers in cancer detection and www.annualreviews.org • Cancer Epigenetics

Chromatin immunoprecipitation (ChIP): A commonly used method to detect binding of histones, modiﬁed histones, or other factors to speciﬁc genomic regions. Chromatin is cross-linked and sheared followed by pull down with speciﬁc antibodies to the histones and their bound DNA. This is further interrogated by PCR ampliﬁcation of speciﬁc regions or microarray analysis (ChIP-chip)

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surveillance. However, various confounding issues, such as the speciﬁcities of the markers for the different tumors, need to be clariﬁed. For example, many of the markers, such as RASSF1 and CDKN2A/p16, appear to be methylated in various tumors or preneoplastic conditions and are therefore not tumorspeciﬁc. Additionally, methods used for sample collection and methylation detection need to be standardized to achieve sufﬁcient reproducibility of the results. Ideally, one marker could be used for the diagnosis of each tumor type. In prostate cancer, hypermethylation of GSTp1 may be promising in that respect (56). In other cases, highly deﬁned panels of genes will probably be used for screening. One example of the latter is a prospective study in which sputum was collected from individuals who were at high risk for lung cancer but were cancer-free upon entering the trial. Methylation status of six genes predicted the occurrence of lung cancer within two years of trial initiation with a speciﬁcity and sensitivity of 65% (57). Although further optimization of this panel is needed to reach sufﬁcient sensitivity and speciﬁcity, this study provides a proof of concept for the prospective use of methylation markers in early detection of cancer. DNA methylation markers can also be used for disease classiﬁcation, and to predict prognosis and response to therapy. For instance, methylation of RASSF1A in many tumors, including lung, breast, and prostate cancers, has been shown to be associated with poor prognosis (58). In another example, neuroblastomas harboring the CIMP phenotype were highly correlated with poor prognosis (59). Metastatic potential can be predicted on the basis of the E-cadherin promoter methylation in breast and oral cancers. In terms of response to therapy, the most compelling example to date is the hypermethylation of the MGMT (O6 -methylguanine methyltransferase) promoter, which increases the sensitivity of glioblastomas to alkylating agents (60). In addition to the study of single genes, large-scale techniques are now generating

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tumor methylation proﬁles, or methylomes, which can be used for molecular classiﬁcation. Furthermore, high-throughput platforms that can analyze the methylation state of a large number of loci in a large number of samples have been developed. One such recently described technology adapts a highthroughput single nucleotide polymorphism (SNP) genotyping system to detect methylation based on genotyping bisulﬁte-converted DNA (60a). By using this technology, ∼1500 CpG sites in ∼400 genes from 96 samples can be analyzed simultaneously. Studies using this technology identiﬁed panels of methylation markers that distinguished lung or bladder cancers from their normal counterparts at high speciﬁcity (61; G. Liang, E. Wolf, P. A. Jones, unpublished results). These panels are promising in terms of their implementation in DNA methylation analyses in large populations.

EPIGENETIC THERAPY Because of their dynamic nature and potential reversibility, epigenetic modiﬁcations are appealing therapeutic targets in cancer. Various compounds that alter DNA methylation and histone modiﬁcation patterns are currently being examined as single agents or in combination with other drugs in clinical settings. Most DNA methylation inhibitors (DNMTi) that have been clinically tested belong to the nucleoside analog family. These drugs are converted into deoxynucleotide triphosphates intracellularly and are incorporated into replicating DNA in place of cytosine. Their main mechanism of action is probably through trapping of the methyl transferases at sites of nucleoside incorporation (3), which depletes the cells of enzymatic activity, resulting in heritable demethylated DNA. Because incorporation occurs during DNA synthesis, only replicating cells are demethylated by DNMTi (62), which may confer the preference for highly proliferating cancer cells. The hypomethylation that ensues over the following cell division

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reactivates various silenced tumor suppressor genes, which is proposed to undermine the antineoplastic properties of the drugs. The prototypes of DNMTi are 5-aza cytidine and 5-aza-2 deoxycytidine. Initially described as cytotoxic agents (63), they were later found to cause DNA demethylation and differentiation and to reactivate silenced genes at much lower doses than those initially used (62). These low doses are now used, mainly for hematological malignancies, leading to better responses and lower toxicity. Both drugs were recently approved by the U. S. Food and Drug Administration (FDA) for the treatment of myelodysplastic syndrome, a preleukemic disease (64). Zebularine is a new addition to the family of nucleoside analogs that has demethylating properties. The drug can be delivered orally, is less toxic than the 5-aza analogs, acts preferentially on cancer cells, and inhibits polyp formation in female APC/MIN-deﬁcient mice (65; C. Yoo, P. A. Jones, unpublished results). However, the need for high concentrations of zebularine and its limited bioavailability in primates have slowed its advancement into clinical trials (66). As discussed above, epigenetic silencing is tightly coupled with histone deacetylation. Various compounds that inhibit HDACs have demonstrated antitumor, growth inhibitory, proapoptotic, and prodifferentiation properties (67). One of the universal targets of HDAC inhibitors (HDACi) is the cell cycle inhibitor p21, which is consistently upregulated by treatment with these drugs in conjunction with histone hyperacetylation at its promoter (68). Several silenced proapoptotic genes, which are members of the death receptor pathway, are also targets of HDACi treatment in leukemic cells, resulting in their promoter hyperacetylation and upregulation (69). Notably, tumor cells are almost always more sensitive to HDACi activity than healthy cells (70). It should be emphasized that in addition to their effects on transcription, the antitumoral activity of HDACi is probably mediated by other mechanisms,

such as disruption of higher-order chromatin structure and DNA repair pathways (67). In the clinic, many phase I trials show that these drugs are well-tolerated, and one of the initial HDACi, suberoylanilide hydroxamic acid (SAHA), has recently been approved by the FDA for the treatment of T cell cutaneous lymphoma. More are being developed and tested in clinical trials for both hematological and solid tumors (71). As histone methylation is also a major player in establishing long-term silencing, drugs targeting the enzymes involved in this modiﬁcation are being developed. For example, 3-Deazaneplanocin A (DZNep) was recently shown to deplete Polycomb group components, inhibit histone H3K27 methylation, and induce selective apoptotic cell death in breast cancer cells (72). In another study, the use of polyamine analogs inhibited the enzyme that removes the active H3K4 methylation mark, resulting in upregulation of aberrantly silenced genes in a cancer cell line (73). The speciﬁcities of these drugs and their potential clinical effectiveness need to be carefully established in further studies. As the interplay between epigenetic pathways is unraveled, the combination of epigenetic drugs with each other or with standard chemotherapies has become a focus of interest. HDACi and DNMTi show synergistic effects on transcriptional activation (74), and initial clinical trials using combinations of both have been promising (75). Further randomized trials are needed to prove their synergy in patients. Both classes of epigenetic drugs might sensitize cells to the action of biological agents such as all-trans retinoic acid, standard chemotherapeutics, or potential immunotherapies. Clinical trials using these combinations are ongoing (75). Despite the promise of epigenetic therapy, several concerns remain, mainly stemming from the nonspeciﬁcity of the drugs. Induction of genomic hypomethylation in mice caused chromosome instability and promoted tumor formation (76, 77), and the question www.annualreviews.org • Cancer Epigenetics

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arises whether the use of hypomethylating drugs will also have carcinogenic effects. One study examining this has not found such effects, although the number of patients was small and the time period short (75). Furthermore, in other mouse models, inhibition of DNMTs prevented tumor development (78). As clinical use of these drugs increases, these concerns will be answered in the coming years. However, the search for more speciﬁc drugs targeting epigenetic modiﬁcations is warranted.

CONCLUDING REMARKS With the recognition of the role of aberrant epigenetic processes in cancer and the rapid advent of new technologies to study them, this is an exciting time for the cancer epigenetics ﬁeld. National and international collaborations are forming to launch a human epigenome project (79). The ultimate aim of this project would be to map all epigenetic modiﬁcations, resulting in a comprehensive

description of these in both normal and diseased cells. Additionally, a pilot project to the Cancer Genome Atlas Project was recently launched, which aims to systematically explore the entire spectrum of genomic changes involved in human cancer, including epigenetic changes such as DNA methylation (80). The data derived from these projects will be able to answer questions such as how many genes are actually affected by epigenetic aberrations in a given tumor. They will also shed further light on the underlying mechanisms. Although screening using epigenetic markers is a promising prospect, speciﬁc and sensitive screening panels are yet to be developed and tested in large prospective clinical studies. It is important to directly compare the efﬁcacy of these panels with classic screening procedures and other evolving screening techniques based on proteomics, mRNA expression, or microRNA arrays. Knowledge of the prevalence and mechanisms of epigenetic modiﬁcations will allow the design of rational intervention strategies to target them.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:267-280. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer,1 Rosemarie H. DeKruyff,2 and Dale T. Umetsu2 1

Immunology Program, Stanford University, Stanford, California 94305; email: [email protected]

2

Division of Immunology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; email: [email protected]; [email protected]

Annu. Rev. Med. 2008. 59:281–92

Key Words

First published online as a Review in Advance on October 15, 2007

allergy, airway hyperreactivity

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.061506.154139 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0281$20.00

Abstract Asthma is an immunological disease with multiple inﬂammatory and clinical phenotypes, characterized by symptoms of wheezing, shortness of breath, and coughing due to airway hyperreactivity (AHR) and reversible airway obstruction. In allergic asthma, the most common form of asthma, airway inﬂammation is mediated by adaptive immune recognition of protein allergens by Th2 cells, resulting in airway eosinophilia. However, in other forms of asthma, inﬂammation is associated with immune responses to respiratory infections and airway neutrophilia. A central feature common to all forms of asthma is AHR, the heightened responsiveness of the airways to nonspeciﬁc stimuli. AHR has been shown recently in animal models of asthma to require the presence of CD1d-restricted, invariant T cell receptor-positive, natural killer T (i NKT) cells. Although allergenspeciﬁc Th2 cells and i NKT cells have many phenotypic similarities (e.g., expression of CD4 and production of Th2 cytokines), they have complementary activities, such as production of Th2 cytokines under different conditions, differential sensitivity to corticosteroids, and responsiveness to different classes of antigen (proteins versus glycolipids). We hypothesize that Th2 cells and i NKT cells interact synergistically to induce asthma but that different forms of asthma result from distinct roles of CD4+ i NKT cells versus Th2 cells.

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INTRODUCTION

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Airway hyperreactivity (AHR): heightened responsiveness of the airways to nonspeciﬁc stimuli such as cold air or smoke

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Bronchial asthma is a major public health problem whose prevalence has increased dramatically during the past two decades. It now affects nearly one in eight individuals in the developed world and a growing number in the developing world (1–3). The increased prevalence of asthma is due in part to rapid changes in the human environment, including changes in the incidence of many infectious diseases and changes in sanitation and air quality (2, 3). Asthma, which affects individuals of all ages, is characterized by airway inﬂammation and airway hyperreactivity (AHR), and by the symptoms of wheezing, shortness of breath, and coughing due to reversible airway obstruction. The most common form of asthma, representing 70%–80% of cases, is allergic asthma. However, there are multiple other forms (e.g., infection-induced, exercise-induced, aspirin-associated), reﬂecting multiple different pathogenic mechanisms and indicating that asthma represents a collection of heterogeneous syndromes. AHR is common to all forms of asthma and distinguishes it from nonasthmatic pulmonary inﬂammatory states such as pneuomonia, bronchiectasis, or sarcoidosis. AHR results in heightened clinical responses to nonspeciﬁc respiratory irritants such as cold air, fumes, or smoke. It can be assessed in humans or animals by measuring pulmonary function during challenge with increasing concentrations of a provocative stimulus such as methacholine or histamine. The degree of AHR predicts the severity of reversible airway obstruction and asthma symptoms. Although airway inﬂammation is associated with AHR, the degree of pulmonary inﬂammation does not always correlate with the severity of asthma (4). Therefore, direct assessment of AHR rather than of inﬂammation may be a more accurate measure of asthma severity, particularly in experimental models of asthma, although measurement of changes in airway inﬂammation (e.g., by measuring exhaled NO or sputum eosinophilia)

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may be helpful in clinical management of asthma. The complex inﬂammation that occurs in asthma is associated with as many as 12 types of inﬂammatory cells and >100 inﬂammatory mediators and gene products (2, 3, 5). In addition, airway epithelial cells, bronchial smooth muscle, ﬁbroblasts, and pulmonary nerves are thought to modulate the inﬂammation in asthma (3). In some forms of asthma, IFN-γ and neutrophils predominate in the airway (4), but in allergic asthma, the inﬂammatory process in the airways is associated predominantly with allergen-speciﬁc adaptive CD4+ Th2 cells, eosinophils, mast cells, and basophils (3). In patients with allergic asthma, CD4+ Th2 cells produce IL-4, IL-5, IL-9, and IL-13. These interleukins have essential roles in asthma, enhancing the growth, differentiation, and recruitment of eosinophils, basophils, mast cells, and IgE-producing B cells.

ADAPTIVE Th2 IMMUNE RESPONSES AND ASTHMA Allergen sensitization, the result of adaptive immunity to common aeroallergens, is a major risk factor for persistent asthma (6, 7). Sensitization results in the development of allergen-speciﬁc IgE as well as allergen-speciﬁc CD4+ Th2 cells. On challenge with allergen, mast cells armed with allergen-speciﬁc IgE respond rapidly with degranulation and release of mediators including histamine and leukotrienes, which increase airway resistance (8, 9). However, mast cells do not by themselves cause AHR, and the increase in airway resistance induced by mast cells is usually transient (8, 9). Allergen challenge also causes CD4+ Th2 cells to produce IL-4 and IL-13, which can act directly on smooth muscles and airway epithelium to induce AHR (10). In animal models, depletion of CD4+ T cells prevents the development of AHR (11). Furthermore, CD4+ Th2 cells are present in the lungs of virtually all patients with asthma (12), suggesting that adaptive immunity, with an interaction of mast cells, Th2

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cells, and eosinophils, plays a critical role in the development of asthma. The importance of CD4+ Th2 cells in asthma is very consistent with the Th1/Th2 paradigm, ﬁrst proposed more than 15 years ago (13), and with the idea that asthma involves atopy, the predisposition toward developing allergies and asthma. Atopic (asthmatic) individuals are genetically predisposed toward the differentiation of T cells into Th2 cells and away from the Th1 phenotype. This Th2 polarization may occur in response to normally harmless exogenous antigens, as a misaligned response to viral and bacterial infections, or secondarily to intrinsic inﬂammatory signals. Moreover, because Th2 cytokines mediate the recruitment and expansion of eosinophils, basophils, and mast cells, allergen-speciﬁc Th2 cells orchestrate the allergic inﬂammatory response that underlies asthma and allergy. The mechanisms that down-modulate the Th2-driven inﬂammation are not fully understood but may involve allergen-speciﬁc Th1 responses or some form of allergen-speciﬁc immune tolerance in which regulatory T cells produce IL-10 and/or TGF-β (14).

NATURAL KILLER T CELLS IN ASTHMA Natural killer T cells (NKT cells) resemble CD4+ Th2 cells in expressing the CD4 antigen and very rapidly producing large quantities of Th2 cytokines. Moreover, NKT cells possess the unique property of linking the innate and adaptive immune systems (15–17). As discussed below, there is strong evidence that NKT cells play an important role in asthma pathogenesis, and the body of evidence is growing as the technology for identifying this novel cell type becomes more widespread (18).

Characteristics of NKT Cells NKT cells express characteristics of both natural killer (NK) and conventional T cells. They can be divided into three types on the

basis of their T cell receptor (TCR) repertoire (16, 19). Type 1 NKT cells express a highly restricted repertoire of invariant αβ TCRs each of which has an invariant α-chain (Vα14-Jα18 in mice and Vα24-Jα18 in humans). These cells, also termed i NKT, are restricted by the MHC-class-I-like cell surface protein CD1d. Therefore, i NKT cells can be speciﬁcally identiﬁed by using CD1d tetramers loaded with the glycolipid α-galactosyl ceramide (α-GalCer). Type 2 NKT cells are also CD1d restricted but have a diverse TCR repertoire and therefore cannot be identiﬁed using α-GalCerloaded CD1d tetramers. Type 3 NKT cells are restricted to MHC (major histocompatibility complex) class I and class II molecules other than CD1d. Of the three types of NKT cells, the contribution of i NKT cells to AHR and asthma is best characterized (18, 20, 21). Unlike conventional T cells, which recognize protein antigens, i NKT and type 2 NKT cells recognize glycolipid antigens presented in the context of CD1d. CD1d is expressed by mucosal epithelial cells of the airways, bronchus, and intestine, as well as by hepatocytes, T cells, B cells, macrophages, and dendritic cells. The recognition of CD1dassociated glycolipids by i NKT cells is highly conserved across phylogeny. For example, αGalCer-loaded murine CD1d tetramers can bind to both the mouse and human invariant TCR of i NKT cells, strongly suggesting a critical, conserved role for these cells in immunity.

Atopy: genetic predisposition to develop allergies and asthma NK: natural killer TCR: T cell receptor

Subsets of i NKT Cells i NKT cells are either CD4+ or are double negative (CD4− CD8− ), and a small subset of human i NKT cells are CD8+ . The human CD4+ i NKT cell subset has been shown to differ from the CD4− CD8− i NKT cell subset, in that CD4+ i NKT cells produce greater amounts of IL-4, IL-13, and GMCSF (Th2 cytokines), whereas CD4− CD8− i NKT cells exhibit greater cytotoxicity and produce only IFN-γ and TNF-α (22, 23).

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Importantly, both CD4+ and CD4− CD8− i NKT cell subsets produce IFN-γ. In mice, CD4+ i NKT cells also produce more IL-4 than do CD4− CD8− i NKT cells and appear to be less effective in tumor immunity (24). Another subset of i NKT cells, which produce IL-17 but not IL-4 and which are NK1.1− , have been described (25), but their speciﬁc function is not yet clear. In addition, subsets of i NKT cells selectively expressing speciﬁc chemokine receptors may exist, which would explain the speciﬁc homing of some subsets of i NKT cells to speciﬁc organs, such as the liver, intestine, or lung. These various subsets of i NKT cells may play important roles in the lungs, either by enhancing the development of AHR in asthmatic patients or possibly by suppressing the development of AHR in normal nonasthmatic individuals.

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Functions of i NKT Cells The presence of a naturally occurring expanded population of cells expressing a TCR that is conserved across phylogeny suggests that the TCR of i NKT cells has speciﬁcally evolved as a pattern-recognition receptor for speciﬁc and important danger signals as part of an innate-like immunity (16, 26). Glycolipid antigens presented by CD1d can be endogenous, for example, when injured or damaged antigen-presenting cells express altered self glycolipids during infection or during any innate or adaptive immunological responses (16). Endogenous glycolipids include the lysosomal glycosphingolipid isoglobotrihexosylceramide (iGb3) and others not yet discovered (27, 28). Glycolipids presented by the CD1d may also be exogenous, as are, for example, bacterial glycosphingolipids found in the gramnegative, lipopolysaccharide-negative Sphingomonadaceae and Rickettsiaceae families, and in Borrelia burgdorferi (28–30). It thus appears that speciﬁc glycolipids can activate i NKT cells, communicating an immunological danger signal that may be of considerable importance, particularly at mucosal surfaces (31). 284

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On activation, i NKT cells produce large quantities of cytokines including IL-4, IL-13, and IFN-γ within minutes to hours of stimulation as a manifestation of innate-like immunity. The rapid production of cytokines by i NKT cells ampliﬁes and regulates adaptive immune responses by enhancing the function of dendritic cells, NK cells, and B cells, as well as conventional CD4+ and CD8+ T cells, thus linking innate and adaptive immunity (26). Moreover, the rapid production of cytokines by i NKT cells regulates the development of antimicrobial, autoimmune, antitumor, and antitransplant immune responses (15, 16, 26), providing either disease-causing or disease-protective effects. In particular, CD1d-restricted i NKT cells appear to play an important role both in the intestinal tract, where they produce IL-4 and IL-13 required for the development of colitis in mice and humans (32), and in the respiratory tract, where they contribute to AHR, as discussed below.

i NKT Cells in the Induction of AHR Over the past four years, several investigators have demonstrated that i NKT cells are required for the development of AHR (20, 21). In CD1d−/− mice, which are deﬁcient in type 1 and type 2 i NKT cells, allergeninduced AHR failed to develop, as measured by whole-body plethysmograph or by direct measurement of airway resistance and dynamic compliance in intubated and mechanically ventilated mice (20, 21). These results were conﬁrmed in another i NKT-celldeﬁcient mouse strain, Jα18−/− mice, which lack the α-chain of the invariant TCR and therefore speciﬁcally lack type 1 i NKT cells (20, 21). Like the CD1d−/− mice, the Jα18−/− mice failed to develop AHR when allergensensitized and challenged, and showed reduced airway eosinophilia (21). Adoptive transfer of puriﬁed wild-type i NKT cells into the Jα18−/− mice prior to allergen challenge fully reconstituted airway inﬂammation and AHR, indicating that i NKT cells were specifically required for the development of AHR

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Table 1 NKT cell-deficient mice do not develop airway hyperreactivity (AHR), but they do develop normal Th2 and IgE responses. However, AHR can be restored in these mice by adoptive transfer of wild-type (WT) NKT cells Wild-type mouse

NKT cell-deficient mouse

NKT cell-deficient mouse + WT NKT cells

Th2 responses

normal

normal

normal

IgE production

normal

normal

normal

AHR

normal

absent

normal

(Table 1). Production of IL-4 and IL-13 by the i NKT cells is evidentally required for the development of AHR, since adoptive transfer of i NKT cells from IL-4−/− IL-13−/− mice into Jα18−/− mice failed to restore AHR. Taken together, these studies demonstrate that i NKT cells producing IL-4 and IL-13 are required for the development of AHR. The requirement for i NKT cells in the development of AHR is curious because, despite the loss of AHR in CD1d−/− and Jα18−/− mice, allergen-speciﬁc Th2-driven responses develop normally in these mice. In addition, although they develop a degree of eosinophilic airway inﬂammation on challenge with allergen, this inﬂammation is clearly not sufﬁcient for the development of AHR. In response to exogenous protein antigens, allergen-speciﬁc Th2 cells can establish an inﬂammatory milieu in the lungs, which we believe leads to the activation of i NKT cells. It appears that the inﬂammation induced by protein antigen-speciﬁc Th2 cells allows the expression of endogenous glycolipids that then activate pulmonary i NKT cells. It should be noted that although Th2 cells may enhance i NKT cell activation, the reverse is also true. Administration of protein antigen with α-GalCer enhances antigen-speciﬁc priming, presumably because the production of IL-4 and IFN-γ by i NKT cells acts as an adjuvant to increase Th0 and Th2 cell development (33). Several additional observations suggest that i NKT cells are critically important for the induction of AHR. First, treatments that anergize or paralyze i NKT cells, for example with strong i NKT cell–activating glycol-

ipids, prevent the subsequent development of allergen-induced AHR (34–36). Second, direct activation of pulmonary i NKT cells by exogenously derived glycolipids is sufﬁcient for the induction of AHR. Thus, when pulmonary i NKT cells were directly activated by the glycolipid antigens α-GalCer or α-glucuronosylceramide (a glycolipid constituent of the membrane of Sphingomonas paucimobilis), AHR was rapidly induced (37). Importantly, the glycolipid-induced AHR response could develop in the absence of conventional CD4+ T cells and adaptive immunity (e.g., AHR increased in MHC class II knockout mice), or of eosinophils (e.g., AHR was unaffected by anti-IL-5 monoclonal antibodies) or B cells (e.g., AHR was only slightly reduced in B cell–deﬁcient JHD mice, which lack the heavy-chain immunoglobulin J region genes). These ﬁndings clearly indicate the importance of i NKT cells in the development of AHR. In conjunction with a report showing that i NKT cells recognize lipids from plant pollens (38, 39), these results suggest that exogenous glycolipids from microbes or from pollens that enter the lung may directly activate i NKT cells and induce AHR. Whereas the requirement for i NKT cells for the development of Th2-biased, allergeninduced AHR has been conﬁrmed by a number of independent research groups (18, 20, 21), the precise role of i NKT cells in nonatopic forms of asthma remains to be determined. For example, in C57BL/6 mice challenged with α-GalCer or its analog PBS 57, airway neutrophilia rather than eosinophilia develops, in association with an NK1.1− i NKT cell subset producing IL-17

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(25). Whether IL-17-producing i NKT cells enhance or inhibit AHR is not yet clear, since administration of recombinant IL-17 appears to inhibit inﬂammation in allergic asthma (40). On the other hand, IL-17 is present in the induced sputum of patients with asthma (41), suggesting that IL-17-producing i NKT cells may contribute to some forms of AHR. i NKT cells may not be required in all forms of AHR. β2-microglobulin−/− mice lack i NKT cells but develop AHR normally when sensitized and challenged with allergen (42, 43). However, although β2microglobulin−/− mice do not express the heterodimeric CD1d molecule (consisting of CD1d and β2-microglobulin chains), they do retain a β2-microglobulin-independent form of CD1d (32) and contain type 1 and type 3 NKT cells. The development of AHR in these mice, which lack both CD8+ T cells and i NKT cells, suggests that Th2 cells may step in under certain circumstances to act as effector cells of AHR, or alternatively that type 2 or 3 NKT cells may substitute for i NKT cells in inducing AHR. In any case, understanding the precise mechanisms by which AHR develops in other forms of asthma may help to clarify the various cell types that can induce AHR development.

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i NKT Cells in Human Asthma The importance of i NKT cells in murine models of asthma suggested that i NKT cells could play an important role in human asthma. However, since mouse models do not replicate all features of human asthma, direct assessment of i NKT cell function in humans with asthma is required. Using CD1d tetramers loaded with α-GalCer, we examined the frequency and distribution of i NKT cells in the lungs of patients with moderate to severe persistent asthma. As controls we used normal nonasthmatic individuals as well as patients with sarcoidosis, an inﬂammatory lung disease also associated with large numbers of pulmonary CD4+ T cells (44). Surprisingly, a large number (∼60%) of the pulmonary 286

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CD3+ T cells in patients with symptomatic asthma were i NKT cells, and the majority of these i NKT cells were CD4+ , whereas <0.1 of peripheral blood CD3+ T cells are i NKT cells, and of these only ∼40%–50% express CD4. Normal individuals have almost no i NKT cells in the lungs, and in patients with sarcoidosis, <1% of the CD3+ T cells in the lungs are i NKT cells. When stimulated with α-GalCer, the pulmonary i NKT cells from patients with asthma rapidly produced IL-4 and IL-13 but not IFN-γ, suggesting that a subpopulation of i NKT cells that was CD4+ and producing Th2 cytokines was selectively recruited or expanded in the lungs. These results also suggest that the i NKT cells present in the lungs of asthmatics have a signiﬁcant pathological role. There are now seven additional published studies on the presence of pulmonary i NKT cells in patients with asthma. Five of these reports conﬁrm that the lungs of asthmatic patients contain more i NKT cells than those of normal controls, although the reported levels of i NKT cells in asthma range from 1% to 10% of T lymphocytes in bronchoalveolar lavage (BAL) ﬂuid or sputum samples (39, 45–48). The two other studies do not examine control BAL ﬂuid from normal individuals but conclude that i NKT cells are not increased in asthma (49, 50); however, these studies report that up to 2% of the T cells in the lungs of asthmatic patients are i NKT cells. This is almost certainly a greater percentage than would be found in normal individuals, whose pulmonary NKT cell numbers are generally <1%. Thus, all eight reports suggest that i NKT cells are indeed present in the lungs of patients with asthma but that the speciﬁc number is highly variable. This is not surprising because asthma severity, like most disease processes, varies considerably, and the number of T cells in asthma can vary more than 100-fold. Moreover, our recent studies (O. Akbari, P. Matangkasombut, E. Israel, D. Umetsu, manuscript in preparation) conﬁrm that the number of i NKT cells in the lungs of patients with asthma of varying severity varies

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considerably (from 1% to 63% of CD3+ cells), roughly correlating with control of asthma symptoms. The speciﬁc number of i NKT cells in the lungs of patients with asthma may not be the critical issue. Like mast cells in anaphylaxis (51), i NKT cells are potent, so even small numbers of i NKT cells may dramatically affect the development of AHR. In naive mice, where the level of i NKT cells in BAL ﬂuid is <1% of the lymphocytes, administration of glycolipids to directly activate i NKT cells rapidly induces AHR. Furthermore, in different murine models of AHR, the number of i NKT cells in the lungs varies from 2% to 50% of T cells (T. Yasumi, P. Matangkasombut, D. Umetsu, manuscript in preparation). Therefore, the important issue now should not be whether NKT cells are present in the lungs in asthma, but rather what mechanisms control the frequency and the function of i NKT cells in the lungs and airway compartment. Understanding the function of pulmonary i NKT cells is important because i NKT cells, while displaying a phenotype similar to conventional Th2 cells (expression of CD4 and production of Th2 cytokines), also display very distinct activities in the pulmonary environment, the most important of which is the capacity to induce AHR. Furthermore, unlike conventional Th2 cells, i NKT cells are resistant to the effects of corticosteroids, the mainstay for the treatment of asthma, and continue to produce cytokines at doses that inactivate Th2 cells (17). The corticosteroid resistance of i NKT cells may explain corticosteroid-resistant asthma, which affects 10%–30% of all patients with asthma. Corticosteroid-resistant asthma accounts for a majority of severe asthmatics, half or more of the health-care costs associated with asthma, and a disproportionate number of deaths (52). In addition, i NKT cells appear to be able to produce Th2 cytokines and alter immunity under conditions in which Th2 cytokine production by conventional T cells would be shut down. For example, in a murine model of

tumor immunosurveillance, IL-13-producing i NKT cells prevented the clearance of a transplanted ﬁbrosarcoma even in the presence of CD4+ T cells producing IFN-γ (53). The regulation of Th2 cytokine production thus differs signiﬁcantly in i NKT cells and conventional CD4+ T cells (16, 26, 54). In fact, transcriptional regulation of IL-4 synthesis in i NKT cells may more closely resemble that of mast cells (54). Another functional difference between i NKT cells and conventional Th2 cells in the lungs is the ability of i NKT cells to increase the repertoire of antigens that can generate pulmonary inﬂammation. i NKT cells respond to glycolipid antigens, allowing pulmonary responses to include nonprotein components of microbes or plant pollens (37, 39). The responses by i NKT cells to such exogenous glycolipids allow the induction of AHR independent of Th2-type allergen sensitization. Alternatively, the interactions between Th2 cells and i NKT cells may amplify airway inﬂammation and AHR. For example, protein allergen exposure may activate allergenspeciﬁc T cells or mast cells, which then alter dendritic cells or other antigen-presenting cells in a way that exposes endogenous glycolipids, which then activate i NKT cells. Investigating the interaction between allergenspeciﬁc Th2 cells and i NKT cells in humans may provide insight into how allergic inﬂammation in general incites the development of asthma and AHR. This relationship between Th2 cells and i NKT cells is important because it may explain why only ∼30%–40% of individuals who develop allergen-speciﬁc Th2 cells (e.g., patients with allergic rhinitis and sensitization to aeroallergen) also develop asthma. This distinction between allergen sensitization and AHR suggests that factors in addition to Th2 cells, such as the accumulation of i NKT cells in the lungs, may be required for the development of asthma and AHR. Thus, further study of i NKT cells in the respiratory tract may greatly improve our understanding of asthma pathogenesis.

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ASTHMA PHENOTYPES, NKT CELLS, AND Th2 CELLS

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To what extent are i NKT cells a common effector mechanism in the development of the different forms of human asthma? Although more studies are required to fully answer this question, we suggest that i NKT cells may play an important but varying role in most types of asthma and AHR, in concert with other types of T cells, such as Th2 cells. As discussed above, asthma is exceedingly heterogeneous not only in etiology but also in severity. Asthma is currently categorized according to clinical history and the timing and general intensity of episodes, on a continuum from mild intermittent through mild persistent, moderate persistent, and severe persistent (5, 55). There is some evidence, however, that severe persistent asthma represents a distinct pathological entity, characterized by corticosteroid resistance and/or neutrophilic airway inﬂammation (56). In addition, asthma can be classiﬁed as atopic or nonatopic (3, 5, 14). In atopic asthma, which affects 60%– 80% of asthmatics, airway inﬂammation is driven by inherited allergic responses to inhaled protein allergens, usually beginning in childhood. In contrast, nonatopic or intrinsic asthma classically develops in adulthood, is sometimes associated with aspirin sensitivity, is less responsive to symptom-based therapy, and is not associated with allergies but rather with recurrent respiratory tract infections. In practice, the speciﬁc etiologies of asthma exacerbations are often hard to deﬁne, and often multiple mechanisms coincide; for example, upper respiratory infections also exacerbate asthma in atopic individuals. We hypothesize that different asthma phenotypes reﬂect the distinct roles of i NKT cells versus Th2 cells. In mild to moderate persistent asthma, i NKT cells may serve as airway effector cells under the ultimate pathoregulation of conventional allergensensitized CD4+ Th2 cells, which are responsive to corticosteroid therapy (Figure 1). In this form, Th2 cells establish the inﬂammatory milieu, which results in expression of en288

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Mild to moderate asthma Allergen

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Severe asthma Microbial or plant glycolipids

NKT cells Figure 1 Possible relationships between Th2 cells, NKT cells, and their function in patients with asthma of different phenotypes. (a) In patients with mild to moderate asthma, allergen activates Th2 cells, which attract NKT cells into the lungs. Th2 cells may also induce the expression of endogenous glycolipids that activate NKT cells. These processes are corticosteroid-sensitive. (b) In patients with severe asthma, exogenous microbial or plant glycolipids enter the lungs and directly activate NKT cells, which may be already present in the lungs in large numbers. This process may be corticosteroid-resistant.

dogenous glycolipids that attract and activate i NKT cells. In contrast, in severe persistent asthma, especially in corticosteroid-resistant asthma, i NKT cells may act as airway effector cells without pathoregulatory oversight by conventional Th2 cells. In this case, i NKT cells could be directly triggered to induce AHR, for example by glycolipids from infectious microbes or by the differential presentation of endogenous glycolipids secondary to airway insult from viruses or other nonspeciﬁc sources.

FUTURE DIRECTIONS The importance of i NKT cells in asthma is a novel and surprising ﬁnding that greatly

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alters our understanding of the pathobiology of asthma. The study of i NKT cells has only recently become possible with the availability of reagents to identify them. Many more experiments and studies are needed to clarify their roles in various forms of asthma. Although many clinical investigators are currently focused on establishing the “correct” number of i NKT cells that are present in the lungs of patients with asthma, it is likely that the ﬁgure will vary considerably, given the potent and complex function of i NKT in asthma. Multiple i NKT subsets appear to exist, and multiple glycolipids appear to bind to the conserved, invariant TCR of i NKT cells, resulting in i NKT cell activation. Thus, the role of i NKT cells may be extremely ﬂexible, such that they can directly induce AHR or

enhance Th2 sensitization to exogenous allergens. Moreover, identiﬁcation of the speciﬁc glycolipid antigens that activate i NKT cells is only beginning; these multiple endogenous and exogenous glycolipids may differentially induce distinct i NKT cell cytokine proﬁles and functions. These multiple functions may explain how this novel cell type may critically regulate the development of distinct forms of asthma. Finally, if our hypothesis regarding i NKT cells in asthma is correct, then therapies that disrupt the activation or effector function of pulmonary i NKT cells may be highly effective in the treatment of multiple forms of human asthma, especially forms that are currently recalcitrant to available therapies, such as corticosteroid-resistant asthma.

DISCLOSURE STATEMENT Dr. Umetsu is a consultant for Innate Immune, Inc.

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:281-292. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson Department of Medicine/Division of Rheumatology, Washington University School of Medicine, St. Louis, Missouri; email: [email protected]

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Key Words

First published online as a Review in Advance on August 16, 2007

factor H (FH), membrane cofactor protein (MCP; CD46), factor I (FI), factor B (FB), complement C3 (C3), transplantation

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.060106.185110 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0293$20.00

Abstract Hemolytic uremic syndrome is a triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. It is one of a group of conditions termed the thrombotic microangiopathies, which are characterized by prominent endothelial cell injury. It may be diarrheal-associated or atypical (aHUS). Evidence for a pathogenic role of the alternative pathway of complement was ﬁrst suggested in 1974. Mutations in the complement regulatory proteins factor H, membrane cofactor protein (CD46), and factor I predispose to aHUS development. Mutations of the activating components factor B and complement C3 have also been reported. Penetrance is ∼50%, suggesting other genetic and environmental modiﬁers are needed for disease expression. Identiﬁcation of mutations is important owing to differences in mortality, renal survival, and outcome of renal transplantation. Current treatment is plasma infusion/exchange, but complement inhibitor therapy provides hope for the future.

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THE HEMOLYTIC UREMIC SYNDROME HUS: hemolytic uremic syndrome TTP: thrombotic thrombocytopenic purpura D+ HUS: diarrheal-associated HUS

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aHUS: atypical HUS CP: classical pathway AP: alternative pathway

The hemolytic uremic syndrome (HUS) was described by von Gasser et al. in 1955 (1). It is deﬁned as the clinical triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure (2). HUS is one of a group of conditions termed the thrombotic microangiopathies. Other examples include thrombotic thrombocytopenic purpura (TTP) and pre-eclampsia (3). Such conditions are characterized by a precipitating endothelial cell injury (4). HUS is subdivided into two major clinical categories. Diarrheal-associated HUS (D+ HUS) is the form characterized by a preceding infection with a Shiga toxin-secreting pathogen, typically E. coli O157:H7. This pathogen causes painful and typically bloody diarrhea, and ∼10% of those affected subsequently develop D+ HUS (5). This subtype is the most common (∼95%), with a reported incidence of 2 cases per 100,000 persons per annum (6). Renal function recovers in the majority of cases (up to 70% in various series) (7). In contrast, atypical HUS (aHUS)—also called D– or nonenteropathic HUS—is rare, with an estimated annual incidence of two cases per million population (6). It may be familial. Although it can occur at any age, aHUS is most commonly seen in early childhood. It is associated with a poor overall prognosis: Up to 50% of cases progress to end-stage renal failure, and 25% of patients may die in the acute phase of the disease (7).

The predominant pathological abnormality in aHUS is found in the renal arterioles and interlobular arteries. Widespread endothelial swelling occurs, with retraction leading to exposure of the basement membrane. The vessel lumens are occluded by red blood cells and platelet rich ﬁbrin thrombi. This preglomerular picture differs from the pathology of D+ HUS, which predominantly affects the glomerular capillaries (reviewed in Reference 8).

THE COMPLEMENT SYSTEM AND ITS REGULATION Complement Activation Complement is an ancient archetypal defense system that promotes the inﬂammatory response and destroys pathogens by altering their membranes (opsonization and lysis). Through these mechanisms it fulﬁlls its three key roles (9): (a) It protects the host against invading pathogens; (b) it bridges innate and adaptive immunity; and (c) it disposes of immune complexes and injured tissues and cells. Complement is activated by three main pathways: the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP) (Figure 1a). The CP is activated by binding of the C1 complex to antibodies bound to antigen. The LP is activated by binding of mannose-binding lectin and the mannose-binding lectin-associated proteases 1 and 2 to mannose groups on the surface of invading bacterial pathogens. The AP, which plays a key role in the pathogenesis of aHUS,

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 1 (a) Diagram of the complement system showing the classical pathway (CP), lectin pathway (LP) and alternative pathway (AP). The ampliﬁcation loop of the AP is emphasized. Factor D (FD) and AP C3 convertase (C3bBb) are serine proteases that cleave at one speciﬁc site to activate their substrates. Excessive activation via this feedback loop secondary to a loss of regulatory activity or a gain of functional activity predisposes to atypical hemolytic uremic syndrome (aHUS). (b) Regulation of the AP C3 convertase by decay-accelerating activity (DAA) and cofactor activity (CA). CA requires a cofactor protein, such as factor H (FH) or membrane cofactor protein (MCP), so that the plasma serine protease factor I (FI) can cleave C3b to iC3b. (c) Cofactor activity. One of these cofactor proteins must bind C3b before FI can inactivate C3b by limited proteolysis. The iC3b fragment generated by this reaction cannot form a C3 convertase. 294

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a. LP CP

AP

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C3

C3b C3bBb(P)

FP

b.

FB

C3bB

FD

DAA C3bBb

C3b + Bb CA

C3b

iC3b + C3f FI C3f

c.

MCP

MCP

Inactivation

Binding C3b

C3b

iC3b

+ Factor I

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FB: factor B FH: factor H DAF: decay-accelerating factor MCP: membrane cofactor protein (CD46)

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is continually activated by a “tick-over” mechanism. In this process, complement C3, the central molecule of the complement cascade, undergoes spontaneous hydrolysis, depositing C3b onto the surface of foreign and host cells in the vicinity. On an activating surface such as a bacterium, C3b joins with factor B (FB), which is then cleaved by factor D to form the C3 convertase, C3bBb. The binding of properdin stabilizes this enzyme. This enzyme complex then cleaves more C3 to C3b to initiate a feedback loop. Unchecked, C3b may also join with the C3 convertase to form the C5 convertase. C5 is cleaved to the anaphylatoxin C5a and C5b, which initiates formation of the lytic membrane attack complex (C5b– 9). Insertion of the membrane attack complex can lyse many viruses and gram-negative bacteria and, through sublytic attack, promote proinﬂammatory changes on host cells. This action is desired on microbes or altered necrotic or apoptotic self cells but undesirable on normal host cells. The latter are protected from complement activation on their surface by a wide array of soluble and membraneassociated complement regulatory proteins, which downregulate complement activation and prevent “bystander damage” of host cells.

these functions, e.g., decay-accelerating factor (DAF) performs only decay-accelerating activity whereas membrane cofactor protein (MCP) performs only cofactor activity. Many of the complement regulatory proteins are found in the RCA (regulators of complement activation) gene cluster on chromosome 1q32 and work together in the regulation of complement. Another important mechanism for regulation of the AP is the means by which host cells are recognized as “self ” or “nonactivators” of complement at times of inﬂammation. Host cells express polyanionic residues such as sialic acid, to which FH can bind via its carboxylterminal domain. C3b deposited on host cells binds FH with a greater afﬁnity than FB in this setting. FH then uses its decay-accelerating and cofactor activity to suppress complement activation. An example is the cleavage by FH plus factor I (FI) of C3b to inactive iC3b. The latter cannot support C3 convertase formation, and thus further complement activation is prevented. The importance of this mechanism of host defense is illustrated by the fact that it has been subverted by many pathogens, which express sialic acid– like residues on their surface, bind FH, and thereby evade AP activation.

Complement Regulation

Early Evidence of a Role for Complement in the Pathogenesis of HUS

Two types of inhibitory activity, termed decay-accelerating activity and cofactor activity, are relevant to the control of the AP (Figure 1b,c). Decay-accelerating activity refers to dissociation of C3 and C5 convertases. In the case of the AP, the decay accelerator protein displaces the catalytic Bb from the target-bound C3b. However, this C3b is free to then bind another FB molecule and reform the convertase. To prevent this, C3b is then inactivated by limited proteolytic cleavage by a protease that irreversibly cleaves C3b in the presence of its cofactor. This is cofactor activity. Many regulators can mediate both functions, e.g., factor H (FH) in the AP; other regulators perform only one of 296

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Historically, a role for the AP of complement has been occasionally noted in the pathogenesis of both forms of HUS. An association between reduced levels of the third complement component (C3) and D+ HUS was reported in 1974 (10). The C3 levels normalized with remission of the disease. Low C3 levels as well as increased levels of FB and C3 breakdown products were reported in the plasma of a few patients with aHUS (11, 12). Granular C3 deposits were also observed in the glomeruli and arterioles from kidney biopsies in the acute phase of disease (13, 14). C4 levels were usually normal and C4 was not

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Frequency and phenotypes of mutations in complement genes in aHUS

Complement protein

Distribution

Frequency in aHUS (%)

Clinical outcome

Risk of recurrence in renal transplant

FH

serum

15–30

poor

high

MCP

cell surface

10–13

good

low

FI

serum

5–12

poor

high

FB

serum

0–3

poor

N/A

C3

serum

N/A

N/A

N/A

∗

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Abbreviations: FB, factor B; FH, factor H; FI, factor I; MCP, membrane cofactor protein; N/A, not available. ∗ There was recurrence in the allograft of the only FB-HUS transplant so far performed.

seen in biopsy specimens, suggesting AP activation. Some patients with recurrent relapsing aHUS had persistently low serum levels of C3, even during remission of disease (12), suggesting continuous overactivation of the AP. The signiﬁcance of an early report of FH deﬁciency in two brothers with familial aHUS (15) was not appreciated for many years (16). It has since been demonstrated that heterozygous deﬁciency of FH predisposes to aHUS. In contrast, homozygous FH deﬁciency is associated with development of partial facial lipodystrophy, type II membranoproliferative glomerulonephritis (17), and recurrent infections with encapsulated bacteria secondary to reduced C3 levels.

COMPLEMENT PROTEINS AND ATYPICAL HUS In recent years, mutations in the complement regulatory proteins FH (16–24), MCP Table 2

(CD46) (18, 25–27), and FI (18, 27–29) have been established as a predisposing genetic factor for the development of aHUS (Tables 1 and 2). Studies characterizing the functional abnormalities in complement regulation caused by these mutations have begun to elucidate the mechanisms by which complement protein mutations are pathogenic and lead to the development of aHUS. In 2007, mutations in two activating components of the AP, namely FB (30) and C3 (31), were also reported in association with aHUS.

Complement Factor H (FH) FH is a serum glycoprotein predominantly synthesized by the liver and composed of 20 complement control protein (CCP) modules. FH is the most important ﬂuid-phase regulator of the AP (32). It acts as a cofactor for FI-mediated proteolytic inactivation of C3b, competes with FB for C3b binding, and

Complement regulatory proteins and aHUS

Complement protein

Mol. wt. (kDa)

Plasma conc. (μg/ml)

FH

150a

242–759

DAA, CA

yes

MCP

60a

–b

CA

yes

FI

90

39–100

CA (protease)

yes

DAF

65a

–b

DAA

no

Function

Haploinsufficiency predisposes to aHUS?

Abbreviations: CA, cofactor activity; DAA, decay-accelerating activity; DAF, decay-accelerating factor; FH, factor H; FI, factor I; MCP, membrane cofactor protein. a These proteins are composed entirely (FH) or largely (MCP and DAF) of a homologous repeating protein module of ∼60 amino acids. The genes encoding all three are in the RCA cluster at 1q32. b MCP and DAF are membrane proteins. MCP is a typical type I transmembrane protein; DAF is tethered to the membrane by a glycolipid (GPI) anchor. www.annualreviews.org • Hemolytic Uremic Syndromes

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FHR1–5: factor H–related proteins 1–5

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accelerates the decay of the C3 convertase into its components. It can also downregulate complement activation on host surfaces by binding to the polyanionic glycosaminoglycans of endothelial cells and exposed basement membranes via its carboxyl-terminal domain (CCP19 and CCP20). The complement regulatory functions, decay acceleration and cofactor activity, are located in CCPs 1–4. The initial formal genetic association between FH and aHUS was made by Warwicker et al. in 1998 (16). They established linkage in three families with aHUS to the RCA gene cluster, containing the FH gene, on chromosome 1q32. This ﬁnding led directly to the discovery of the ﬁrst mutations in FH as a predisposing cause of aHUS (16). Following this report, mutations in FH were described in ﬁve large cohorts of aHUS patients; FH mutations account for up to 30% of reported cases (17– 22). The majority of FH mutations (60%– 70%) are heterozygous missense mutations that cluster in the C-terminal region of FH. The genes for FH and FH-related proteins (FHR1–5) are all tightly linked in the RCA cluster, which arose by several large genomic duplications. These duplications have resulted in a high degree of sequence identity between the gene for FH and genes for FHR1–5 (33, 34). These large genomic duplications predispose to gene conversion events and genomic rearrangements (35). Unambiguous evidence of a role for gene conversion in generation of FH mutants in aHUS was provided by Heinen et al. (23). In their panel, 44% of all FH mutations associated with aHUS could have arisen by gene conversion. Large genomic rearrangements in FH have also been described (24). Functional studies of FH mutants in aHUS have demonstrated, for the most part, decreased binding to glycosaminoglycans and cultured endothelial cells. They have also shown a decreased binding capacity for the FH ligand, C3b. This is in keeping with recent renal biopsy data from an aHUS patient with a C-terminal mutant showing reduced FH binding to damaged renal endothelium Kavanagh

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compared to wild-type FH (36). The elucidation of the structure of CCPs 19–20 by nuclear magnetic resonance (37) and X-ray crystallography (38) has further enhanced our understanding of the impaired FH binding to host surfaces seen in aHUS. In addition to mutations in FH, Caprioli et al. (39) and others (40, 41) have demonstrated an association between FH single nucleotide polymorphisms (SNPs) and aHUS. One of these polymorphisms, c.-257T, is located in a putative NF-κB binding site of the FH promoter. Whether this has a role in determining expression at the time of inﬂammation or infection, or whether the SNPs are in linkage disequilibrium with other susceptibility alleles, remains to be determined. In addition to genetic defects, FH autoantibodies have been reported to predispose to aHUS (6% of cohort) (42). These antibodies resulted in an acquired defect in the regulatory function of FH.

The Factor H–Related Genes (FHRs) In addition to FH, the RCA cluster contains ﬁve FH-related genes (FHR1–5). A haplotype containing a deletion of FHR1 and FHR3 is common and is probably due to nonallelic homologous recombination. This haplotype has been demonstrated to increase the risk of aHUS (43). FHR1 and FHR3 both lack intrinsic cofactor and decay-accelerating activity; however, both bind C3b and heparin, suggesting a regulatory function for C3b. In addition, FHR3 does have cofactor-enhancing activity (44). Serum from patients with this deletion showed an impaired ability to protect erythrocytes from complement activation (43). Nevertheless, it is not yet clear whether the absence of FHR1 and FHR3 is responsible for the increased risk of aHUS per se or whether this deletion is in linkage disequilibrium with other susceptibility alleles in FH. FHR5 is the only factor H–related protein that possesses intrinsic cofactor activity (45). Recent analysis of FHR5 demonstrated that polymorphisms in this gene were more

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common in a panel of aHUS patients than in a control population (46). Further evaluation is required to assign causality to this ﬁnding.

reduce transcription activity in reporter gene assays (40, 41).

Factor I (FI)

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Membrane Cofactor Protein (MCP; CD46) MCP is a widely expressed membrane inhibitor of complement activation (47). It is on the surface of most human cells with the notable exception of erythrocytes, and is highly expressed in the kidney, particularly on endothelium. MCP protects host cells from complement attack by serving as a cofactor for FI. It is an intrinsic complement regulator, protecting the cell to which it is bound by the proteolytic inactivation of deposited C3b and C4b on host cells. The gene for MCP, like that for FH, resides in the RCA cluster at 1q32. Linkage analysis implicated MCP as a candidate gene, and this led to the discovery of mutations in MCP in three families in 2003 (48). Since this initial description, more than 20 mutations in MCP have been described in aHUS (18, 25–27, 49). Mutations in MCP account for 10%–13% of aHUS cases (18). Most mutations in MCP are heterozygous, although ∼25% are either homozygous or compound heterozygous (49). The majority of mutations cluster in the four extracellular complement control protein domains of MCP, which contain the region critical for complement regulation. Around 75% of mutants described have reduced surface expression on peripheral blood mononuclear cells (type I mutation). In the remaining 25%, the mutant is expressed normally but has absent or decreased complement regulatory activity (type II mutation) (49, 50). These mutants in MCP affect the ability of the protein to downregulate the AP of complement via cofactor activity. In addition to mutations in MCP, a significant association between a speciﬁc SNP haplotype block in MCP, termed the MCPggaac haplotype, has been reported (41). This risk haplotype contains SNPs in the MCP promoter region that have been demonstrated to

CR1: complement receptor 1

FI is an 88-kDa serum glycoprotein predominantly synthesized in the liver. It is a speciﬁc serine protease that cleaves the α chains of C3b and C4b in the presence of a cofactor protein. These cofactors are FH for C3b, C4 binding protein (C4BP) for C4b, and MCP and complement receptor 1 (CR1) for both. By inactivating these proteins and preventing the formation of the C3 and C5 convertases, FI downregulates both the AP and the CP. Unlike the genes for MCP, FH, CR1, and FHR1–5, the FI gene does not reside in the RCA cluster on 1q32 but is located on chromosome 4q25. Mutations in FI have been reported in patients with aHUS (18, 27–29). FI is a heterodimer. It consists of a noncatalytic heavy chain linked by a disulﬁde bond to a catalytic light chain. Mutations in FI appear to be a less common cause of aHUS than FH or MCP mutations, accounting for 5%–12% of aHUS cases. All FI mutations so far described are heterozygous. Approximately half of the FI mutants associated with aHUS are type I mutants and result in low serum levels of FI. Functional analysis of the type II mutations that occur in the light-chain serine protease domain of FI has revealed loss of CP and AP cofactor activity (51).

Decay-Accelerating Factor (DAF; CD55) DAF is located in the RCA cluster. This widely expressed complement regulator accelerates the decay of either C4b2a or C3bBb and the corresponding C5 convertases but lacks cofactor activity. In 120 aHUS patients in the two cohorts so far screened (30, 52), only one mutation has been found in DAF (I197V), and it did not impair the activity of the protein (52). This is surprising because DAF is a potent inhibitor of AP C3 convertase, is www.annualreviews.org • Hemolytic Uremic Syndromes

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expressed on endothelial cells, and works synergistically with MCP (49, 50, 53).

mutations predispose to aHUS indicates that tight control of AP activation is required.

Complement Receptor 1 (CR1; CD35)

C3 Mutations

CR1, a C3b/C4b binding protein also known as the immune adherence receptor, is a membrane-bound complement regulator with AP and CP cofactor and decay-accelerating activity. It is expressed on all erythrocytes, B cells, polymorphonuclear leukocytes, monocytes, follicular dendritic cells, and glomerular podocytes but is not found on glomerular microvascular endothelium. In one reported cohort of aHUS patients, no mutations were described (54).

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COMPLEMENT COMPONENTS AND ATYPICAL HUS: GAIN-OF-FUNCTION MUTATIONS Factor B (FB) Mutations The initial genetic causes of aHUS described above have the common effect of loss of cofactor activity in complement regulatory proteins. Recently, a completely novel mechanism was reported: Goicoechea de Jorge et al. found gain-of-function mutations in FB associated with aHUS (30). Mutations in FB are rare, accounting for 0%–3% of aHUS cases in the panels examined (30, 56). FB is the zymogen that carries the catalytic site of the complement AP convertase (C3bBb). Mutations in FB were found in two families. One mutant showed enhanced formation of the C3bB proenzyme, likely to produce a more active enzyme in vivo. The other mutant formed a C3bBb enzyme more resistant to decay by the complement regulators DAF and FH, which will also cause increased enzyme activity in vivo. This provides evidence that gain-of-function mutations predispose to aHUS and reinforces the importance of the AP in the pathogenesis of aHUS. The fact that both loss- and gain-of-function 300

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C3 is the most abundant serum complement protein and is critical to all three complement activation pathways. A recent abstract suggests that heterozygous mutations in C3 predispose to aHUS (31). Analogous to FB, the expectation is that these mutations would produce a gain of function.

MOLECULAR DIFFERENTIATION OF ATYPICAL HUS AND TTP Thrombotic thrombocytopenic purpura (TTP), like HUS, is a thrombotic microangiopathy. Clinical differentiation has historically been based on predominant neurological involvement for TTP and predominant renal involvement for HUS. Recent advances allow discrimination based on the molecular biology of the conditions. TTP is caused by mutations in the gene for ADAMTS13 (a disintegrin and metalloprotease with eight thrombospondin-1-like domains), which codes for a von Willebrand factor (vWf )–cleaving protease, or by antibodies against this protein (57). Unlike aHUS, TTP has not been linked to mutations in complement proteins. However, there is a case report of TTP in two sisters, one of whom had unusually severe renal involvement requiring long-term dialysis while the other had solely neurological features. The sister with renal involvement had both ADAMTS13 and FH mutants, whereas the sister with only neurological features had only an ADAMTS13 mutant (58). This suggests that the presence of the FH mutant modulates expression of disease phenotype with regard to renal pathology. The importance of a predisposing endothelial cell injury in the two conditions is supported by data from recent mouse models of aHUS (59) and TTP (60, 61), described below.

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LESSONS FROM COMPLEMENT REGULATORY PROTEIN INVOLVEMENT IN ATYPICAL HUS AND AMD

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Why Does Overactivation of Complement Lead to aHUS? An acquired or inherited deﬁciency of complement regulatory proteins of the AP has been associated with a marked increase in susceptibility to renal disease (9). Theories about the particular susceptibility of the renal endothelium to complement-mediated damage in aHUS relate to the fenestrated glomerular endothelium, a lack of endogenous membrane-bound complement regulators on the glomerular basement membrane, and the high shear stress to which the kidney is exposed during times of endothelial cell injury. In aHUS many of the currently identiﬁed predisposing triggers to disease development are recognized causes of endothelial injury. Examples include infections such as HIV and S. pneumoniae, chemotherapeutic agents such as mitomycin, and pregnancy (2). There are ﬁve main mechanisms by which abnormalities of complement regulation predispose to the development of aHUS in the presence of an endothelial cell insult: 1. Insufﬁcient complement regulatory protein may be available at the time of injury owing to a failure of protein production caused by type I mutations. 2. The protein may have a functional abnormality. For example, mutations in the C terminus of FH prevent binding to sialic residues on exposed glomerular basement membrane following endothelial cell injury. This allows complement activation to proceed and a prothrombotic environment to develop. 3. Autoantibodies to a complement protein may prevent its normal function. 4. A mutation may stabilize a convertase and inhibit complement regulation,

promoting complement activation, as seen with one FB mutant. 5. Polymorphisms within one (or more) complement proteins and/or regulatory elements may have signiﬁcant effects on activation or regulatory capacity and inﬂuence levels of complement protein at times of inﬂammation and stress.

AMD: age-related macular degeneration

The importance of polymorphisms has recently been demonstrated by studies in age-related macular degeneration (AMD), described below, and by a report of a 25% long-term survival advantage in patients with the C3 Slow (C3S) polymorphism who receive a renal allograft containing one or two C3 Fast (C3F) alleles from the donor rather than a kidney from a C3S donor (62). This common polymorphic variant clearly confers major differences in risk in terms of the long-term development of chronic allograft nephropathy.

Age-Related Macular Degeneration and Complement Although we have described the link between aHUS and complement regulatory proteins, a review of this ﬁeld would be incomplete without at least a brief discussion of AMD. AMD is the leading cause of irreversible visual loss in the developed world. The role of complement in AMD emerged from genetic associations between speciﬁc haplotypes of the complement FH gene in the RCA cluster. Hageman et al. identiﬁed a major risk FH haplotype, encoding the Y402H polymorphism, and protective haplotypes for AMD (63). Functional analysis has demonstrated that the 402H risk variant binds less avidly to both heparin oligosaccharides and C-reactive protein (64). This suggests an impaired targeting of FH to retinal pigment epithelium with a resultant loss of complement regulation at that site. Interestingly, Bruch’s membrane of the eye shares many features with the glomerular basement membrane. It too has no endogenous membrane-bound complement regulators and therefore relies on FH binding through the C terminus to protect www.annualreviews.org • Hemolytic Uremic Syndromes

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it from complement-mediated damage. Although FH risk alleles are also seen in aHUS, the risk factors for AMD and aHUS are different. For example, the 402H risk allele for AMD is not a susceptibility factor for aHUS. Also, the protective effects conferred by some FH haplotypes in AMD have been linked to a common deletion of the FHR1 and FHR3 genes—a deletion that increases the risk of aHUS (65, 66). Further support for the involvement of the complement cascade in AMD is provided by a related discovery that haplotypes of two additional complement components, FB and C2 on chromosome 6p21, confer protection or increase the risk for AMD (67).

control protein modules, is replaced as a transgene (FH−/− .FH16–20). The homozygote develops a spontaneous renal thrombotic microangiopathy at 12–16 weeks of age, analogous to human aHUS. Plasma levels of FH16–20 were ∼64% of wild-type FH levels. Deposition of C3, in the absence of immunoglobulin, was seen within the glomerular capillary loops, on the mesangium, and on renal vessel walls. However, the heterozygous mouse (50% wild-type FH; 50% FH16– 20), which is analogous to a human with a heterozygous FH carboxyl-terminus mutant, does not develop HUS spontaneously (59). A primary endothelial damaging agent may be required in this situation.

LESSONS FROM MOUSE MODELS

ADAMTS13 Knockout Mouse (ADAMTS13−/− )

Two mouse models relevant to the pathogenesis of aHUS have now been reported. These models provide a means to experimentally examine both the mechanisms underlying HUS development and treatment options.

The ADAMTS13 knockout mouse on a CASA/Rk background, Adamts13B/CN−/− , develops a systemic thrombotic microangiopathy analogous to human TTP, but only when exposed to Shiga toxin, the agent that causes D+ HUS in humans (60). This suggests that in the context of a predisposition to platelet aggregation and ultra-large vWf multimer formation caused by ADAMTS13 deﬁciency, an additional endothelial cell insult, such as Shiga toxin, is required to precipitate platelet thrombus formation. These results were supported by a report of a second ADAMTS13 knockout mouse, in which the gene knockout alone did not cause a systemic TTP-like illness (61). However, complete deﬁciency of ADAMTS13 did predispose to thrombus formation when blood from the homozygous knockout animals was placed under shear stress rather than under static conditions, again mimicking conditions of endothelial cell injury.

Factor H Knockout Mouse (FH−/− ) The homozygous FH knockout mouse (FH−/− ), like FH-deﬁcient humans (68) and Norwegian Yorkshire pigs (69), develops spontaneous classical type II membranoproliferative glomerulonephritis (70). This can be prevented by crossing the mice with homozygous FB knockout mice (FB−/− ), indicating that uncontrolled C3 activation and formation of the C3 convertase C3bBb in vivo is essential for development of the renal phenotype. The heterozygous animal (FH+/− ) also has evidence of increased complement activation and C3 turnover (70).

Factor H Transgenic Mouse (FH−/− .FHΔ16–20) The FH transgenic mouse was developed on the background of the FH knockout mouse model. A mutant form of FH, delCCP16–20, which lacks the ﬁve C-terminal complement 302

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PENETRANCE OF ATYPICAL HUS Incomplete penetrance of ∼50% is described for mutations in FH, FI, MCP, and FB.

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Several explanations, not mutually exclusive, may account for this observation. It is being increasingly demonstrated that the manifestation of disease may require a combination of mutations, risk haplotypes, and SNPs (71). Even where this combination exists, many individuals do not present with disease until middle age. This suggests that a precipitating cause is needed to unmask these latent complement regulatory defects. In a recent series of patients with mutations in MCP, aHUS was precipitated in all cases by infection (18). In FH-HUS, 70% of cases were preceded by infection, and pregnancy and drugs each accounted for 4% (18). Among FI-HUS cases, 40% were preceded by pregnancy and 60% were precipitated by infection (18).

prognosis. Most patients with FI-HUS and FB-HUS develop ESRF.

THERAPEUTIC CONSIDERATIONS Plasma Infusion/Plasma Exchange Following the introduction of plasma exchange and plasma infusion, the aHUS mortality rate fell from 50% to 25% (7). To this day, plasma infusion/plasma exchange remains ﬁrst-line therapy. An improved understanding of the role of complement in aHUS provides a sound rationale for a therapy to replace deﬁcient or defective complement proteins with functioning substitutes.

Renal Transplantation GENOTYPE/PHENOTYPE CORRELATIONS IN ATYPICAL HUS There is a great deal of heterogeneity in the severity of disease. Overall, the outcome in aHUS is poor; 50% of patients develop endstage renal failure (ESRF), and up to 25% die in the acute phase (8). However, some patients have a mild disease with no long-term sequelae. Others have recurrent episodes with intervals of no disease activity. An explanation for some of this variation begins to emerge when the disease is analyzed by mutation type and a genotype/phenotype correlation can be seen. Patients with mutations in FH have more severe disease than those with mutations in MCP (18). For those with FH-HUS, 70% develop ESRF or die, whereas >80% with MCP-HUS remain dialysis-independent. Thus, although MCP-HUS is usually associated with a better prognosis than FH-HUS, there are exceptions. It is likely that these will turn out to be due to different genetic and environmental modiﬁers of disease expression (Table 1). Although numbers are small, mutations in FI and FB seem to be associated with a poor

A genotype/phenotype correlation can also be observed with regard to outcome after transplantation. The two distinct groups are those with mutations in serum complement regulators (FH or FI) and those with MCP mutations (Table 1). About 80% of transplant recipients with FH-HUS have had recurrent disease (72). Similarly, all seven FI-HUS patients receiving renal transplants have had recurrent disease (18, 28, 29, 72). Because FH and FI are synthesized in the liver, transplantation of a kidney will not correct this plasma protein deﬁciency. In the one patient with a FB mutation who has undergone renal transplantation, there was recurrence of aHUS. Because FB is a serum factor, one would predict a recurrence after renal transplantation, as seen in FI-HUS and FH-HUS. Screening of further large cohorts is now required to give a more accurate picture of the recurrence rate of FB-HUS. In contrast, MCP is a transmembrane regulator, and renal allografts will therefore be protected by wild-type MCP from the donor. Indeed, the recurrence rate in MCP-HUS is substantially lower (∼10%) (18, 26, 72). Screening for mutations in these genes should www.annualreviews.org • Hemolytic Uremic Syndromes

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therefore allow patients and clinicians to make informed decisions regarding listing for transplantation, based on the risk of recurrence and its not inconsiderable consequent morbidity and mortality (73).

Living Related Donor Renal Transplantation

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Transplantation of a kidney from a living related donor also carries a poor prognosis, and in four reported cases, the donors themselves have gone on to develop HUS within a year of donation (74). Living related donor transplantation is not recommended because of the risks of recurrence in the recipient and of de novo disease in the donor. If live donation is considered, it is essential that the donor and recipient undergo FH, MCP, FB, and FI genotyping to identify hitherto unsuspected carriers, given the high reported rates of incomplete penetrance for mutations in all genes (reviewed in Reference 73). This screening will not, however, eliminate the risk for the donor in those forms of aHUS with an unknown genetic basis.

Complement Inhibitor Therapy One potential treatment for aHUS is the introduction of replacement soluble complement regulators, e.g., high-dose FH concentrate (not yet available). A second is the use of antibodies against key activating components of complement; for example, C5 should have beneﬁcial effects in ameliorating the downstream damage mediated by the anaphylotoxin C5a and preventing the formation of a membrane attack complex on host cell surfaces. This proposal presumes, though, that this part of the cascade is responsible for setting up the procoagulant state and not C3b or C3a. Experimental data suggest that preventing C5 activation in FH−/− mice ameliorates spontaneous and experimental glomerulonephritis (76). It would be useful to test this approach in the FH transgenic mouse FH−/− .FH16–20, which develops aHUS. It is unclear what complement fragment (C3b, C3a, C5a, C5b-9) or fragments are responsible for promoting thrombosis, and such studies should help to clarify this issue.

SUMMARY Liver/Kidney Transplantation Because FH, FI, and FB are synthesized in the liver, combined liver/kidney transplantation is a potential treatment. Three initial reports (reviewed in Reference 2) describing this procedure for FH-HUS noted a poor outcome, with two deaths. A more recent report using plasma exchange preoperatively was successful (75). Unless functioning FH is already present in the recipient, complement activation will cause graft failure (75).

Mutations in the complement regulatory proteins FH, MCP, and FI of the AP predispose to the development of aHUS. These mutations all cause a loss of cofactor activity. Recently reported mutations in FB and C3 cause increased activation of the AP. The identiﬁcation of mutations has clinical significance for predicting survival, renal recovery, and transplantation outcome. Understanding of the pathogenesis of aHUS may lead to the development of opportunities for treatment using complement inhibitors.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

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22. Caprioli J, Bettinaglio P, Zipfel P, et al. 2001. The molecular basis of familial hemolytic uremic syndrome: mutation analysis of factor H gene reveals a hot spot in short consensus repeat 20. J. Am. Soc. Nephrol. 12:297–307 23. Heinen S, Sanchez-Corral P, Jackson M, et al. 2006. De novo gene conversion in the RCA gene cluster (1q32) causes mutations in complement factor H associated with atypical hemolytic uremic syndrome. Hum. Mutat. 27:292–93 24. Venables J, Strain L, Routledge D, et al. 2006. Atypical haemolytic uraemic syndrome associated with a hybrid complement gene. PLoS Med. 3:e431 25. Fremeaux-Bacchi V, Moulton EA, Kavanagh D, et al. 2006. Genetic and functional analyses of membrane cofactor protein (CD46) mutations in atypical hemolytic uremic syndrome. J. Am. Soc. Nephrol. 17:2017–25 26. Richards A, Kemp EJ, Liszewski MK, et al. 2003. Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome. Proc. Natl. Acad. Sci. USA 100:12966–71 27. Esparza-Gordillo J, Goicoechea de Jorge E, Buil A, et al. 2005. Predisposition to atypical hemolytic uremic syndrome involves the concurrence of different susceptibility alleles in the regulators of complement activation gene cluster in 1q32. Hum. Mol. Genet. 14:703– 12 28. Kavanagh D, Kemp E, Mayland E, et al. 2005. Mutations in complement factor I (IF) predispose to the development of atypical HUS. J. Am. Soc. Nephrol. 16:2150–55 29. Fremeaux-Bacchi V, Dragon-Durey M-A, Blouin J, et al. 2004. Complement factor I: a susceptibility gene for atypical haemolytic uraemic syndrome. J. Med. Genet. 41:e84 30. Goicoechea de Jorge E, Harris CL, Esparza-Gordillo J, et al. 2007. Gain of function mutations in complement factor B are associated with atypical hemolytic uremic syndrome. Proc. Natl. Acad. Sci. USA 104:240–45 31. Fremeaux-Bacchi V, Regnier C, Blouin J, et al. 2007. Protective or aggressive: paradoxical role of C3 in atypical hemolytic uremic syndrome. Mol. Immunol. 44: 172 (Abstr.) 32. Atkinson J, Liszewski M, Richards A, et al. 2005. Hemolytic uremic syndrome. An example of insufﬁcient complement regulation on self tissue. Ann. NY Acad. Sci. 1056:144–52 33. Male D, Ormsby R, Ranganathan S, et al. 2000. Complement factor H: sequence analysis of 221kb of human genomic DNA containing the entire fH, FHR1 and FHR3 genes. Mol. Immunol. 37:41–52 34. Zipfel PF, Jokiranta TS, Hellwage J, et al. 1999. The factor H protein family. Immunopharmacology 42:53–60 35. Lupski J, Stankiewicz P. 2005. Genomic diorders: molecular mechanisms for rearrangements and conveyed phnotypes. PLoS Genet. 1:e49 36. Vaziri-Sani F, Holmberg L, Sjoholm A, et al. 2006. Phenotypic expression of factor H mutants in patients with atypical hemolytic uremic syndrome. Kidney Int. 69:981–88 37. Herbert A, Uhrin D, Lyon M, et al. 2006. Disease-associated sequence variations congregate in a polyanion-recognition patch on human factor H revealed in 3D structure. J. Biol. Chem. 281:16512–20 38. Jokiranta TS, Jaakola V-P, Lehtinen M, et al. 2006. Structure of complement factor H carboxyl-terminus reveals molecular basis of atypical haemolytic uremic syndrome. EMBO J. 25:1784–94 39. Caprioli J, Castelletti F, Bucchioni S, et al. 2003. Complement factor H mutations and gene polymorphisms in haemolytic uraemic syndrome: the C-257T, the A2089G and the G2881T polymorphisms are strongly associated with disease. Hum. Mol. Genet. 12:3385– 95

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76. Pickering M, Warren J, Rose K, et al. 2006. Prevention of C5 activation ameliorates spontaneous and experimental glomerulonephritis in factor H-deﬁcient mice. Proc. Natl. Acad. Sci. USA 103:9649–54

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:293-309. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

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Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys1,2 and Joseph V. Bonventre1,2,3 1

Renal Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; 2 Harvard Stem Cell Institute and 3 Harvard-MIT Division of Health Sciences and Technology, Boston, Massachusetts 02115; email: [email protected]; joseph [email protected]

Annu. Rev. Med. 2008. 59:311–25

Key Words

First published online as a Review in Advance on October 3, 2007

repair, progenitor cells, acute renal failure

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.061506.154239 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0311$20.00

Abstract The potential role of mesenchymal stem cells (MSCs, also called mesenchymal stromal cells) in endogenous repair and cell-based therapies for acute kidney injury (AKI) is under intensive investigation. Preclinical studies indicate that administered MSCs both ameliorate renal injury and accelerate repair. These versatile cells home to sites of injury, where they modulate the repair process. The mechanisms responsible for their protective and regenerative effects are incompletely understood. Some have reported that MSCs are capable of direct engraftment into injured nephrons under certain circumstances. This is highly controversial, however, and even those who argue there is engraftment acknowledge that the primary means of repair by these cells most likely involves paracrine and endocrine effects, including mitogenic, antiapoptotic, anti-inﬂammatory, and angiogenic inﬂuences. There is a good deal of interest in MSCbased approaches for the treatment of human kidney injury, thanks to positive preclinical results, the strong clinical need for novel therapies to treat AKI, the ease of isolation and expansion of MSCs, and encouraging preliminary clinical trial results in other ﬁelds. This review summarizes current knowledge and identiﬁes gaps in our understanding of MSC biology that will need to be ﬁlled in order to translate recent discoveries into therapies for AKI in humans.

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INTRODUCTION

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Adult stem cell: cell present in postembryonic tissue that can self-renew and generate all or a subset of the cell types of the tissue from which it was derived Dedifferentiation: process in which a terminally differentiated cell reverts to an earlier developmental stage, often after injury or neoplastic transformation BMDC: bone marrow–derived cell BM: bone marrow HSC: hematopoietic stem cell Mesenchymal: derived from embryonic mesoderm, the middle layer of an embryo that forms connective tissues (bone, cartilage, fat) and blood-related organs MSC: mesenchymal stem cell or mesenchymal stromal cell

Stem cells play fundamental roles in the selfrenewal of adult tissues throughout life. Some tissues are characterized by ongoing loss of cells, including the hematopoietic system, intestine, and skin, and adult stem cells are responsible for replenishing these cells to maintain tissue homeostasis. Other organs, such as kidney and lung, have a much lower rate of cellular turnover but are capable of proliferating and repairing after an injury (1). Some injured tissues can be reconstituted by the recruitment, proliferation, and differentiation of epithelial stem cells, but it remains unclear if the kidney follows this paradigm for epithelial stem cell–based homeostasis and repair after injury (2). Basal tubule cell turnover in kidney is exceedingly low, and the turnover that can be detected appears to occur by division of terminally differentiated tubular epithelial cells (3). Soon after injury, by contrast, there is diffuse tubular cell proliferation. This may reﬂect the intrinsic ability of surviving epithelial cells to adapt to the loss of neighboring cells by dedifferentiation and proliferation, and ultimate replacement of the cells that have died as a result of the insult (Figure 1). Based on the high proliferative capacity of injured kidney, one longstanding model holds that tubular cells themselves are the source of nephron repair (1). Studies on the role of bone marrow– derived cells (BMDCs) have challenged this model of dedifferentiation followed by proliferation and redifferentiation of existing tubular cells after injury. Bone marrow (BM) con-

tains at least two populations of stem cells: the hematopoietic stem cells (HSCs), which give rise to all differentiated blood cell types, and mesenchymal stromal cells (MSCs), which give rise to mesenchymal cell types including chondrocytes, osteocytes, and adipocytes. Although it has long been appreciated that BMderived inﬂammatory cells home to injured kidney, recent studies have suggested that BMDCs directly participate in renal injury and repair. Mesenchymal stem cells in particular have been reported to protect against experimental renal injury as well as accelerate the repair process in rodent models. Some reports (reviewed below) indicate that MSCs directly replace dead tubular epithelial cells, whereas other observations suggest that MSCs regulate the endogenous reparative machinery without transdifferentiation into tubular cells. Overall, the emerging evidence describing MSC modulation of acute kidney injury (AKI) has stimulated a reappraisal of the cellular mechanisms behind renal injury and repair and has generated considerable excitement about the prospects for novel cell therapies to treat human kidney diseases.

THE MESENCHYMAL STEM CELL MSCs are undifferentiated adult cells that can be isolated from a variety of tissues but primarily BM stroma. The embryonic lineage of these cells is mesodermal; they emerge from mesenchymal cells that give rise to connective tissues such as bone, cartilage, and fat

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 1 Epithelial dedifferentiation and proliferation following ischemia-reperfusion injury. The outer medulla, containing the S3 segment of the proximal tubule, is most susceptible to acute injury owing to its high metabolic demands and the nature of the vasculature. After an ischemic insult, surviving epithelia in the S3 segment of the proximal tubule lose their brush border and display a typical ﬂattened appearance along the basement membrane (+). Some tubules contain casts containing sloughed necrotic and apoptotic cells (∗ ). Mitotic ﬁgures can also be seen (arrow). The robust proliferative response after injury is reﬂected by the high percentage of tubular epithelial cells that express the proliferation marker Ki67 (brown nuclear stain) in the injured kidney, compared to absent KI67 expression in uninjured kidney. These sections are from mouse kidney subjected to 30 min of ischemia-reperfusion injury and sacriﬁced 72 h later. PAS, period acid-Schiff. Scale bar, 20 μM. 312

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Transdifferentiation: a switch in the fate of a cell, usually a differentiated cell, into a different differentiated cell type Acute kidney injury (AKI): clinical syndrome characterized by a rapid fall in glomerular ﬁltration rate, often due to ischemic or toxic renal injury. Previously known as acute renal failure

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as well as blood supply–related organs such as the vasculature and hematopoietic system. MSCs are deﬁned by adherence to plastic in culture, multipotentiality (ability to differentiate into different cell types), expression of typical surface markers such as CD73, CD90, and CD105, and the absence of expression of hematopoietic lineage markers (4). MSCs reside not only in BM but also in fat and vasculature and may be present in all adult tissues, including kidney (5). There is no proof that MSCs are clonal, self-renewing stem cells, and for this reason, many deﬁne MSCs as “multipotent mesenchymal stromal cells” (6). The functional properties of MSCs make them unique. These multipotent stem cells can differentiate to cells of the mesenchymal lineage such as osteocytes, adipocytes, and chondrocytes, and potentially other cell types. Directed differentiation can be achieved by culturing MSCs in deﬁned conditions (7). MSCs are easily cultured, unlike embryonic stem cells (which require feeder cells and special growth medium) and other adult stem cells. Because MSCs can be expanded, it is not difﬁcult to obtain clinically useful numbers of cells so they have been among the ﬁrst cells to be used for cellular therapies in humans. Finally, MSCs possess immunomodulatory properties that make them especially attractive for potential use in treating human disease characterized by autoimmunity or inﬂammation, including graft-versus-host disease, multiple sclerosis, and Crohn’s disease (8).

BONE MARROW PLASTICITY AND RENAL REPAIR The current interest in MSCs for treatment of AKI grew in part from the observations that BM-derived cells could develop into hepatocytes (9, 10). This ﬁnding, later reported in humans (11), led to intensive research on the plasticity of BM-derived cells. Evidence for engraftment of BM-derived cells was soon reported in other tissues, including 314

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lung, gastrointestinal tract, and skin. Krause et al. demonstrated that a single transplanted HSC could provide hematopoietic reconstitution for a lethally irradiated recipient and that this single hematopoietic cell could also engraft nonhematopoietic tissues including lung, liver, gastrointestinal tract, and skin (12). These surprising results were followed by studies of renal biopsies from male patients transplanted with female kidneys. Two groups reported the presence in the allografts of Y-chromosome-positive tubular epithelial cells—varying from <1% up to 20% of cells examined (13, 14), with similar results found in mice (15). Follow-up studies have led to a reevaluation of the physiologic relevance of the initial observations concerning BM-derived cells transdifferentiating into renal epithelia. It has been proposed that the early results could be due to cell fusion or possible artifactual detection of lineage markers. The inability to repeat some of these ﬁndings in other labs has also raised doubts (16, 17). Not all issues are resolved but several conclusions are possible. The method of marking and detecting the BM lineage is critical. Bacterial β-galactosidase transgene activity may be problematic owing to high expression of endogenous kidney β-galactosidases and possible leakage of the enzyme by damaged cells, with subsequent uptake by neighboring cells. Green ﬂuorescent lineage markers, such as enhanced green ﬂuorescent protein, are also subject to misleading artifacts because of the high intrinsic autoﬂuorescence of the postischemic kidney. High-resolution marker detection in kidney sections is especially important; three-dimensional deconvolution or confocal microscopic techniques are required to distinguish true cellular staining from closely apposed and overlying cells and nuclei (18). BM-derived leukocytes trafﬁc to the renal interstitium after renal injury, and a superimposed leukocyte nucleus may be mistaken for an epithelial cell nucleus without sufﬁciently high-resolution imaging. Cell overlay and intrinsic autoﬂuorescence have

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also complicated interpretation of BMDCs’ contribution to myocardial regeneration. It is difﬁcult to track cell fate in vivo, particularly in injured tissues (19, 20). Recent studies of mice with BM transplants harboring several different lineage markers have indicated that BM-derived cells only rarely contribute to the renal epithelial lineage under physiologic conditions (at most 0.1% but probably less) (21, 22, 27). The importance of cell fusion as a possible explanation for earlier results is emphasized by a recent study that convincingly showed a 20%–50% fusion of tubular epithelia with BM-derived cells in a mouse model characterized by long-term, intense genetic pressure (23). Clearly the mechanism and relative importance of cell fusion under normal conditions require further study.

MSCs AMELIORATE RENAL INJURY AND ACCELERATE REPAIR Although we conclude that endogenous BMderived cells or exogenously administered MSCs do not directly replace renal epithelia to a signiﬁcant extent during renal repair, several lines of evidence indicate that exogenously administered MSCs do modulate the kidney repair and regenerative response. Intravenous injection of the lineagenegative BM fraction prior to injury, part of which contains MSCs, blunts the initial rise in BUN after ischemia reperfusion injury (IRI) (24), whereas whole BM has no protective effect (21). Injection of puriﬁed MSCs almost completely protects against the cisplatin-induced rise in BUN, whereas injection of puriﬁed HSCs has virtually no protective effect (25). Similar protection from injected MSCs was found in a glycerol-induced pigment nephropathy model (26) and in a model of IRI (22, 27). Importantly, infused MSCs have been shown to enhance recovery of rodents subjected to IRI even if administered 24 h after the injury, suggesting active participation of these cells in the repair

process (27, 28, 52). Taken together, there is good evidence that administered MSCs protect against AKI and accelerate the recovery phase in toxic and ischemic rodent models.

IRI: ischemia reperfusion injury

DO EXOGENOUS MSCs DIRECTLY ENGRAFT INTO INJURED TUBULES? Morigi et al. (25) and Herrera et al. (26) reported that exogenous MSCs can engraft into injured tubules and proposed that the ability to transdifferentiate explained their protective effect. Yokoo et al. directly injected exogenous MSCs into developing kidney and after subsequent embryo and organ culture observed MSC incorporation into glomerulus, tubule, and interstitium, ﬁndings that seem to support the possibility of direct engraftment (29). In contrast, other studies (18, 21, 22, 27) showed protection from injury by exogenous MSCs but very little or no tubular incorporation. Some of the discord may be explained by different injury models and protocols (30); however, the caveats described previously regarding proof of tubular incorporation of BMDCs also apply to studies of injected MSCs. The nature of the MSC marker, careful three-dimensional microscopic analysis, and the possibility of cell fusion all must be taken into account. In a follow-up study, Herrera et al. (31) reported much lower tubular incorporation of MSCs (∼2.5%) than in an earlier report (∼20%) that had relied on the same glycerol-induced renal injury model but a different ﬂuorescencebased method of tracking injected MSCs. In our opinion, the data indicate that the effects on renal repair of exogenous MSCs are not explained by direct repopulation of the tubule. The timing of renal epithelial cell proliferation seems too rapid to be explained by transdifferentiation of extrarenal cell types into epithelial cells. In most studies, the protective effect of injected MSCs is observed within 24–48 h. When careful lineage analysis has been done, the numbers of MSC- or

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BMDC-derived epithelial cells appear to be so low (0.1% or less) that they could not have functionally contributed to repairing the nephron, at least by direct engraftment. Vogetseder and colleagues have argued that in the uninjured kidney, the small amount of epithelial proliferation present occurs by division of terminally differentiated cells (3, 32). Lin et al. have presented preliminary evidence that at least a subset of genetically tagged tubular epithelial cells proliferate after injury (33). Both observations suggest that reparative cells in renal injury derive from within the kidney. In summary, there is a growing consensus that both endogenous BMDCs and exogenously administered MSCs can give rise to renal epithelial cells only rarely, if at all, and that cell fusion may explain some of the results interpreted as direct replacement of epithelial cells. The rarity of transdifferentiation to kidney epithelia indicates that direct tubule repopulation by BMDCs or administered MSCs does not have physiologic relevance to renal repair from injury in vivo.

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HOMING OF EXOGENOUS MSCs The mechanisms by which MSCs promote kidney repair remain unclear, but an important aspect of the therapeutic effects of MSCs is their apparent ability to home to injured organs. After exogenous MSCs labeled with iron-dextran were administered to rats following IRI, magnetic resonance imaging located these cells primarily in the renal cortex. These cells remained associated with kidney three days after IRI, and histologically they were localized to glomerular capillaries (28). In a more detailed analysis, ﬂuorescently labeled MSCs were localized by two-photon microscopy to both glomeruli and peritubular capillaries within 10 min of intra-arterial injection into rats subjected to IRI 24 h before (27). The relative importance of MSC homing to glomerular versus peritubular capillaries is not known. MSCs have been detected in both compartments in both acute 316

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and chronic injury models (34). Either location may be efﬁcacious, with peritubular MSCs poised to signal to adjacent tubular epithelia and glomerular MSCs potentially able to secrete factors that are ﬁltered into the tubular lumen, where they may bind to and directly regulate and/or facilitate proliferation of damaged epithelial cells. Another unresolved question is whether MSCs bound to the renal microvasculature are capable of migrating into the renal interstitium. No direct evidence supports this possibility, but it has not yet been examined rigorously. Recent studies have begun to dissect the signals that regulate MSC homing. An attractive candidate has been the chemokine SDF1 (stromal-derived factor-1), which binds to its receptor CXCR4, which is expressed in distal tubule, and is upregulated after renal injury (35). CXCR4 is expressed in MSCs. Its expression is upregulated by hypoxia, and the SDF-1/CXCR4 pair is known to regulate HSC migration. Furthermore, hypoxic preincubation of MSCs appears to increase engraftment in vivo (36). Another promising candidate as a regulator of homing is plateletderived growth factor (PDGF), a growth factor known to be secreted from the basolateral aspect of human epithelial cells (37). Cultured MSCs express PDGF receptors and potently migrate in response to exogenous PDGF. This migratory response is enhanced by preincubation of MSCs with tumor necrosis factor (TNF) (38). A recently described candidate for regulation of MSC homing is CD44, which is expressed on MSCs and required for renal localization of injected MSCs after glycerol-induced renal injury. The receptor for CD44, hyaluronic acid, is upregulated in kidney after injury, and CD44-negative MSCs show reduced migration to injured kidney as well as decreased protection from injury (31). Elucidating the precise mechanisms controlling MSC migration to injured kidney may have important clinical consequences, since effective delivery of these cells to damaged tissue may be critical for therapeutic efﬁcacy.

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EVIDENCE THAT MSCs REPAIR KIDNEY BY PARACRINE AND ENDOCRINE MECHANISMS If MSCs do not directly repopulate repairing tubules, then paracrine and/or endocrine mechanisms must explain their therapeutic effects in kidney injury. Given the importance of inﬂammation in the pathophysiology of acute kidney injury (39), it is very important to consider the immunomodulatory properties of MSCs and the role these may play in renoprotection (40). MSCs are immunologically privileged, and allogeneic MSCs do not induce a proliferative T cell response. The mechanisms for this tolerance include low surface expression of both major histocompatibility complex (MHC) class I and II molecules, lack of expression of major costimulatory molecules such as CD40, CD80, and CD86, and direct inhibition of dendritic cell alloantigen-induced differentiation and activation, among others (41). MSCs also exert anti-inﬂammatory inﬂuences on T cells. Coculture of MSCs with either Th1, Th2, or natural killer (NK) cells decreases their secretion of proinﬂammatory cytokines such as TNFα and IFN-γ and increases their secretion of suppressive and tolerance-promoting cytokines such as IL-10; this effect is largely mediated by MSC production of the eicosanoid prostaglandin E2 (PGE2) (42). T cells are important in both immune-mediated and ischemic kidney disease, so the ability of MSCs to regulate T cell function is probably relevant to their therapeutic effects in AKI (43). In support of this notion, higher levels of antiinﬂammatory cytokines have been found in kidney extracts from MSC-treated animals after IRI (44). Pro-inﬂammatory stimuli such as IFN-γ promote the immunosuppressive effects of MSCs, including protection from NK cell–mediated cytolysis, enhanced secretion of hepatocyte growth factor (HGF) and TGF-β, enhanced induction of indoleamine 2,3dioxygenase (IDO), an enzyme that inhibits T cell proliferation by depleting the essential lymphocyte proliferation cofactor tryp-

tophan (45, 46). Other potentially important immunosuppressive actions of MSCs include suppression of B lymphocyte proliferation and antibody production, inhibition of dendritic cell activation, and possibly the induction of regulatory T cells (41). These mechanisms are summarized in Figure 2. MSCs can secrete a broad array of growth factors in addition to cytokines, including granulocyte-colony stimulating factor, stem cell factor, leukemia-inhibitory factor, macrophage-colony stimulating factor, IL6, and IL-11 (47). Secretion of bioactive molecules important for hematopoietic differentiation is not surprising, since MSCs provide the marrow stroma that supports HSC differentiation. Recent studies have identiﬁed other growth factors and chemokines secreted by MSCs, such as vascular endothelial growth factor (VEGF), basic ﬁbroblast growth factor, monocyte-chemoattractant protein-1, HGF, and insulin-like growth factor-1 (IGF-1) (47, 48). Some of these polypeptides enhance epithelial proliferation, modulate inﬂammation, or promote angiogenesis, and are therefore good candidates for therapeutic efﬁcacy in renal injury. IGF-1 and HGF, for example, are epithelial mitogens and morphogens that also promote renal blood ﬂow and are known to protect against ischemic injury in mice (49, 50). Studies have begun to address the speciﬁc paracrine factors secreted by MSCs that might explain their beneﬁcial effects in AKI. Togel et al. (51) found signiﬁcant levels of VEGF, HGF and IGF-1 in MSC-conditioned media, which was capable of enhancing endothelial cell proliferation and differentiation. When MSCs were infused just prior to IRI, these cells quickly homed to the renal microvascular circulation, and endogenous cell apoptosis was decreased in regions that contained MSCs. These authors propose that the ability of MSCs to home to injured microvasculature and inhibit apoptosis is an important aspect of MSC-induced renoprotection (51). Whether the therapeutic effects of MSCs can

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HGF: hepatocyte growth factor

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TNF-α, IFN-γ DC

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+ PGE2 NO NK cell

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CD4+ T cell

Figure 2 Mechanisms of immunomodulation by MSCs. The immune suppressive effects of MSCs may be ampliﬁed after exposure to proinﬂammatory stimuli such as TNF-α or IFN-γ. Although NK cells can lyse MSCs by activating lectin NKG2D ligands expressed on MSCs, pre-exposure to IFN-γ protects MSCs from cytolysis, and MSCs inhibit IL-2-induced NK cell proliferation. After proinﬂammatory stimuli, MSCs process soluble antigen and present it to CD4+ T cells. Soluble factors are very important in mediating anti-inﬂammatory effects of MSCs. PGE2, nitric oxide, HGF, IL-10, TGF-β, and IDO all exert inhibitory effects on immune cells in a paracrine fashion. Adapted from Reference 40 with permission.

be entirely ascribed to their ability to home to injured tissues and secrete trophic mediators, i.e., to deliver growth factors, is an important open question. Some doubt about the importance of tissue homing has been raised by a recent report in which the intraperitoneal injection of MSCs also conferred protection from IRI in the absence of MSC trafﬁcking to kidneys (52). Whether the higher local concentrations of paracrine factors released from intrarenal MSCs versus lower systemic concentrations released from extrarenal MSCs are critical factors in their therapeutic effect requires careful follow-up studies. Figure 3 summarizes the paracrine mechanisms for the therapeutic effects of MSCs in AKI.

RECRUITMENT OF ENDOGENOUS BONE MARROW MSCs The observation that exogenously administered MSCs can protect against renal injury 318

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naturally leads to the question of whether endogenous MSCs might be recruited to participate in the repair process as well. It has been hypothesized that endogenous BM-derived MSCs may circulate in much the same fashion as HSCs, and some studies have reported that MSC-like cells can be puriﬁed from blood, albeit in very low numbers (53). Whether endogenous MSCs may be recruited from their BM niche and home to sites of injury is an unresolved issue. Although it is very clear that BM-derived cells, primarily inﬂammatory cells, trafﬁc to the interstitium of injured kidney, it is unknown whether a subset of these cells comprises MSCs. Genetic lineage analysis in chimeric mice has proven that BMDCs trafﬁc to the interstitium of injured kidney (22, 54), but whether these interstitial BMDCs include MSCs has not been ascertained to date (55). It is worth noting that the proportion of MSCs compared to nonMSCs in whole BM is very low (0.01% or less), so it is very likely that the great majority of BM-derived cells in injured kidney are

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exogenous

endogenous?

Acute Kidney Injury

mitogenic

MSC

anti-apoptotic

pro-angiogenic

Injured tubule

anti-inflammatory Figure 3 Model for paracrine actions of MSCs on the injured tubule. After an acute kidney injury, injected MSCs home to injury sites and may be recruited from endogenous niches (bone marrow or kidney) as well. MSCs bind to glomerular and/or peritubular capillary endothelium and both protect the kidney from further injury and accelerate repair. Paracrine mediators play important roles in repair, including VEGF, IGF, HGF, PGE2 and other soluble factors that exert mitogenic, antiapoptotic, proangiogenic, and anti-inﬂammatory effects.

inﬂammatory cells such as monocytes, macrophages, neutrophils, and lymphocytes.

KIDNEY MSCs An alternative possibility to homing of BMderived MSCs is activation of an endogenous

kidney MSC population. Gupta et al. (56) isolated a population of cells from adult rodent kidney that expressed markers of MSCs (CD90, CD44), expressed Oct4 but not cytokeratin, self-renewed in culture, and incorporated into the renal epithelium. The authors called these cells multipotent renal

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progenitor cells and suggested that they were candidate renal stem cells. Bussolati et al. (57) isolated a CD133+ population from human kidney and found CD133+ cells in the interstitium and within tubular cells. These cells did not express hematopoietic markers but expressed MSC markers (CD29, CD90, CD44, and CD73). When these cells in Matrigel were injected subcutaneously into SCID mice, they developed into tubular structures that expressed proximal and distal tubular markers. When cultured in vitro with VEGF, they expressed endothelial markers, and when these differentiated cells were injected subcutaneously in Matrigel, they were reported to form vessels that connected to the endogenous mouse vessels. When injected into mice with glycerol-induced AKI, these cells homed to the kidney and reportedly integrated into proximal and distal tubules. Sagrinati and coworkers isolated a population of CD133+ CD24+ parietal epithelial cells from human adult kidney that could be induced in vitro to express markers of proximal and distal tubular cells, osteogenic cells, adipocytes, and neurons (58). Finally, Plotkin & Goligorsky (5) isolated a multipotent clonal cell line from kidney and showed that these cells could differentiate into erythropoietinproducing ﬁbroblasts in hypoxic culture. These cells migrated to a peritubular and interstitial location, but not a tubular location, after injection into postischemic kidney. Although it is not yet clear what type of cells each group has isolated, one interpretation of these intriguing studies is that kidneyspeciﬁc MSCs exist. Whether these cells participate in repair of injured kidney, either indirectly or through direct epithelial engraftment, requires further study. It is also possible that kidney MSCs may contribute to some of the adverse longer term effects of injury, such as interstitial ﬁbrosis through activation of ﬁbroblasts and/or differentiation into ﬁbroblasts.

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THE NEXT STEP: CLINICAL TRIALS FOR MSCs IN ACUTE KIDNEY INJURY? BM-derived cell therapies for several human diseases are already being tested. Most attempts have been made in the ﬁeld of myocardial infarction, where several small phase I and phase II trials have reported modest improvements in both physiologic and anatomic parameters after intracoronary delivery of various populations of BMDCs, including MSCs (59). Calls for large randomized trials of cell therapies are now being made (60), and the ﬁrst phase I trial of MSCs in AKI is scheduled to begin shortly. This safety study will enroll cardiac surgical patients at high risk for developing AKI. Patients who are scheduled to undergo on-pump coronary artery bypass grafting or valve surgery and who possess renal risk factors such as pre-existing renal disease, diabetes, age >60 years, and redo surgery will be enrolled. Because autologous MSCs will need to be expanded in vitro, most surgeries will be elective, but one arm of the study will involve the administration of allogeneic MSCs to patients in need of emergent surgery (C. Westenfelder, personal communication). An important advance has been the storage of well-characterized human MSCs by Prockop’s group at Tulane University, where banked MSCs are available to investigators (http://www.som.tulane.edu/ gene therapy/distribute.shtml). With the development of biomarkers for earlier detection of AKI (61), it is possible that we can one day identify patients at an early stage of AKI, select matched MSCs, thaw them, and infuse within 24 h of the renal insult. MSCs have had a very good safety record in human studies to date. Unlike pluripotent embryonic stem cells, MSCs do not form teratomas in animals when injected in vivo. In the human trials reported so far, no major adverse effect has been attributed to the injection of this cell type. Nevertheless, little

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long-term follow-up information about the consequences of administered MSCs is available. In a recent study, Kunter et al. (34) injected MSCs intrarenally into rats in a model of glomerulonephritis. Although there was a beneﬁcial therapeutic effect, on day 60 ∼20% of the glomeruli of MSC-treated rats contained large adipocytes derived from maldifferentiation of MSCs with pronounced surrounding ﬁbrosis. Ectopic osteogenic maldifferentiation of MSCs has also been observed in a cardiac cryoablation injury model (62). One possible explanation for the observations of Kunter and colleagues is the high number of MSCs injected. These investigators injected 2 × 106 MSCs into the renal artery of rats, whereas Westenfelder and others have injected 1 × 105 cells into the suprarenal aorta of mice. Even after normalization for kidney size, Kunter et al. injected signiﬁcantly more MSCs, and the resulting high local cellular concentration may predispose toward certain pathways of MSC differentiation.

AMNIOTIC FLUID–DERIVED STEM CELLS Multipotent stem cells isolated from amniocentesis specimens—termed amniotic ﬂuid–derived stem cells (AFSs)—hold promise for use in cellular therapy (63). AFSs represent ∼1% of all amniotic ﬂuid cells and are characterized by expression of the cell surface marker c-kit, as well as other surface antigens also expressed by MSCs (e.g., CD73, CD90, CD105). Like MSCs, and unlike embryonic stem cells, they do not form teratomas in vivo. AFSs differ from MSCs in two important ways. First, they are signiﬁcantly more broadly multipotent than MSCs and may in fact be pluripotent. Second, they are clonal, and therefore are a true stem cell population. Whether these properties of AFSs will make them a better candidate for cellular therapies in kidney injury needs to be investigated. Their accessibility makes them a very attractive candidate for regenerative medicine. The prospect of banking amniocentesis specimens for future AFS isolation and use in autologous cell therapies, or matching histocompatible donor cells with recipients, represents an important potential advance in regenerative medicine.

SUMMARY POINTS 1. Kidney epithelial cells show very high proliferation after acute injury but very low proliferation in the basal state. 2. Bone marrow (BM)-derived cells can incorporate into repairing renal epithelium, but these events are rare, may be explained by cell fusion, and are not the primary physiologic mechanism for nephron regeneration. 3. MSCs are multipotent cells derived from BM and perhaps in the adult kidney itself. They are easily expanded from BM in culture, facilitating their use in cell therapies. 4. MSCs are capable of homing to injured kidney when injected intravenously soon after injury. 5. MSCs can accelerate functional repair of injured nephrons, most likely through paracrine and endocrine mechanisms. The physiologic signiﬁcance and long-term consequences of transdifferentiation and/or fusion with epithelial cells are unclear.

FUTURE ISSUES 1. What molecular signals regulate homing of MSCs to injured tissues? 2. Do endogenous MSCs exist in the kidney and, if so, how do they participate in repair and longer-term complications of injury such as ﬁbrosis?

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3. If MSCs accelerate repair primarily through paracrine mechanisms, which secreted factors underlie these effects? 4. Do endogenous MSCs circulate, and can they be mobilized pharmacologically to accelerate kidney repair? 5. Do other stem cell types (such as amniotic ﬂuid–derived stem cells; see sidebar) or kidney-speciﬁc MSCs possess similar repair capabilities? 6. Are there long-term risks, such as maldifferentiation or transformation, associated with administered MSCs?

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DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We thank all the members of the Humphreys and Bonventre labs for valuable discussions and Christof Westenfelder for thoughtful comments on the manuscript. This work is supported in part by NIH grants DK73628 to B.D.H. and DK72381, DK39773, and DK74099 to J.V.B.

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RELATED RESOURCES: http://www.som.tulane.edu/gene therapy/distribute.shtml http://stemcells.nih.gov/info

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:311-325. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez Arizona Respiratory Center and Mel and Enid Zuckerman College of Public Health, University of Arizona, Tucson, Arizona 85724; email: [email protected], [email protected]

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Key Words

First published online as a Review in Advance on September 10, 2007

interaction, complex disease, CD14

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.060406.213232 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0327$20.00

Abstract Asthma risk has a clear hereditary component but, unexpectedly, the majority of reported associations between genetic variants and asthma have not been consistently replicated across studies. Methodological ﬂaws have been indicated as a possible explanation for these inconsistencies. However, an alternative explanation is that the effects of genetic variants depend on other factors whose frequency and distribution vary, both across individuals and across populations. Within this framework, we review recent advances in asthma genetics and conclude that a paradigm shift is needed, because a static model in which the DNA sequence is associated with disease risk in a linear fashion fails to consider the interdependence of the diverse components of asthma risk. We propose an integrated approach, linking sequence variation to speciﬁc phenotypic manifestations of the disease by taking into account concurrent inﬂuences from biological systems and environmental factors that interact within speciﬁc developmental windows of opportunity.

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GENETICS OF COMPLEX DISEASES: ASTHMA AS A PARADIGM

Phenotype: the expression of a speciﬁc trait, as determined by genetic and environmental inﬂuences

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The identiﬁcation via positional cloning of the genetic variants that cause known monogenic diseases (e.g., cystic ﬁbrosis) was among the major achievements of biomedical science in the twentieth century. Yet, to date, neither linkage nor association studies have proved as successful in dissecting the genetic components of more “complex” diseases, which do not display classical Mendelian forms of inheritance but do show strong evidence of genetic involvement. These complex conditions encompass cancer as well as respiratory and cardiovascular diseases (among others) and account for most of the public health burden in many areas of the globe. The vast majority of genetic associations are not consistently replicated across studies of complex diseases (1, 2). This lack of replication appears even more striking when one considers that, in principle, genetic association studies should be relatively robust to some of the main limitations of traditional epidemiological studies, including the ambiguous temporal sequence between exposure and outcome and the risk of unobserved confounding (3, 4). Methodological ﬂaws—such as inﬂated risk of type I error, lack of statistical power, and publication bias—have been indicated as a possible explanation for these inconsistencies, and recent meta-analyses suggest that false positives and false negatives do account for a sizable fraction of conﬂicting genetic associations (1, 5). However, reducing the entire issue to a technical matter would oversimplify the problem. Conﬂicting ﬁndings are not limited to poorly designed studies; they have also emerged from genetic association studies that were well designed, properly powered, based on strong a priori hypotheses, and consequently robust to major methodological ﬂaws. We need to explore alternative explanations by critically evaluating the implicit assumptions behind the replication studies.

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One of these assumptions is that the main genetic effects on disease risk are homogeneous across different populations. However, this assumption is unreliable if the effects of genetic variants depend on the presence of (and interaction with) other factors whose frequency and distribution vary across individuals and populations. Studies of genetic and environmental factors as determinants of complex phenotypes in animal models support the existence of strong gene-byenvironment interactions and suggest that genetic inﬂuences cannot be studied separately without considering the concurrent inﬂuence of the environment (6, 7). For example, recent studies in the mouse have conclusively demonstrated that gene-by-environment interactions explain a proportion of the variance of complex phenotypes that may be higher than that of either main effect (genetic or environmental) considered separately (7). Within this framework, we review recent advances in asthma genetics. We do not intend to present a comprehensive review of asthma susceptibility genes, which can be found elsewhere (8–10). Instead we review recent evidence that supports the need for a paradigm shift in studying the genetics of asthma and other complex diseases. We advocate a shift from a static model, in which the DNA sequence is associated with disease risk in a linear fashion (Figure 1a), to an integrated approach, in which sequence variation is linked to speciﬁc phenotypic manifestations of the disease by taking into account concurrent inﬂuences from biological systems and environmental factors that interact within speciﬁc developmental windows of opportunity (Figure 1b).

ASTHMA GENETICS: MULTIPLE GENES FOR MULTIPLE PHENOTYPES Even in a simple linear model such as the one depicted in Figure 1a, investigators will succeed in detecting an association between a genetic variant and the disease of interest only if

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a

Disease

b

Biological systems

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epigenetics, epistasis, haplotypic blocks, cell environment...

Phenotype1 Windows of opportunity

Environmental factors

Disease

Phenotype2 Phenotypen

lifestyle, exposures, infections, diet, medications, occupation...

Figure 1 Two different approaches to studying genetic components of human disease. (a) In a static model, DNA sequence is associated with disease risk in a simple linear fashion. (b) An integrated approach links sequence variation to speciﬁc phenotypic manifestations of disease by taking into account concurrent inﬂuences from biological systems and environmental factors that interact within speciﬁc windows of opportunity.

the “right” gene is tested against the “right” phenotype. This is by no means a trivial issue in asthma research.

Finding (All) the Asthma Genes To date, genetic association studies on asthma have used a candidate gene approach. This means that the genes to be tested were selected a priori on the basis of their location, function, or both. Genome screens have linked asthma-related traits to several chromosomal regions, including 2q, 5q, 6p, 12q, and 13q (11). Among them, chromosome 5q holds particular interest because it is one of the regions most consistently replicated in linkage studies, it corresponds to a region of common evolutionary ancestry that has been linked to airway eosinophil inﬁltration in mouse models (12), and it contains a cluster of genes—including those encoding the β2 -

adrenoceptor (ADRB2) (13–20), CD14 (21– 30), IL-4 (31, 32), and IL-13 (33, 34)—that are likely to be involved in asthma pathophysiology and, therefore, represent suitable positional and functional candidate genes for this disease. Interestingly, a recent thorough review of the literature found these four genes to be among the top ten asthma candidate genes in terms of number of positive replication studies (9). Other potential candidate genes have been identiﬁed in chromosomal regions implicated in asthma predisposition because of signiﬁcant genetic linkage with asthma. For example, chromosome 6p contains the extensively studied human leukocyte antigen (HLA) genes, including HLADRB1 and HLA-DQB1 (35), as well as TNF and LTA (36, 37). Similarly, chromosome 12q harbors, among others, VDR (38, 39), STAT6 (40, 41), and NOS1 (42, 43). The products encoded by all these genes are involved in www.annualreviews.org • Asthma Genetics

ADRB2: β2 adrenergic receptor HLA: human leukocyte antigen TNF: tumor necrosis factor LTA: lymphotoxin α VDR: vitamin D receptor STAT6: signal transducer and activator of transcription 6 NOS: nitric oxide synthase

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Haplotype: sequence of alleles located on the same chromosome in a speciﬁc genomic region

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ADAM33: A disintegrin and metalloproteinase domain 33 GPRA: G protein–related receptor for asthma

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asthma pathways, and their genetic variation has been repeatedly associated with asthmarelated traits. It is tempting to limit the search to genes that, like these, are both located in asthmalinked chromosomal areas and mechanistically involved in asthma pathways. However, evidence has emerged that questions the speciﬁcity (i.e., the ability to capture only true asthma genes) and sensitivity (i.e., the ability to capture all the true asthma genes) of this approach. First, although many of these positional-functional candidate genes have been replicated in association with asthma in multiple studies, they are by no means free of the inconsistency that plagues most genetic associations. The validity of this argument may not be evident at ﬁrst because of the large number of replication studies on these genes, which obviously inﬂates the number of positive replication studies. However, when one considers criteria for consistency that are independent of how frequently a speciﬁc gene has been tested—such as the ratio of positive to negative reports, consistency in the direction of the genetic association, and consistency in the speciﬁc polymorphisms/haplotypes linked to asthma—striking discrepancies across studies become evident. Second, the positional-functional candidate gene approach can miss asthma genes that are not identiﬁed (a) as functional, because their function is unknown or cannot be easily associated with asthma pathways, or (b) as positional, because their effects on asthma risk are too small to be detected in linkage studies (which have relatively low power compared with association studies) (44). ADAM33 (45) and GPRA (46) are two examples of the former group, as they were identiﬁed via pure positional cloning before the function of their encoded proteins was associated with asthma pathways. However, many (if not most) asthma genes are likely to belong to the latter group. Indeed, given our limited knowledge of asthma pathophysiology, it is not unreasonable to believe that a sizable fraction of asthma genes may have Guerra

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not been tested at all in association studies to date. Thus, in their search for the asthma genes, scientists face on one side a puzzlingly mounting number of inconsistent genetic associations and on the other the intrinsic limitations of the methodological approaches that have been used so far. The result is that, as of today, not only do we not know how many of the tested genes are truly asthma genes, but we also do not know how exhaustive is the list of candidate genes identiﬁed heretofore. Greater methodological rigor and larger sample sizes for replication studies have been advocated to minimize false positives and false negatives. Similarly, the anticipation of large genome-wide association studies (47) is fueled by the expectation that they will overcome some of the limitations of linkage and candidate gene association studies by maximizing statistical power and capturing inﬂuences of novel genes and pathways on disease risk in a hypothesis-free fashion. We argue that rigorous methods and new genomic strategies, albeit necessary, will not be sufﬁcient to provide deﬁnitive answers on the genetics of asthma and that future studies, no matter how rigorous the methodology or large the sample size, will fall short in identifying the asthma genes (let alone explaining the functional nature of these associations) if they do not embrace a new, integrated approach that recognizes the inﬂuence of additional factors on the link between genetic variation and disease. Figure 1b shows a model for such an approach. We propose the inclusion of three “modifying” factors: 1. Interactive inﬂuences from the environment 2. Interactive inﬂuences from biological systems 3. Developmental processes (i.e., windows of opportunity). In addition to these factors, the importance of a precise characterization of the phenotype (rather than the disease) of interest is highlighted in the proposed model. We discuss

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phenotypic characterization below and cover each of the three modifying factors in the second part of this review.

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Phenotypic Characterization All complex diseases are to some extent heterogeneous. Heterogeneity is manifest in many domains—age at onset, severity, response to treatment, intermediate phenotypes, natural history, and so on. Asthma is no exception. Indeed, this disease shows extreme heterogeneity. It can have its onset at any age; it recognizes a large number of speciﬁc and unspeciﬁc triggers; it can take any form from a mild, transient phenotype to a severe, persistent, life-threatening illness; and it is associated with a large number of intermediate phenotypes, including atopy, bronchial hyperresponsiveness, eosinophilia, and others, which can be assorted in any combination. The implications of this heterogeneity for genetic research are remarkable, as different phenotypes and clinical manifestations are likely to recognize different pathways and different genes. The case of ADAM33, located on chromosome 20p13, is illustrative. The original linkage signal detected between physicianconﬁrmed asthma and 20p13 became substantially stronger when the phenotype was further characterized by the presence of bronchial hyperresponsiveness (BHR), but not when a combined phenotype of asthma plus elevated total IgE was used (45). The preferential link of ADAM33 with the BHR phenotype was conﬁrmed in association analyses in the same original report (45) and is indirectly supported by previous mapping of BHR to a region of common evolutionary ancestry on mouse chromosome 2 (48). Other preferential genetic associations with speciﬁc asthma-related phenotypes have emerged from association studies, some of which are discussed in the following sections, as well as from multipoint linkage analyses of regions linked to asthma. One of them (chromosome 12q), for instance, was found to har-

bor three distinct loci that were separately linked to asthma, BHR, and forced expiratory volume in one second (FEV1) in a subset of families identiﬁed through the Childhood Asthma Management Program (49). Another potential source of phenotypic variability in asthma is that disease inception and disease progression appear to be inﬂuenced by different factors. Some environmental exposures may even have opposite effects on asthma initiation and progression. For example, exposure to endotoxin (50) and pets (51) has been suggested to exert protective effects against asthma-related traits early in life, whereas such exposure triggers symptoms and airway inﬂammation once the disease has developed. The potential implications of these developmental-speciﬁc processes on genetic research are discussed later in this review. Here, it will sufﬁce to note that, if the mechanisms involved in the inception of asthma differ from the mechanisms involved in its clinical course, genes may be found that are associated with the severity of the disease even in the absence of any detectable effect on asthma susceptibility (and vice versa). The β2-adrenergic receptor gene, ADRB2, might be one of these genes. ADRB2 has been extensively studied in asthma for several reasons. First, it is located in a chromosomal region (5q) repeatedly linked to asthma. Second, its two bestcharacterized polymorphisms are missense mutations in codons 16 and 27 (Arg16Gly and Gln27Glu), which are both functionally important (17) and representative of the most common haplotypic blocks in the gene (15). Third, its encoded protein is expressed on several lung cells, including bronchial smooth muscle cells, and plays a key role in controlling bronchial tone and response to the most commonly used asthma medication (i.e., albuterol). Yet, a meta-analysis suggested no direct effects of ADRB2 on susceptibility to asthma or BHR per se, but it did ﬁnd direct inﬂuences of the Gly16 allele on asthma severity (18). A recent large prospective study reported that the same polymorphism, but opposite www.annualreviews.org • Asthma Genetics

BHR: bronchial hyperresponsiveness IgE: immunoglobulin E FEV1: forced expiratory volume in 1 sec Alleles: alternative forms of a given polymorphic genetic locus

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allele (Arg16), was associated with persistence of asthma symptoms from childhood to adult life (19). This evidence suggests that, although inconsistencies across studies on asthma genetics cannot be entirely explained by their phenotypic heterogeneity, a detailed characterization of the phenotype of interest needs to be an essential part of every association study and is likely to result in increased statistical power, better comparability across replication studies, and new insights into the multiple genetic and molecular pathways involved in this disease.

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INTERACTIVE INFLUENCES FROM THE ENVIRONMENT Before we examine possible interactive inﬂuences on asthma susceptibility, the biological and epidemiological meanings of “interactive” should be distinguished. It can be argued that any factor that affects disease susceptibility does so as part of complex biological interactions. For example, in order for genetic variants to be able to inﬂuence disease susceptibility, their DNA sequence must be placed in the context of the many other cellular elements that are required for DNA organization, recombination, transcription, translation, etc. Similarly, once a genetic variant has altered the quality or quantity of its encoded protein, that protein will be part of complex biological networks at the cell, organ, and systemic levels. However, although these biological interactions illustrate the complexity of identifying genetic effects, they do not limit in principle our ability to detect an association between a genetic variant and a disease. Indeed, they are part of the scientiﬁc rationale behind any genetic association study that seeks to identify the downstream effects of genetic variants on disease susceptibility. In contrast, from an epidemiological standpoint, interaction is deﬁned on the basis of effect modiﬁcation and is present when the main effect of factor A on disease risk differs signiﬁcantly across different levels of ex332

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posure to factor B. This interaction is deﬁned as a gene-by-environment interaction if A is a genetic factor and B is an environmental factor (or vice versa). The impacts of these potential gene-by-environment interactions on association studies are crucial because our ability to detect signiﬁcant genetic effects will depend entirely on the presence or absence of speciﬁc environmental factors in the study population. Interactions are usually quantitative. In those cases, the association between factor A and disease risk has different magnitude—but the same direction—across different levels of exposure to factor B. However, interactions can also be qualitative, when the direction of the association between factor A and disease risk depends on factor B. In those cases, the same factor A will be associated with increased or decreased risk of disease according to the level of exposure to factor B. Recent work on two key innate immunity genes (TLR2 and CD14) provides evidence of both quantitative and qualitative interactions in asthma. Innate immunity genes are involved in the organism’s ﬁrst line of defense against microbes. Since the late 1980s (53), epidemiological observations suggested that exposure to microbial products early in life could confer protection against atopic diseases by triggering precoded maturational mechanisms of the immune system. This idea was later dubbed the hygiene hypothesis. It is thus not surprising that, as innate immunity receptors with speciﬁcity for different microbial products were characterized at the molecular level, variation in their genes was also studied in relation to asthma-related traits (54). TLR2 is one of these receptors and has speciﬁcity for a wide variety of components of the cell membrane of gram-positive and gramnegative bacteria. In vitro studies have shown that monocytes from healthy adult volunteers carrying two copies of the T allele at position –16934 in the ﬁrst intron of TLR2 express signiﬁcantly more membrane TLR2 than monocytes from carriers of the A allele, suggesting a functional role of this polymorphism (55). In rural communities of farmers,

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children carrying the TLR2/–16934T allele were found to be protected against asthma, hay fever, and atopy as compared with children carrying the AA genotype (56). However, these associations were not present among children from the same communities who were not living on farms and, therefore, were exposed to lower levels of the microbial products that are abundant in the animal-farming environment. This difference suggests the existence of quantitative interactions that modulate the effects of TLR2/–16934 on asthma and atopy. In this framework, CD14 may arguably represent the most interesting example of qualitative interaction in asthma genetics. CD14 is a pattern recognition receptor with speciﬁcity for endotoxin [lipopolysaccharide (LPS), a component of gram-negative bacteria] and is expressed both anchored on the membrane (mCD14) of several cell types, including mature myeloid cells, and in soluble form (sCD14) in human ﬂuids, including blood and breast milk. In 1999, the ﬁrst singlenucleotide polymorphism (SNP) of the CD14 gene was identiﬁed at position –159 of the 5 ﬂanking region (21). Since then, the functional nature of this SNP has been demonstrated by linking the CD14/–159T allele to increased sCD14 levels in plasma (21) and breast milk (57), increased mCD14 expression on antigen-presenting cells (58), and increased gene expression in functional studies (59). CD14/–159T was originally associated with protection against atopic sensitization (21). However, the relation of CD14/–159 to atopic sensitization and asthma has been inconsistent across subsequent studies. For example, consistent with the original CD14 report (21), in a Dutch urban population adults carrying the T allele were found to have on average a lower number of positive skin tests than subjects homozygous for the C allele (22). However, two large epidemiological studies from Germany failed to ﬁnd any association between CD14/–159 and IgE levels or the prevalence of atopic diseases (23, 24). Even more strikingly, reports emerged regarding

subjects supposedly heavily exposed to endotoxin that questioned the very direction of the association between CD14/–159 and atopy. These reports suggested a protective role for the C rather than the T allele (25, 26). Such conﬂicting ﬁndings support the existence of qualitative gene-by-environment interactions and suggest that the same allele can exert protection or enhance risk for asthma-related traits depending on the study population and its level of endotoxin exposure (Figure 2a). Final proof of the existence of such interactions in asthma genetics will require interdisciplinary approaches and convincing experimental evidence of their biological bases. However, three recent studies that have investigated interactive effects of CD14/–159 and endotoxin load within the same population have provided important support to this scenario (27–29). In two of these studies, CD14/ –159T was protective against atopy at low levels of endotoxin load, whereas it was associated with an increased risk for atopy at high levels of endotoxin exposure (27, 28). Similarly, in the third study, endotoxin load was inversely associated with allergic sensitization among ﬁve-year-old children carrying the CC genotype, whereas no association was evident among their peers carrying the T allele (29) (Figure 2b). What have we learned from the CD14 case? Probably the most important lesson is that dissecting the complex interplay between genetic variants and environmental factors will not be possible by studying either of these main effects independent of the other. Much as we would not study the effects of a drug-metabolizing enzyme on disease progression independently of whether patients are or are not treated with that medication, genetic association studies will increasingly need to include assessments of environmental exposures that interact with speciﬁc genetic variants as an essential part of their design. This will prove a challenging task. In association studies, environmental exposures are not controlled by the investigators, can change dramatically in time and space, and are www.annualreviews.org • Asthma Genetics

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Risk of atopic disease

a

Qualitative interactions between CD14/–159 and endotoxin load in affecting risk of atopic disease. (a) A hypothetical model in which the genetic effects of CD14/–159T will be opposite at low and high levels of exposure to endotoxin. At middle ranges of exposure to endotoxin, CD14/–159 will have no effect on disease risk. (b) Fitted predicted probability curves for allergic sensitization associated with CD14/–159 genotypes in relation to environmental endotoxin load among ﬁve-year-old children from the Manchester Asthma and Allergy Study (from Reference 29).

T allele C allele

Level of exposure to endotoxin Low

b

High

1.0

Predicted probability for sensitization

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Figure 2.

CC CT TT

0.8

0.6

0.4

0.2

0 1.0

7.4

54.6

403.4

22,000

162,755

1.2 x 106

Endotoxin load (EU m–2) highly sensitive to residual confounding. Yet, it is highly unlikely that we will understand the genetics of asthma until we understand the complexity of these interactions. In this framework, pharmacogenetics studies can be considered as a special type of geneby-environment studies, in which medication use represents the environmental exposure. Clinical applications of genetic research on

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asthma management are indeed likely to come ﬁrst from pharmacogenetics studies rather than observational genetic association studies for several reasons. First, in clinical trials, exposure to medication is controlled to a large extent by the investigators within protocols that include drug dose and treatment duration, whereas in association studies the measurement error in assessing environmental

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Table 1 Polymorphisms in genes encoding proteins involved in regulation, metabolism, and/or response to asthma medications that have been associated with in vivo pharmacogenetic effects (modified from Reference 56a) Gene

Medication class

β2-adrenoceptor (ADRB2)

β2 -agonists

Polymorphism Arg16Gly

Pharmacogenetic effects Arg16 associated with increased acute response to albuterol independent of asthma Arg16 associated with reduced response to regularly scheduled treatment with β2-agonists in patients with asthma

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glucocorticoid receptor (GR)

glucocorticoids

corticotrophin-releasing hormone receptor 1 (CRHR1)

leukotriene C4 synthase (LTC4S)

leukotriene modiﬁers

5-lipoxygenase (ALOX5)

exposures is likely to be quite large most of the time. Second, ﬁndings from pharmacogenetics studies have strong potential for immediate and straightforward clinical applications, such as designing personalized pharmacological treatments based on patients’ genetic background with the ultimate goal of maximizing beneﬁts and minimizing side effects. A complete discussion of pharmacogenetics studies conducted on asthma is beyond the scope of this review and can be found elsewhere (see Reference 56a and Table 1). Here, we summarize some ﬁndings from studies that have investigated the effects of the ADRB2 Arg16Gly polymorphism on clinical response to β-agonists. This polymorphism has been extensively investigated in in vitro functional studies and has shown opposite effects on acute versus chronic response to albuterol in clinical studies. In a population-based cohort of 11-year-old children, subjects homozygous for Arg16 had ﬁvefold increased odds of re-

Thr164Ile

Ile164 associated with reduced vasodilator response to agonists

Asp363Ser

Ser363 associated with higher sensitivity to exogenously administered glucocorticoids

Haplotype tagging SNPs rs1876828, rs242939, and rs242941

Diplotype GAT/GAT associated with enhanced response to inhaled corticosteroids in multiple asthmatic populations

−444/A→C

C allele associated with larger FEV1 improvements in response to zaﬁrlukast and pranlukast

Tandem repeats of Sp1-binding motifs

Wild-type repeat number (i.e., ﬁve) associated with FEV1 improvements in response to a selective inhibitor of ALOX5 (unknown effects on response to leukotriene receptor antagonists)

sponding to albuterol than subjects homozygous for Gly16, and this association appeared to be independent of asthma status (14). In contrast, ﬁndings from the NIH-funded BetaAdrenergic Response by Genotype (BARGE) study, the largest pharmacogenetic clinical trial on asthma, supported deleterious effects of the Arg16 allele on response to regularly scheduled treatment with albuterol, both in terms of lung function and symptoms (16). A potential explanation for these conﬂicting results has been proposed in the frame of the so-called dynamic model (52). According to this model, alleles (such as Gly16) that are associated with increased downregulation by endogenous catecholamines may be less responsive to acute exposure to exogenous β-agonists but relatively resistant to the tachyphylactic effects of regular βagonist use. Findings from the BARGE and other future prospective studies on ADRB2 have the potential to impact signiﬁcantly the www.annualreviews.org • Asthma Genetics

Tachyphylactic: the condition of having decreased response to a medicine over time, requiring larger doses to produce the original effective response

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clinical use of β2 -agonists in patients with asthma.

INTERACTIVE INFLUENCES FROM BIOLOGICAL SYSTEMS

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We think of the environment as the totality of circumstances that surround us and/or with which our body interfaces. Yet, the soma itself can be considered another type of environment, an internal one that provides the biological systems that are required to translate the information contained in the DNA sequence into meaningful biological events. Why should this internal environment be any less important than the external environment in inﬂuencing the relation of our genes to disease? As discussed in the previous section, a complex sequence of biological interactions is required for a genetic variant to impact downstream disease risk and, therefore, it provides the fundamental assumption of any genetic association study. However, when key elements of these biological systems differ substantially across individuals and populations, they can inﬂuence a gene-disease relationship differently in different individuals, impacting the validity and power of replication genetic studies much like we have seen in the case of gene-by-environment interactions. The simplest example is probably represented by the interaction between polymorphisms at different loci. Indeed, studies using haplotypes have shown stronger power in deﬁning molecular (57) and clinical (15) phenotypes as well as disease risk (37), compared with single SNP analyses, although the latter can sometimes offer statistical advantages. Interactions can also occur between variants that are located on different genes (epistasis). This has been shown in plants. For example, two quantitative trait loci located within a short 210-kb genomic segment in Arabidopsis thaliana were recently found to exert antagonistic epistatic interactions—that is, an allele at one locus could have opposite effects on the phenotype under study depending on which allele was 336

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present at the other locus (60). Thus, in the case of epistatic interactions, main genetic effects can be completely missed or erroneously interpreted if the effects of one locus are considered separately from the concurrent effects of the other. Examples of interactions between genes involved in similar molecular pathways are also found in asthma research. For instance, IL-4 receptor alpha (IL-4Rα) plays a key role in modulating both IL-4 and IL-13 receptor binding (61). Both cytokines are produced by Th2 cells and are involved in the production of IgE, an important asthma-related phenotype. Interestingly, in a Dutch population, the combination of risk genotypes for SNPs in both IL-4Rα and IL-13 genes was found to confer a risk for asthma that was signiﬁcantly higher than that expected under a simple multiplicative model (34). Only carriers of risk genotypes for both genes (not carriers of risk genotypes for only one gene) were at increased risk of asthma compared with carriers of no risk genotype. These ﬁndings have important implications for replication studies across different geographic areas or ethnic groups, as the power of these studies in detecting main effects of these variants will be strongly affected by the corresponding allele frequencies in different study populations. SNP-by-SNP and gene-by-gene interactions are only two of many possible types of interactive effects exerted by biological systems. We still know very little about the potential inﬂuence of epigenetics on susceptibility to complex diseases, but processes of gene regulation are also very likely to modulate the functional impact of variation in DNA sequence, and their measurement should be considered in future functional and association studies. The cell environment is another important modulator of the functional impact of genetic variation. For example, CD14/–159 was found to affect signiﬁcantly gene expression in monocytic Mono Mac 6 cells, but not in liver HepG2 cells (59), possibly because of the different milieux of transcription factors in these two cell types. Similarly, another SNP located 391 bp upstream of CD14/–159 was

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found to affect sCD14 levels in opposite directions in plasma and breast milk, suggesting that the same SNP exerted different effects in hepatocytes than in mammary epithelial cells (57). To add a further layer of complexity, it is likely that different clinical manifestations of asthma are mediated to some extent by different biological systems. For example, asthma symptom attacks that are triggered by allergen exposure will involve different pathways than exacerbations that are infectious in origin. The implication is that the inﬂuence of biological systems themselves needs to be studied within pathway-speciﬁc phenotypes to maximize our ability to detect genetic determinants of disease. Biological systems are also known to differ between males and females, and the potential modifying effect of sex on genetic associations is receiving increasing attention. Recently, heritability and genome-wide linkages for 17 quantitative traits (including four asthmarelated phenotypes) were compared between males and females, and signiﬁcant sex interactions were detected for the majority of them (62). Whether these interactions are related to biological or social differences (i.e., internal versus external environment) between sexes remains to be determined, but these ﬁndings indicate the importance of considering potential sex interactions in genetic association studies. Indeed, the overall impact of multiple biological systems—including haplotype effects, epistatic relationships, and pathwayand cell-speciﬁc events—should be increasingly evaluated in future genetic epidemiological research.

WINDOWS OF OPPORTUNITY Inception of many complex diseases occurs preferentially within windows of opportunity, that is, within limited developmental and/or temporal intervals during which genetic and environmental exposures exert their maximum effect on disease risk. For instance, many children who develop asthma show early markers of allergy and Th2 polarization that

appear to be related to alterations of developmental processes of the immune system in the ﬁrst years of life. Because these alterations are likely related to the presence or absence of speciﬁc environmental exposures, we conclude that, in order to understand the impact of genetic variants on asthma susceptibility, we must take into account not only the level but also the timing of exposure to environmental stimuli. Once again, this concept can be best illustrated by studies on CD14. Consistent with the hygiene hypothesis, microbial exposure early in life has been suggested to reduce risk of atopic sensitization and asthma, and studies in animals have shown that exposure to endotoxin prior to (but not after) sensitization abolishes Th2-type responses linked to asthma phenotypes (63). In addition, plasma sCD14 levels at birth correlate directly with early production of IFN-γ and inversely with the risk for recurrent wheezing by the age of 1 year (64), indicating the importance of adequate CD14-mediated responses to endotoxin early in life for immune maturation and protection against subsequent asthma phenotypes. However, once asthma has occurred, endotoxin exposure is a well-established trigger of disease symptoms and an aggravating factor of disease severity (50). These results suggest that activation of the endotoxin receptor system needs to occur at the right time during the sensitization process to exert its protective effects on asthma risk. Consistent with this scenario, in a longitudinal cohort from Belmont, New South Wales, CD14/–159T (the allele associated with increased gene expression) was found to be protective against atopy during the school years, but this protection disappeared as the children entered adulthood (30). Indeed, among adult farmers occupationally exposed to high levels of endotoxin, the same allele was associated with an increased risk of wheezing and reduced lung function (65). To date, the potential modifying effects of developmental processes on asthma genetics have been rarely studied. Yet, emerging evidence strongly suggests that, as in the case of www.annualreviews.org • Asthma Genetics

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gene-by-environment and gene-by-gene interactions, genetic effects on asthma risk may vary in magnitude, and even in direction, with age and disease stage.

CONCLUSIONS

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In a dispassionate analysis, the most appropriate word to describe the literature on asthma genetics is “inconsistent.” This inconsistency applies to the presence, the magnitude, and even the direction of genetic effects across studies on asthma-related traits. In this review, we have discussed three possible reasons for these inconsistencies: methodological ﬂaws, phenotypic heterogeneity, and the existence of modifying factors. Although at present we cannot determine what proportion of inconsistent ﬁndings is accounted for by each of these issues, future genetic association studies will need to take all of them into account if ﬁnal answers on the genetic components of

asthma and other complex diseases are to be found. Association studies using rigorous statistical approaches and applying genome-wide searches on large populations have been advocated. These studies will certainly be necessary, but they will not be sufﬁcient to resolve the discrepancies that have characterized genetic studies thus far, unless they are coupled with a detailed characterization of the phenotype and with the dissection of interactive effects from environmental factors, biological systems, and developmental processes. In this framework, a different word describes the evidence in asthma genetics: “multifactorial.” To capture this multifactorial nature, we need to decrease the emphasis on analytical approaches based on simple linear genedisease relationships (Figure 1a) and move toward an integrated approach (Figure 1b) that reﬂects the heterogeneity of this complex disease.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS This work was supported by the National Heart, Lung, and Blood Institute, the American Thoracic Society, the Alpha1 Foundation, the American Heart Association, and the Barry and Janet Lang Donor Advised Fund. Dr. Guerra is the recipient of a Parker B. Francis Fellowship.

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:327-341. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

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The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease∗ Stavros Garantziotis,1,2 John W. Hollingsworth,1 Aimee K. Zaas,1 and David A. Schwartz2 1

Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710; email: [email protected], [email protected], [email protected]

2

National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709-2233; email: [email protected], [email protected]

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Key Words

First published online as a Review in Advance on September 10, 2007

innate immunity, toll-like receptors, genomics, gene-environment interactions

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.061206.112455 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0343$20.00 ∗ The U.S. Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

Abstract Toll-like receptors (TLRs) enable innate immune recognition of endogenous and exogenous prototypic ligands. They also orchestrate innate and adaptive immune response to infection, inﬂammation, and tissue injury. Given their signiﬁcance in the immune response, it is not surprising that genetic variations of TLRs can affect their function and by extension affect the response of the organism to environmental stimuli. The genetics of TLRs provides important insights in gene-environment interactions in health and disease, and it may enable scientists to assess patients’ susceptibility to diseases or predict their response to treatments.

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TLR: toll-like receptor LPS: lipopolysaccharide

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PAMP: pathogen-associated molecular pattern

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INTRODUCTION

BIOLOGY OF TLR SIGNALING

The ability of mammals to recognize and engage invading pathogens and to limit and restore tissue injury is essential for their survival. Innate immunity consists of cells (e.g., neutrophils and macrophages) and humoral factors (e.g., complement and lectins) that inherently recognize pathogen- and injuryassociated molecular patterns and are the ﬁrst responders to an infectious or noxious agent. The central role of innate immunity in the infectious response is ﬁrmly established, but innate immunity is also increasingly recognized as an essential component in diseases and biological processes that were previously thought completely unrelated, such as cancer, sterile tissue injury, and transplant rejection. In the past decade, many novel innate immune receptors called toll-like receptors (TLRs) were found, which mediate innate immune recognition and responses. Coincident with the discovery and description of the TLRs came the discovery of genetic variations (polymorphisms) that inﬂuence the afﬁnity of TLRs to their ligands and/or downstream signaling of TLR-mediated innate immune responses. Thus, the study of TLRs pioneered the investigation of interactions between genetic variations and environmental stimuli in pathogenesis. Here we review the evidence for the effects of TLR genetic variations in human disease.

Infectious microorganisms contain elements that have remained unaltered during evolution because they are essential for survival. Examples of these conserved molecules are cell wall components such as lipopolysaccharide (LPS) and peptidoglycan, ﬂagellae and cilia, and DNA or RNA elements. These pathogenassociated molecular patterns (PAMPs) are recognized by pattern recognition receptors on cell membranes and in the cytoplasm. Thus, innate immune systems target structurally conserved PAMPS and allow immediate response to invading microbes without needing the intricate gene rearranging that characterizes the adaptive immune response (1). TLRs are pattern recognition receptors that recognize proteins and toxins released by microorganisms and endogenous ligands, such as heat shock proteins and hyaluronan. Although this is somewhat speculative, it is likely that the TLRs are capable of responding to a variety of environmental toxins, and in fact may have evolved in response to exogenous cues, microbial and otherwise. TLRs are transmembrane receptors with extracellular leucine-rich repeat domains and an intracellular TIR (Toll/IL-1 receptor) homology domain. Most TLRs bind to one or more PAMPs (Table 1). Eleven TLRs have been described in humans and 13 in mice. In humans, TLR genes are found on ﬁve different

Table 1

Human TLRs and their respective PAMPs

TLR

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PAMP

Microorganism

TLR1

TLR2 cofactor?

Neisseria, Borrelia, Mycobacterium

TLR2

lipoprotein, peptidoglycan, HSP60

gram-pos., Mycobacterium, spirochetes, mycoplasm

TLR3

dsRNA

viruses

TLR4

endotoxin (lipopolysaccharide)

gram-neg. and respiratory syncytial virus

TLR5

ﬂagellin

bacteria

TLR6

peptidoglycan and zymosan

fungi

TLR7

imidazoquinoline in vitro

unknown in vivo

TLR8

imidazoquinoline in vitro

unknown in vivo

TLR9

bacterial DNA (unmethylated CpG motif)

bacteria

TLR10

prokaryotic DNA

bacteria

TLR11

proﬁlin-like protein

Toxoplasma gondii, uropathogenic E. coli

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Figure 1 MyD88-dependent TLR-signaling. Binding of ligand to TLR leads to TLR association to adaptor proteins via the TIR domain. With the participation of IRAK-1 and IRAK-4, MyD88 induces TRAF6-mediated activation of NF-κB. NF-κB dissociates from its inhibitor IκB and is translocated to the nucleus, where it induces cytokine gene activation.

chromosomes: chr. 4 (TLR1, TLR2, TLR3, TLR6, and TLR10), chr. 9 (TLR4), chr. 1 (TLR 5), chr. 10 (TLR7, TLR8), and chr. 3 (TLR9). TLR11 may be on chr. 14. TLR sequences are highly conserved throughout evolution, which suggests that they are crucial to immune responses and makes genetic variations more likely to have functional relevance. TLR signaling is mainly mediated through the intracellular protein myeloid differentiation factor 88 (MyD88), but MyD88independent pathways also exist (Figure 1). In vertebrates, the TLR signaling cascade occurs via signaling complexes that include IL-1 receptor associated kinase (IRAK) and tumor necrosis factor receptor–associated factor 6 (TRAF6). The cascade culminates in nuclear factor kappa B (NFκB)-mediated pro-

motion of transcription of proinﬂammatory cytokines such as TNFα, IL-1, IL-6, and IL-8 (Figure 1).

TLR POLYMORPHISMS AND INFECTIOUS DISEASE SUSCEPTIBILITY A critical role for TLRs is recognition of potential pathogens. The recognition that host genetic variation alters host susceptibility to infections has led to multiple studies evaluating TLR polymorphisms in susceptibility to infections. Knockout studies in mice have deﬁned the pathogen speciﬁcity of many TLRs, but ascertaining the role of subtle differences in TLR function related to polymorphisms requires a human cohort as well as mechanistic

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proof of functional differences between the variant and common receptor types. A listing of all TLR polymorphisms and studies evaluating association with disease susceptibility or progression is beyond the scope of this review, but key examples are described below.

TLR4

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Human TLR4 was the ﬁrst characterized mammalian TLR (2), and the interactions between TLR4, its effector molecules, and LPS comprise the best-described signaling pathway in mammalian innate immunity. TLR4 recognizes and binds LPS via accessory molecules known as lipopolysaccharide binding protein, CD14, and MD-2. The functionality of TLR4 was characterized by genetic analysis of a mouse strain known to be LPS-hyporesponsive. Early research discovered that homozygosity for a single locus (Lps d ) produced the LPS hyporesponsive state. Positional cloning led to the discovery that the gene responsible for Lps d is TLR4, and that mutations in this receptor lead to LPS hyporesponsiveness (3). Subsequently, more TLR4 mutant murine strains were discovered, and TLR4 knockout mice conﬁrmed this association. Building on these murine data, the cloning and sequencing of human TLR4 identiﬁed two missense single-nucleotide polymorphisms (SNPs): Asp299Gly and Thr399Ile. The Asp299Gly substitution results in replacement of a conserved aspartic acid residue with glycine at amino acid 299, and alters the extracellular domain of the receptor. We found an additional missense mutation [replacing a nonconserved threonine with an isoleucine at amino acid 399 (Thr399Ile) in the extracellular domain of TLR4] cosegregating with the Asp299Gly substitution (4). These polymorphisms had an allele frequency of ∼7% in the original study (Caucasian subjects), although the allele frequency is substantially lower in other populations (5). The original subjects with the variants Asp299Gly and Thr399Ile were observed to 346

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be hyporesponsive to inhaled LPS, and in vitro evaluations showed defective responses to LPS by cells possessing the Asp299Gly variant (4). These ﬁndings remain somewhat controversial because in vitro studies have not consistently conﬁrmed defective LPSrelated signaling related to the Asp299Gly polymorphism (6). Conﬂicting results may reﬂect small study size, heterogeneity of the underlying phenotype, or focus on SNP-speciﬁc genotyping instead of evaluating for the multiple other known polymorphisms of TLR4 (7). Human TLR4 contains many rare missense mutations, and it is possible that these unusual mutations inﬂuence disease susceptibility, as was shown for a cohort of patients with meningococcal sepsis (8). Many studies have assessed the role of common human TLR4 polymorphisms in susceptibility to systemic infections and systemic inﬂammatory syndromes involved in host-bacterial interactions. Because of the known involvement of TLR4 in the response to gram-negative LPS, susceptibility to gram-negative infection has been studied extensively. Association studies have linked TLR4 polymorphisms to increased susceptibility to gram-negative osteomyelitis (9), gram-negative sepsis (10), and necrotizing pancreatitis (11). TLR4 is also involved in host recognition of yeast (12), so TLR4 polymorphisms have been evaluated in patient cohorts with candidemia. Notably, the Asp299Gly polymorphism was shown to be overrepresented in patients with candidemia as compared to controls, and this heightened susceptibility was potentially related to increased IL-10 production (13). Again, additional mechanistic studies would lend support to this association. Plasmodium falciparum, the predominant cause of severe malaria worldwide, has recently been shown to interact with several TLRs. P. falciparum glycosylphosphatidylinositol (GPI) was reported to induce signaling via both TLR2 and -4, and hemozoininduced immune activation was reported to involve TLR9 (14, 15). A case-control study

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of 870 Ghanian children found that TLR4Asp299Gly and TLR4-Thr399Ile conferred 1.5- and 2.6-fold increased risks of severe malaria, respectively (16). This example illustrates how TLR polymorphisms affect disease progression rather than overall disease susceptibility, as frequencies of TLR4 variants were increased only slightly in parasitemic controls but signiﬁcantly in severe malaria patients. Finally, TLR4 polymorphisms have been associated with susceptibility to respiratory syncytial virus (RSV) infection in several small cohorts (17). These evaluations were undertaken because RSV activates TLR4 via the viral fusion (F) glycoprotein (18). RSV bronchiolitis can vary from mild to severe respiratory disease requiring hospitalization. A major factor in determining disease severity is the host immune response. Thus, alterations in the interaction of RSV with TLR4 may contribute to host outcome following infection. However, controversy exists regarding the role of these polymorphisms and disease susceptibility, as other studies have demonstrated a lack of association of the Asp299Gly polymorphism with development of RSV bronchiolitis in children (19).

TLR2 TLR2 plays a critical role in the response to gram-positive organisms by responding to the PAMP lipoteichoic acid. Additional ligands for TLR2 include components of mycobacteria and fungi, as well as viral envelope proteins. The most-studied TLR2 polymorphism is Arg753Gln, which results in a decreased immune response to stimulation with the prototypic TLR2 ligand bacterial lipopeptide (20). Located within the intracellular TIR domain, this SNP occurs in 9%–10% of Caucasians (21) and has been associated with tuberculosis (22). The TLR2 SNP (Arg677Trp) increases the risk of lepromatous leprosy (23) and tuberculosis (24). A highly polymorphic GT repeat has been found 100 bp upstream of the translation start site in the TLR2 gene and affects promoter activity (25).

TLR2 polymorphisms have been studied extensively to evaluate their role in susceptibility to common bacterial infections. Some studies have shown that deﬁciencies in TLR2 may increase susceptibility to Staphylococcus aureus infection (21). However, in vitro data demonstrate that only one copy of wild-type TLR2 is necessary for full response to lipoteichoic acid (20), so this particular polymorphism does not have a large effect on susceptibility to S. aureus (25). The involvement of TLR2 polymorphisms in pneumococcal disease has not been shown conclusively; one small study demonstrated a lack of association of common TLR2 polymorphisms with invasive pneumococcal disease (26). TLR2 also responds to the viral envelope protein of cytomegalovirus (CMV) (27). Primary CMV infection is a relatively universal phenomenon, but CMV, like other herpesviruses, remains latent in the host and can return to its replicative state during times of immune suppression. Thus, control of CMV disease is a major problem in solid organ transplant recipients. Kijpittayarit et al. (28) evaluated the role of Arg753Gln in control of CMV disease in a cohort of 92 liver transplant recipients. Homozygosity, but not heterozygosity, for the TLR2 Arg753Gln polymorphism was associated with CMV disease. CMV disease occurred in 3 (60%) of 5 patients who were homozygous for the TLR2 Arg753Gln polymorphism, in 1 (14%) of 7 patients who were heterozygous for this polymorphism, and in 20 (25%) of 80 patients who did not have the this polymorphism. The difference between homozygous and non-homozygous individuals was statistically signiﬁcant.

TLR5 TLR5 is known to respond to bacterial ﬂagellin. Sequencing of TLR5 in healthy donors demonstrated a cytosine–thymidine transition at base pair 1174 that changes the arginine at amino acid 392 to a stop (TLR5392STOP). This change is predicted to prematurely truncate TLR5 in the extracellular

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COPD: chronic obstructive pulmonary disease

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domain and cause the loss of the transmembrane domain and the entire signaling cytoplasmic tail (29). In a case-control study of persons involved in an epidemic of Legionnaire’s disease, combined with in vitro lung epithelial studies, it was demonstrated that this TLR5 stop codon polymorphism is dominant and is associated with susceptibility to infection by a ﬂagellated organism in humans (29). Despite responding to a relatively common bacterial antigen (ﬂagellin), this polymorphism was not found to be associated with susceptibility to enteric fever with S. typhi (30).

TLR9 TLR9, which responds generally to the PAMP of bacterial (or unmethylated) DNA, is also involved in host response to HIV infection. Two SNPs in TLR9 (1635A/G and +1174G/A) in linkage disequilibrium with each other were associated with the rapidprogressor phenotype (31). As in the case of TLR4 and malaria, this TLR polymorphism inﬂuences disease progression rather than acquisition. Controlling the initial steps of hostpathogen interactions is a key function of TLRs. The complexity and variation of host response to invading microbes is inﬂuenced by genetically encoded variation in TLR activity, or perhaps PAMP afﬁnity. The myriad of genetic associations between TLR polymorphisms and infection susceptibility provide insights into the basic biology of the immune response, as well as the opportunity to design individually targeted prophylactic or therapeutic interventions. Indeed, TLR agonists are already in preliminary trials for vaccination and antibacterial use (32).

TLR1 Like other TLRs, TLR1 enables host responses to a variety of bacteria, including pathogenic species of mycobacteria. I602S, a common polymorphism within TLR1, is associated with aberrant trafﬁcking of the receptor to the cell surface and diminished re348

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sponse of blood monocytes to bacterial agonists. Cells expressing the variant TLR1 are less responsive to mycobacterial stimulation than are cells with wild-type TLR1. Among a cohort of white Europeans, the 602S allele is associated with a decreased incidence of leprosy, suggesting that Mycobacterium leprae subverts the TLR system as a mechanism of immune evasion (33).

TLRs IN PULMONARY DISEASES Current data suggest that TLR signaling can modify both allergic asthma and chronic obstructive pulmonary disease (COPD). Activation of TLRs can be either beneﬁcial or detrimental depending on many host factors, as well as dose, duration, and intensity of exposure to TLR ligands. Multiple epidemiologic studies have associated childhood exposure to TLR ligands with protection against developing allergic asthma later in life (the “hygiene hypothesis”); for example, individuals living on farms have a reduced risk of developing hay fever or asthma (34). The most extensively studied TLR is TLR4. Recent work demonstrated that levels of the TLR4 ligand LPS in the bedding of school-aged children are inversely proportional to the incidence of hay fever and atopic asthma (35). However, in apparent contrast to those observations, exposure to higher levels of LPS has also been associated with exacerbations of airway disease (36). In addition, in patients with documented dust mite allergy, the LPS levels in the home environment are more closely related to exacerbations of asthma and wheeze than the levels of speciﬁc antigen (37, 38). These observations suggest that the timing and level of TLR activation could regulate the ultimate effects on clinical disease. Given the apparent opposing effects of LPS, it is perhaps not unexpected that many studies have not revealed an effect of the common functional TLR4 polymorphism (Asp299Gly) on the overall incidence of allergic asthma. However, individuals having this polymorphism have a blunted airway

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response (4) and reduced systemic inﬂammation (39) in response to inhaled LPS. Consistent with these observations, a study of asthma speciﬁcally associated with LPS in house dust showed that people with the TLR4 polymorphism Asp299Gly had a decreased risk of bronchoreactivity (40). These observations are consistent with the hypothesis that LPS can exacerbate existing airway inﬂammation and that individuals with the Asp299Gly polymorphism have diminished pulmonary responses to LPS. However, other studies found that asthmatic individuals with the Asp299Gly polymorphism have an increased incidence of atopic asthma (41). Each of these associations with common polymorphisms of TLR4 is consistent with the ability of LPS to exacerbate existing asthma and to decrease atopy, respectively, and suggests that the Asp299Gly polymorphism could be predictive of airway and atopic responses in only a speciﬁc subset of the population. Genetic approaches to identify associations between airway disease and activation of the innate immune system are not limited to TLR4. Polymorphisms of the accessory LPSbinding molecule CD14 are associated with increased levels of soluble CD14 and an enhanced biological response to LPS, and have been associated with both a decrease in total serum IgE in asthmatic children (42) and a decrease in lung function among LPS-exposed farmers (43). The observation among children is consistent with the hygiene hypothesis; attenuation of allergic symptoms coincides with enhanced response to LPS. The enhanced biological response among farmers occupationally exposed to high levels of LPS would be expected to manifest as decreased lung function, as was observed. When the level of LPS exposure in the home is considered, the effect of CD14 is remarkably consistent (44, 45). In subjects exposed to low concentrations of LPS, the T allele for CD14/-159 (associated with increased soluble CD14) was protective. However, at high concentrations of LPS, the opposite was observed. These studies highlight the importance of gene-environment in-

teractions in a complex disease such as allergic asthma. Given the broad range of environmental challenges leading to asthma, it is not surprising that apparent divergent phenotypes are observed with modiﬁcation of TLRdependent signaling associated with common functional polymorphisms. Growing evidence suggests that TLRs other than the TLR4/CD14 complex play a role in airway disease. For example, common polymorphisms of TLR2 among European farmers were associated with protection from asthma, atopy, and hay fever (46). This observation would suggest that a blunted response to TLR2 agonists is protective against the subsequent development of allergic asthma. Although this is inconsistent with the hygiene hypothesis, the exposure dose to ligands of TLR2 among farmers, as well as the functional signiﬁcance of this polymorphism of human TLR2, remains poorly understood. Similarly, polymorphisms of TLR6 have been associated with a decreased risk of asthma in African-Americans (47). Finally, polymorphisms of TLR10 have been associated with an increased risk of asthma in two separate cohorts (48). Despite the limitations of genetic association studies, these observations provide insight into the role of TLRs in the development and progression of human airway disease. Unlike TLR2 and TLR4, signaling through TLR9 appears to effectively prevent the development of atopic airway disease and can reverse established eosinophilic inﬂammation (for review see Reference 49). Thus, unmethylated CpG DNA, which binds TLR9, attenuates multiple components of the allergic phenotype, including eosinophilic airway inﬂammation, serum IgE, Th2 cytokines, airway hyperresponsiveness, subepithelial ﬁbrosis, and goblet cell metaplasia (50). Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG or immunostimulatory sequence-DNA (ISS-ODN) have a similar suppressive effect in murine models of allergic asthma and could have important

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therapeutic potential in human allergic disease (51). In 2004, clinical studies of a TLR9-agonist therapeutic compound (CpG-ODN) for allergic asthma were initiated, and a second compound is currently undergoing preclinical evaluation. Furthermore, immunostimulatory sequences (ISS-ODN), either alone or combination with allergens, are currently under investigation for therapeutic applications in allergic asthma. Results from these clinical trials are unavailable at this time but are expected within 2–3 years. The role of TLR agonists or antagonists to pharmacologically manipulate the immune system and prevent the development or progression of airway disease is an expanding area of investigation. Given the complexity of the asthma phenotype, it is likely that timing and dose of the TLR agonist/antagonist will greatly affect the impact on airway disease. In addition to allergic airway disease, it is likely that TLRs play a critical role in the development of COPD. This is supported by the epidemiologic observation that occupational exposure to LPS is associated with decrements in lung function (52). Consistent with the role of TLR4 signaling in the development of COPD, some recent work demonstrates a low frequency of the Asp299Gly functional polymorphisms of TLR4 in patients with COPD (53), although this ﬁnding has not always been replicated. Seemingly in contrast to these observations, recent observations demonstrate that in the absence of signiﬁcant LPS exposure, TLR4 signaling plays a critical role in protecting the lungs of mice from the development of emphysema (54). Once again, the intensity of TLR signaling may determine the observed phenotype. Clear understanding of the biological and pathological role of innate immune signaling in the lungs will require comprehensive understanding of gene-environment interactions. Given the observations outlined here, it seems reasonable that low-level activation of TLRs could prove protective in COPD, whereas high levels of activation may prove harmful.

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TLRs IN CARDIOVASCULAR DISEASE Atherosclerosis is an inﬂammatory process, and innate immunity has been shown to participate in the development and rupture of atherosclerotic plaques (55). TLR4 polymorphisms that render the receptor less responsive to its ligands would therefore be expected to hinder the development and progression of atherosclerosis. Indeed, the Asp299Gly polymorphism has been associated with decreased atherosclerosis (56), decreased risk for acute coronary events (57), and an improved response to statin treatment (58). The exact mechanism of this beneﬁcial effect is unknown; however, TLRs are expressed on several cells that participate in the atherosclerotic plaque, such as macrophages, dendritic cells, endothelia, smooth muscle cells, and lymphocytes. TLR4 may therefore mediate the inﬂammatory response to atherogenic stimuli, and hyporesponsive TLR4 polymorphisms may ameliorate this inﬂammatory response, thereby protecting the vessels from plaque progression. Consistent with this hypothesis, Asp299Gly carriers have lower serum levels of circulating proinﬂammatory cytokines, such as IL-6, ﬁbrinogen, and soluble VCAM1 (56). Furthermore, TLR4 polymorphisms may play a role in the subsequent modulation of adaptive immunity, which is activated in atherosclerosis (59). As the involvement of innate immunity in atherosclerosis is better understood, more genetic factors are likely to be discovered to inﬂuence both the susceptibility to cardiovascular disease (56, 60) and the response to treatment (58).

TLRs IN CANCER Inﬂammation is a double-edged sword in cancer. On one hand, chronic inﬂammation is associated with carcinogenesis, and cancer is a complication of chronic inﬂammatory conditions such as Crohn’s disease, chronic cystitis, and hepatitis (61). TLR activation leads to production of NF-κB, which is associated

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with carcinogenesis and chemoresistance (for a comprehensive review see Reference 62). On the other hand, the immune system is necessary for the elimination of malignant cells, and immunosuppressed patients are at risk for the development of cancer (63). Immunotherapy (i.e., use of the patient’s own immune system to combat cancer, with the aid of vaccination or adjuvants) has been gaining attention as a potential treatment option in cancer. Indeed, we are only now beginning to understand how cancer cells evade elimination by modifying the innate and adaptive immune responses of the host (64). Activation of the immune system may therefore be a viable approach to cancer therapy. Clinical data regarding the effect of TLRs on cancer pathogenesis and treatment response are scant. TLR4 is important in the clearance of H. pylori. The hyporesponsive Asp299Gly TLR4 polymorphism was found to be associated with an 11-fold increased risk of hypochlorhydria in patients with H. pylori infection, and with a more than twofold risk of noncardia gastric carcinoma in all carriers (65). The polymorphism had no effect on the prevalence of H. pylori infection in the population, underscoring the gene-environment interaction in cancer pathogenesis and suggesting that the ability of patients to clear H. pylori infections may affect their subsequent cancer risk. The same polymorphism was inversely associated with the risk of nonHodgkin’s lymphoma (66) and gastric lymphoma (67) in two different populations. In other studies, two separate sequence variants of TLR4 were associated with a ∼25% increase in prostate cancer risk in a Caucasian population (68, 69). However, both polymorphisms were in untranslated regions of the gene and have no known functional significance; they may simply be markers of still unidentiﬁed functional polymorphisms with which they are in linkage disequilibrium. The same Caucasian population examined in one of the above studies also showed an association of multiple haplotype-tagging SNPs at the TLR6-TLR1-TLR10 gene cluster on chr.

4 with a 20%–38% increased risk of prostate cancer. These SNPs were in linkage disequilibrium, which suggests a founder variant, thought likely to be in TLR1 or TLR6 (70). Because chronic prostatitis is a risk factor for prostate cancer, TLR variants promoting inﬂammation may predispose their carriers for carcinogenesis. Researchers have identiﬁed TLR4 as an important mediator of the anticancer actions of the chemotherapy drug OK-432 (71), and TLR2- and TLR4-mediated maturation of dendritic cells is important for the anticancer actions of bacillus Calmette-Guerin in the treatment of bladder cancer (72). Chronic lymphocytic leukemia (CLL) and some types of lymphoma cells strongly express TLR7 and TLR9 on their surface. Several pilot studies are under way for treatment of such patients with TLR7 and TLR9 agonists; the hypothesis is that TLR activation may promote cell death or make cells more amenable to immune recognition (73). The TLR7 agonist imiquimod is currently approved for the treatment of basal cell carcinoma in the United States (74), and several other TLR agonists are being investigated (mostly as adjuvants) in cancers of the colon, lung, lymphatic system, skin, and breast (32). In conclusion, TLR cancer biology is a promising target for epidemiological studies of risk assessment, as well as for the design of novel treatment options for this devastating disease.

TLRs IN INFLAMMATORY BOWEL DISEASE Inﬂammatory bowel disease (IBD) is currently thought to result from an abnormal immune response to gut ﬂora and is strongly associated with innate immune activation. Hyporesponsive polymorphisms in the cytoplasmic innate immune activator NOD2, which binds to muramyl dipeptide on gram-positive and -negative bacteria and aids in the intracellular killing of Salmonella, have been associated with the occurrence of Crohn’s disease in humans (75). TLR4 recognizes

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the gram-negative wall component LPS, and therefore was a strong candidate gene for the pathogenesis of IBD. The hyporesponsive Asp299Gly TLR4 polymorphism was found to be associated with the presence of Crohn’s disease in patients from Belgium, Germany, and Greece, but not from Scotland or the Netherlands. Another German study found an association between ulcerative colitis and the Thr399Ile polymorphism, but not the Asp299Gly TLR4 polymorphism; no association with Crohn’s disease was observed (these studies are reviewed in Reference 76). Given the fairly strong experimental evidence from animal models for the role of TLR4 in the pathogenesis of IBD (77), the observed discordant reports may reﬂect methodological or population differences. TLR5 recognizes ﬂagellin, a common antigen on gut bacteria (Table 1) and a dominant antigen in IBD (78); its signaling is modulated by NOD2 (79). TLR5 was therefore a plausible candidate gene for susceptibility testing, and in fact a dominant-negative TLR5 polymorphism (described above) appears to be protective against Crohn’s disease (80). Polymorphisms in TLR1, -2, and -6 do not affect incidence but may inﬂuence the extent of IBD (81). In aggregate, these reports suggest that the innate immune response to bowel ﬂora is important for the pathogenesis of IBD, and that genetic variation in innate immune response genes, including the TLRs, contributes to susceptibility to IBD.

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INNATE-ADAPTIVE IMMUNITY CROSS-TALK: TLRs IN AUTOIMMUNITY AND TRANSPLANTATION The innate and adaptive immune responses interact in a variety of ways. Recent evidence suggests that innate immunity modulates adaptive immune responses at least partly by enhancing antigen presentation through dendritic cells (82) and suppressing regulatory T cell activity (83). Innate immunity can therefore inﬂuence disease processes 352

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that were long assumed to be the exclusive domain of adaptive immunity, such as autoimmune diseases and transplant allograft rejection. Autoimmune diseases are genetically complex and are inﬂuenced by endogenous and exogenous stimuli. Systemic lupus erythematosus (SLE) is the prototypic autoimmune disease, characterized by the production of antibodies against nucleosomal antigens. A novel model of pathogenesis postulates a twophase model, with initial TLR-independent uptake and presentation of self-antigens by dendritic cells and a later TLR-dependent ampliﬁcation of the adaptive response mediated by IFN-α/β (84). Exogenous bacterial and viral components can promote a direct activation of autoimmunity by molecular mimicry, or indirectly aggravate an autoimmune predisposition by inducing TLR activation on lymphocytes. Furthermore, a myriad of endogenous TLR ligands are now being described, such as DNA, RNA, hyaluronan, heparan, ﬁbrinogen, and heat-shock proteins (reviewed in Reference 85), which can directly bind TLRs on lymphocytes and antigen-processing cells and thus promote the autoimmune process. Consistent with this hypothesis, the TLR5-392STOP polymorphism described above was recently associated with protection against SLE (86). Interestingly, this association was most pronounced in individuals who were seronegative for doublestranded DNA antibodies, indicating that innate immune responses to a bacterial exposure may be pathogenic in only a subpopulation of SLE patients. Allograft rejection is the major obstacle to successful organ transplantation. Recent human and animal research supports a role for TLR signaling in allograft rejection. In animal models, absence of the TLR messenger MyD88 leads to tolerance of mismatched skin grafts (87). In an animal bone marrow transplant model, noninfectious lung injury (idiopathic pneumonia syndrome) can be prevented by using TLR4-deﬁcient animals (88). TLR activation may therefore promote

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alloimmune graft injury by inducing adaptive immune activation. Human data support this hypothesis: Carriers of the LPShyporesponsive TLR variant Asp299Gly are protected from acute and chronic rejection after lung and kidney transplant (89–91).

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FUTURE DIRECTIONS The past decade has seen enormous progress in understanding the role of innate immunity in infection and tissue injury, atherosclerosis, inﬂammatory airway and bowel disease, cancer development and defense, and autoimmune and alloimmune disease. More areas of involvement are likely to be discovered in the future; for example, TLR signaling is now recognized in the biological process of

Alzheimer’s disease (92). Therapeutic modiﬁcation of TLR signaling may therefore be a novel approach to many diseases and is being actively pursued (32). TLR sequence polymorphisms and epigenetic effects (93) inﬂuence both susceptibility to and progression of these diseases, and they may ultimately be used for more individualized diagnosis and treatment. Innate immune biology is a young ﬁeld of medical research that holds much promise for risk stratiﬁcation and novel therapeutic approaches. The innate immune receptors are involved in a multitude of biological processes, and their study has uncovered individual susceptibility factors to numerous diseases. Further research will undoubtedly lead to important translational applications.

SUMMARY POINTS 1. Toll-like receptors (TLRs) mediate recognition of endogenous and exogenous molecular patterns and orchestrate innate and adaptive immune responses to these stimuli. 2. TLRs are very important constituents of the immune system, and have therefore been genetically conserved throughout evolution. 3. Genetic changes in TLR sequence have functional repercussions in the immune response and innate immune signaling. 4. Innate immunity (including TLRs) participates in a wide array of disease processes such as infection, airway disease, atherosclerosis, cancer, inﬂammatory bowel disease, autoimmune diseases, and allograft rejection. 5. Investigation of the genetics of TLRs has enabled scientists to understand how genes modify the host response to environmental and endogenous stimuli. 6. In the future, TLR genetics may help physicians pursue individualized prophylaxis and treatment.

FUTURE ISSUES 1. The extent to which innate immunity is involved in many pathological processes (e.g., atherosclerosis, carcinogenesis and immune response to cancer cells, degenerative diseases) is still unknown. 2. Some TLRs, such as TLR10 and TLR11, have unknown or insufﬁciently described ligands. New ligands are being described for other TLRs.

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3. Timing, dose of the TLR ligand, and cell type involved may signiﬁcantly inﬂuence the TLR-mediated response to a speciﬁc ligand, so therapeutic interventions may achieve conﬂicting results.

DISCLOSURE STATEMENT

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Dr. Schwartz has an active patent on variants of TLR4 and their importance in a variety of diseases that are affected by innate immunity. Dr. Zaas is on the Speakers Bureau of Pﬁzer Chemicals, serves as a consultant for Merck and Co., and receives grant support from Enzon Pharmaceuticals.

ACKNOWLEDGMENTS The authors gratefully acknowledge support from the National Institute for Environmental Health Sciences (ES11961); the National Heart, Lung, and Blood Institute (HL91335); the National Institute of Allergy and Infectious Diseases (AI58161 and 5AI065837); and the intramural research program at the National Heart, Lung, and Blood Institute, as well as the American Thoracic Society/American Society of Transplantation (T-05-004).

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:343-359. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Advances in Antifungal Therapy Carole A. Sable,1 Kim M. Strohmaier,2 and Jeffrey A. Chodakewitz2 1

Novexel SA, Romainville, France; email: [email protected]

2

Merck Research Laboratories, West Point, Pennsylvania

Annu. Rev. Med. 2008. 59:361–79

Key Words

First published online as a Review in Advance on October 29, 2007

echinocandins, voriconazole, posaconazole, galactomannan, β-glucan

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.062906.071602 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0361$20.00

Abstract The prevalence of invasive fungal infections (IFIs) has increased over the past three decades owing to the increasing numbers of immunocompromised hosts. These infections are associated with signiﬁcant morbidity and mortality. Recent signiﬁcant advances in antifungal therapy include the broad-spectrum triazoles (voriconazole and posaconazole) and a new class of antifungals, the echinocandins (caspofungin, micafungin, and anidulafungin). New treatment strategies, such as combination therapy and pre-emptive therapy, are being investigated. There have also been signiﬁcant improvements in diagnostics; the galactomannan enzyme immunoassay and the β-glucan test are now part of the EORTC/MSG criteria for diagnosis of IFI. Despite these advances, there remain a number of unanswered questions regarding optimal management of serious fungal infections, and research continues to discover and develop new therapies and evaluate new management strategies.

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INTRODUCTION HSCT: hematopoietic stem cell transplant IFI: invasive fungal infection

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MIC: minimum inhibitory concentration

During the past 30 years, there has been a dramatic increase in the number of immunocompromised patients associated with the increase in the frequency of solid-organ and hematopoietic stem cell transplants (HSCTs), more aggressive chemotherapy, the advent of AIDS, and advances in critical care. There has been a coincident rise in the prevalence of opportunistic fungal infections. Candida and Aspergillus spp. remain the most common causes of invasive fungal infections (IFIs) in immunocompromised hosts, but infections due to other fungi, such as zygomycetes, Fusarium, Scedosporium, and the black molds have been seen more frequently, particularly in severely immunosuppressed patients (1). IFIs have historically been associated with high morbidity and mortality, partly because of the limitations of available antifungal therapies and difﬁculties in making a rapid and accurate diagnosis. Since the late 1950s, the standard of care for treatment of serious fungal infections had been amphotericin B, an IV-only agent with signiﬁcant toxicity. The 1990s saw the introduction of lipid formulations of amphotericin B, as well as the triazoles ﬂuconazole and itraconazole. Although these agents offered clear advantages over amphotericin B, they were limited by formulation, spectrum of activity, and/or the development of resistance. In the past decade, there have been major advances in therapy. Broader-spectrum triazoles (voriconazole and posaconazole) and the new echinocandin class of antifungals (caspofungin, micafungin, and anidulafungin) have been introduced, and noninvasive diagnostic methods have improved. This review focuses on recent developments in the diagnosis and treatment of IFIs.

NEW ANTIFUNGAL THERAPIES Since 2001, several new antifungal agents have received regulatory approval for a broad range of indications. One of the challenges 362

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in evaluating the relative efﬁcacy and safety of these new antifungals has been the lack of direct comparative trials. Studies have typically compared a new drug to an older agent (e.g., amphotericin B, ﬂuconazole) and have differed in study design, patient population, diagnostic criteria, and endpoint definitions. An additional challenge has been that the spectrum of activity is frequently deﬁned by in vitro susceptibility tests with little, if any, clinical data. This is problematic because of variations in in vitro susceptibility testing methods and the lack of approved deﬁnitions for susceptible versus resistant pathogens (MIC breakpoints) for the new agents, except for voriconazole. Interpretation of in vitro data is further complicated by the lack of correlation between minimum inhibitory concentration (MIC) and clinical outcome for the echinocandins. As a result, it is difﬁcult to make direct comparisons among agents.

Echinocandins Echinocandins inhibit the synthesis of (1,3)β-D-glucan synthetase, which is encoded by FKS1 and FKS2. The echinocandins exert their antifungal effect through noncompetitive inhibition of (1,3)-β-D-glucan biosynthesis, which causes destabilization of the fungal cell wall, cell lysis, and death. The three currently available echinocandins, namely caspofungin, micafungin, and anidulafungin, exist only in IV formulations. All exhibit linear pharmacokinetics, are highly protein bound (97%–99%), and are not dialyzable. Metabolism and drug interactions for the echinocandins are summarized in Table 1. The echinocandins are active against a broad range of Candida species and Aspergillus species, including those that are resistant to azoles and polyenes. They have activity against the cyst form of Pneumocystis carinii (but not the trophozoite) and have shown variable activity against Penicillium spp. The echinocandins are not active against Cryptococcus spp. and have limited in vitro activity

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Metabolism and drug interactions of new antifungal agents

Drug

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Metabolism

Drug interactions

Caspofungin (5)

Slowly metabolized in the liver through nonenzymatic peptide hydrolysis and N-acetylation into inactive metabolites No apparent metabolism through CYP450 system

Based on LFT elevations in a phase I study, use of caspofungin with cyclosporine should be limited to patients for whom the potential beneﬁt outweighs the potential risk Tacrolimus serum levels reduced ∼20% by caspofungin and should be monitored during concomitant use Dose may need to be increased from 50 to 70 mg daily when used with efavirenz, nevirapine, phenytoin, dexamethasone, rifampin, carbamazepine, or other inducers of metabolism

Micafungin (12)

Metabolized in the liver by arylsulfatase, catechol-O-methyl-transferase, and to a lesser extent by CYP (primarily CYP3A)

Increases AUC of sirolimus by 21% and the AUC and Cmax of nifedipine by 18% and 42%, respectively Monitor for sirolimus or nifedipine toxicity and reduce dosages if necessary

Anidulafungin (17)

Degraded chemically in the bloodstream to inactive products

No clinically relevant drug interactions observed

Voriconazole (35)

Extensive metabolism by hepatic CYP2C19, CYP3A4, and CYP2C9

Do not use with terfenadine, astemizole, cisapride, pimozide, quinidine, sirolimus, rifampin, carbamazepine, long-acting barbiturates, high-dose ritonavir, efavirenz, rifabutin, or ergot alkaloids Voriconazole dose adjustment may be needed when used with phenytoin, protease inhibitors, or NNRTIs Dose adjustments required for cyclosporine, tacrolimus, phenytoin, and omeprazole; may be needed for methadone, oral contraceptives, warfarin, HIV protease inhibitors, NNRTIs, benzodiazepines, statins, calcium channel blockers, sulfonylureas, and vinca alkaloids

Posaconazole (42)

Primarily metabolized via UDP glucuronidation Substrate for p-glycoprotein

Do not use with ergot alkaloids, terfenadine, astemizole, cisapride, pimozide, halofantrine, or quinidine. Avoid concomitant use with cimetidine, rifabutin, or phenytoin May increase plasma levels of cyclosporine, tacrolimus, sirolimus, midazolam, vinca alkaloids, statins, and calcium channel blockers

Abbreviations: AUC, area under the curve; Cmax, ˙maximum concentration of drug; LFT, liver function test; NNRTI, non-nucleoside reverse transcriptase inhibitor; UDP, uridine diphosphate.

against Fusarium spp., Rhizopus spp., Mucor spp., Scedosporium spp., and Pseudallescheria boydii (2–4). Thus, their spectrum of activity is relatively narrow. Caspofungin (CANCIDAS®) was approved in the United States and European Union in 2001 as salvage therapy for invasive aspergillosis (IA). It has subsequently been approved for the treatment of esophageal candidiasis (EC), candidemia, and other invasive Candida infections (intra-abdominal abscesses, peritonitis, and pleural space infections) and as empirical therapy for presumed

fungal infections in febrile neutropenic patients (5) (Table 2). In a randomized double-blind phase III study of 175 patients with EC, the efﬁcacy of caspofungin was comparable to that of ﬂuconazole (81% versus 85%) with a similar safety proﬁle (6). In a randomized double-blind phase III study of 224 patients with invasive candidiasis (IC), primarily candidemia (7), caspofungin met the deﬁnition of noninferiority to amphotericin B (73% versus 62%) with signiﬁcantly fewer adverse events and less nephrotoxicity. Efﬁcacy has also been seen in less common www.annualreviews.org • Advances in Antifungal Therapy

IA: invasive aspergillosis EC: esophageal candidiasis IC: invasive candidiasis

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Clinical studies of new antifungal agents

Study Design

Treatment

Results

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Caspofungin Randomized, double-blind, phase III; 175 pts with EC (6)

Cas 50 mg vs Flu 200 mg for 7–21 days

Overall response rate (symptom resolution + signiﬁcant endoscopic improvement 5–7 days after end of treatment) = 81% for Cas vs 85% for Flu; relapse rate = 28% for Cas vs 17% for Flu ( p = 0.19)

Randomized, double-blind, phase III; 224 pts with IC (7)

Cas 70/50 mg/day vs AmB 0.6–1.0 mg/kg for 14 days after last positive Candida culture

Favorable overall response (resolution of symptoms/signs + conﬁrmed eradication) at end of IV therapy = 73% for Cas vs 62% for AmB overall, and 72.5% vs 62.5% in pts with candidemia. Fewer drug-related AEs in Cas group (28.9% vs 58.4%, p < 0.001)

Open-label; 48 pts with nonblood IC (8)

Cas 50–100 mg/day as primary or salvage therapy

Favorable response (complete resolution of symptoms + eradication of Candida or radiographic resolution) = 81% overall

Open-label; 90 pts with refractory or intolerant IA (9)

Cas 50 mg/day (after 70 mg on day 1) for median of 28 days

Complete or partial response, as assessed by independent expert panel, in 45% of patients

Randomized, double-blind, phase III; empirical therapy in 1095 pts with febrile neutropenia (11)

Cas 50 mg/day (after 70 mg on day 1) vs L-AMB 3 mg/kg/day for 72 h after resolution of neutropenia (or ≥7 days after resolution of neutropenia + symptoms in pts with IFI)

Overall success rate = 33.9% for Cas vs 33.7% for L-AMB on 5-part composite endpoint (fever, baseline IFI, breakthrough IFI, survival, early discontinuation); Cas showed higher success rates for: baseline fungal infection (51.9% vs 25.9%, p = 0.04), survival ≥7 days after therapy (92.6% vs 89.2%, p = 0.05), and discontinuation (10.3% vs 14.5%, p = 0.03)

Randomized, double-blind, phase III; 523 pts with EC (13)

Mica 150 mg/day vs IV Flu 200 mg/day for 1 to 33 days

Endoscopic cure (grade 0 on 0–3 scale) = 88% in both groups; clinical cure (complete resolution of symptoms) = 92% in both groups; overall cure = 85.8% for Mica vs 85.3% for Flu

Randomized, double-blind, phase III; prophylaxis in 882 pts undergoing HSCT (14)

Mica 50 mg once/day vs Flu IV 400 mg once/day, during pre-engraftment neutropenia

Treatment success (absence of documented or suspected IFI through end of prophylaxis + absence of documented IFI through 4 wk posttreatment) in 80.0% vs 73.5% ( p = 0.03); breakthrough infection in 1.6% vs 2.4%; suspected infection in 15.1% vs 21.4%

Randomized, double-blind, phase III; 580 pts with IC (15)

Mica 100 and 150 mg/day vs Cas 50 mg/day (after 70 mg on day 1) for at least 10 days

Complete or partial clinical response + positive mycological response at end of IV therapy: 76.2% for Mica 100 mg, 74.0% for Mica 150 mg, and 73.5% for Cas

Open-label; salvage or primary therapy in 225 pts with IA (16)

Mica 75–325 mg/day alone or in combination with other antifungal agents, for 7 to 90 days

Complete or partial response at end of therapy in 80/225 (35.6%) overall, 15/34 (44.1%) on monotherapy, and 65/191 (34.0%) on combination therapy

Micafungin

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(Continued )

Study Design

Treatment

Results

Randomized, double-blind, phase III; 504 pts with EC (18)

Ani 50 mg/day (after 100 mg on day 1) vs Flu 100 mg/day (after 200 mg on day 1) for 14–21 days

Endoscopic cure (endoscopic grade of 0) or improvement (decrease of ≥1 grade from baseline) = 97.2% for Ani vs 98.8% for Flu; relapse rate = 36% for Ani vs 11% for Flu ( p < 0.0001)

Randomized, double-blind, phase III; 245 pts with IC (19)

Ani 100 mg/day (after 200 mg on day 1) vs Flu 400 mg/day (after 800 mg on day 1) for 14–42 days

Global response (clinical cure or improvement + microbiological success) 75.6% for Ani vs 60.2% for Flu at end of therapy; 64.6% vs 49.2% at 2-wk follow-up

Randomized, double-blind, phase III; 391 pts with EC (36)

Vori 200 mg b.i.d. vs Flu 200 mg q.d. for 14–42 days

Successful response (normal endoscopy at end of therapy or ≥1 grade improvement in endoscopic score) 98.2% for Vori vs 95.0% for Flu

Randomized, phase III; 370 pts with IC (37)

Vori 3 mg/kg IV q12 h for at least 3 days (after 6 mg/kg on day 1), then 200 mg b.i.d. p.o. vs AmB 0.7–1.0 mg/kg q.d. for 3–7 days, then Flu 400 mg q.d. for 2 wk after last positive blood culture

Successful outcome (mycological eradication + clinical cure or improvement) at 12 wk after end of treatment = 41% in both groups; successful outcome at end of therapy = 65.5% for Vori and 71.3% for AmB/Flu

Randomized, open-label, phase III; primary therapy in 277 pts with IA (38)

Vori 4 mg/kg b.i.d. (after 6 mg/kg b.i.d. on day 1) vs AmB 1– 1.5 mg/kg q.d.

Successful outcome in 53% of Vori group vs 32% of AmB group (<0.0001) and better survival at wk 12 (71% vs 58%)

Randomized, open-label; empirical therapy in 837 febrile neutropenic pts (41)

Vori 3 mg/kg b.i.d. for at least 3 days (after 6 mg/kg b.i.d. on day 1), then 200 mg p.o. b.i.d. vs L-AmB 3 mg/kg q.d.

Success in 5-part composite endpoint (26% vs 30.6%; 95% CI—10.6, 1.6%) Documented breakthrough fungal infections 1.9% vs 5%

Randomized, controlled phase III; 329 pts with EC (43)

Posa 200 mg q.d. for 1 day, then 100 mg q.d. for 13 days vs Flu 200 mg q.d. for 1 day, then 100 mg q.d. for 13 days

Complete or partial resolution of lesions and symptoms = 92% for Posa vs 93% for Flu; relapse rate at 4 wk after end of therapy = 29.0% for Posa vs 35.1% for Flu

Randomized, double-blind, phase III; prophylaxis in 600 pts with HSCT and GVHD (44)

Posa 200 mg t.i.d. vs Flu capsules 400 mg q.d.

Incidence of proven or probable IFI at day 112 = 5.3% for Posa vs 9.0% for Flu; proven/probable IA = 2.3% for Posa vs 7.0% for Flu, p = 0.006); lower mortality in Posa group (1% vs 4%, p = 0.046)

Randomized, open-label, phase III; prophylaxis in 602 pts with neutropenia (45)

Posa 200 mg t.i.d. vs Flu 400 mg q.d. or Itra 200 mg b.i.d.

Fewer patients in Posa group with proven/probable IFI (2% vs 8%, p < 0.001) or IA (1% vs 7%, p < 0.001) during treatment; longer survival in Posa group ( p = 0.04)

Open-label salvage therapy in 107 pts with IA vs external controls (46)

Posa 200 mg q.i.d. or 400 mg b.i.d. for up to 372 days (median 56 days)

Global response at end of treatment (assessed by data review committee) 42% for Posa vs 26% for controls ( p = 0.006)

Open-label compassionate use in 91 pts with zygomycosis (47)

Posa 200 mg q.i.d. or 400 mg b.i.d. for at least 30 days

Complete or partial response in 55 (60%) of patients 12 wk after start of therapy

Anidulafungin

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Voriconazole

Posaconazole

Abbreviations: AEs, adverse events; AmB, amphotericin B; EC, esophageal candidiasis; GVHD, graft-versus-host disease; HSCT, hematopoietic stem cell transplant; IA, invasive aspergillosis; IC, invasive candidiasis; IFI, invasive fungal infection; L-AmB, liposomal amphotericin B; pts, patients.

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forms of IC (endocarditis and osteomyelitis) in small numbers of patients (8). Caspofungin has been shown to be effective as salvage therapy1 for IA (9, 10), with favorable response rates of ∼45%. Empirical therapy with caspofungin was comparable to liposomal amphotericin B in a randomized double-blind study of 1095 patients with persistent fever and neutropenia (11). The caspofungin group had lower rates of nephrotoxicity, infusion-related events, and drug-related adverse events. Micafungin (MYCAMINE®) was approved in the United States in 2005 for the treatment of EC and for the prevention of Candida infections in patients undergoing HSCT (12) (Table 2). In a randomized double-blind phase III study of patients with EC, the endoscopic cure rate was comparable for micafungin 150 mg/day and ﬂuconazole 200 mg/day (13). Micafungin was also been shown to be effective as prophylaxis during the pre-engraftment period of neutropenia when compared with ﬂuconazole (80% versus 73.5%) in 882 patients undergoing HSCT (14). In a randomized trial involving 580 patients with IC (75% with candidemia), micafungin 100 and 150 mg/day was noninferior to caspofungin (15). This was the ﬁrst clinical study to compare two echinocandins. The efﬁcacy of micafungin alone or in combination with other antifungal agents was evaluated in an open-label noncomparative study of 225 patients with IA (16). Most patients in this study received combination therapy; however, a favorable response was seen in 15 of 34 patients (44%) who received micafungin alone. Micafungin is not yet approved for the treatment of IC or IA.

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Salvage therapy was deﬁned as therapy for patients whose IA was either refractory to or intolerant of standard antifungal therapy. Refractory cases were deﬁned by progression or failure to improve after at least 7 days of therapy. Intolerance was deﬁned by renal insufﬁciency or signiﬁcant infusion-related adverse events. Standard antifungal therapy was an amphotericin B formulation, itraconazole, or voriconazole.

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Anidulafungin (ERAXIS®) was approved in the United States in 2006 for the treatment of EC, candidemia, and other Candida infections (intra-abdominal abscess and peritonitis) (17) (Table 2). Anidulafungin was compared with ﬂuconazole in a randomized double-blind phase III study of 504 patients with EC (18). The endoscopic success rate at the end of therapy was similar for anidulafungin and ﬂuconazole (97.2% versus 98.8%), but the relapse rate at the two-week followup was higher for anidulafungin (36% versus 11%, <0.001). In a randomized doubleblind phase III study of 261 patients with IC (89% with candidemia) (19), anidulafungin was superior to ﬂuconazole at the primary endpoint, the end of IV therapy (75.6% versus 60.2%).

Echinocandin in vitro susceptibility testing and resistance. Decreased susceptibility to echinocandins has been evaluated most extensively for caspofungin (20–22). Amino acid substitutions in two regions of FKS1p have been identiﬁed in resistant isolates, including the region between amino acids 641 and 648 and at 1357 or 1361. These mutations are dominant, and both heterozygote and homozygote fungi demonstrate decreased susceptibility. As mentioned above, rare clinical isolates with these resistance mutations have been identiﬁed (21, 22), and cross-resistance among echinocandins has been seen. Efﬂux pumps do not appear to confer resistance to echinocandins (22, 23). Although there are no approved testing methods or interpretive breakpoints for echinocandins, the best way to distinguish caspofungin-resistant mutants from susceptible isolates is to use RPMI-1640 medium with the MIC endpoint read as partial inhibition of growth at 24 h. In contrast to susceptible isolates, which have MICs ≤2 μg/ml, mutants with resistance to caspofungin typically have MICs ≥4 μg/ml. Echinocandins retain activity against isolates that are resistant to azoles and amphotericin B.

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Safety profiles. Overall, the echinocandins are very well tolerated, and a favorable safety proﬁle is one of the key attributes of this class. Similar types of adverse events and laboratory abnormalities are seen across the class. The most common adverse events reported are phlebitis, rash, ﬂushing, fever, chills, headache, nausea, vomiting, diarrhea, and abdominal pain; the most common laboratory abnormalities include elevated transaminases, alkaline phosphatase, and bilirubin. Adverse events tend to be mild and not related to dose (2–5, 12, 17). Use in children. All of the echinocandins are being studied in children; none is currently approved for pediatric use. The pharmacokinetics of caspofungin were studied in children and adolescents (2–17 years old) with neutropenia (24). Dosing based on body surface area (50 mg/m2 /day) resulted in plasma levels comparable to those in adults receiving 50 mg/day. Preliminary results of a prospective open-label study of caspofungin in 28 children with EC, IC, or IA (25) showed response rates similar to those seen in adults. Caspofungin was generally well tolerated in this population. The pharmacokinetics of micafungin were studied in 77 children with febrile neutropenia (26) and in 18 premature infants weighing >1000 g (27). In both studies, micafungin was well tolerated. In a randomized double-blind study, micafungin 2 mg/kg/day was compared with liposomal amphotericin B (L-AmB) in 98 pediatric patients with IC (28); favorable responses were observed in 73% (35/48) of the micafungin patients and in 76% (38/50) of the L-AmB patients. In a noncomparative study of 58 pediatric patients with IA, micafungin alone or in combination with another antifungal produced a favorable response in 26 patients (44%) (29). The pharmacokinetics of anidulafungin were studied in 25 children and adolescents (2–17 years old) with neutropenia at high risk for IFI (30). In this population, anidulafungin dosages of 0.75 and 1.5 mg/kg/day were generally well toler-

ated, and concentration proﬁles were similar to those in adult patients receiving 50 or 100 mg/day, respectively.

Second-Generation Triazoles The second-generation triazoles, i.e., voriconazole and posaconazole, block the synthesis of ergosterol through the inhibition of CYP450-dependent lanosterol 14αdemethylase, leading to destruction of the fungal cell membrane and the accumulation of toxic sterol precursors. Both have excellent in vitro activity against most Candida spp., including some ﬂuconazole-resistant isolates, and are considered fungicidal against Aspergillus spp. They also have in vitro activity against other yeasts (e.g., Cryptococcus neoformans and Trichosporon spp.), ﬁlamentous fungi (e.g., Pseudallescheria boydii, Scedosporium apiospermium, and Penicillium spp.), and dimorphic fungi (e.g., Blastomyces dermatitidis, Coccidioides immitus, Histoplasma capsulatum, and Sporothrix schenckii) (31–34). Voriconazole has good in vitro activity against Fusarium spp. (31, 32), and posaconazole is the only agent to show consistent in vitro activity against zygomycetes (33, 34). Voriconazole, available in both IV and oral formulations, exhibits nonlinear pharmacokinetics and is rapidly and completely absorbed. Systemic absorption of oral voriconazole is reduced by ∼22% in the fed versus fasted state but is not affected by agents that increase gastric pH. Voriconazole is extensively distributed into tissues and is ∼58% protein bound (31, 32). Posaconazole is available only as an oral suspension and displays doseproportional pharmacokinetics up to a dose of 800 mg/day. Exposure is increased by administration with food, and bioavailability is signiﬁcantly increased when posaconazole is given as divided daily doses. Posaconazole is >95% protein bound (33, 34). Metabolism and drug interactions for these agents are summarized in Table 1.

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Voriconazole (VFEND®) was approved in the United States and European Union in 2002 for the treatment of IA and refractory cases of scedosporiosis and fusariosis, and subsequently for the treatment of EC, candidemia in non-neutropenic patients, and disseminated Candida infections (35) (Table 2). In a randomized double-blind phase III study (36), oral voriconazole was as effective as oral ﬂuconazole in 391 patients with EC (98% versus 95%). In a randomized open-label phase III trial in 370 non-neutropenic patients with IC (37), IV voriconazole followed by oral voriconazole was as effective as IV amphotericin B followed by ﬂuconazole at the end of antifungal therapy (70% versus 74%) as well as 12 weeks later (41% response in both groups). In a randomized open-label phase III study of 277 patients with IA, voriconazole was superior to amphotericin B as primary therapy, with a successful outcome in 53% versus 32% of patients ( p < 0.0001) and better survival at week 12 (71% versus 58%) (38). Retrospective studies suggest that voriconazole may be useful in CNS aspergillosis (39) and bone aspergillosis (40). Efﬁcacy in fusariosis (9/21; 43%) and scedosporiosis (15/24; 63%) is based on small numbers of patients (35). Voriconazole was also compared to L-AmB as empirical therapy in 837 febrile neutropenic patients but failed to demonstrate noninferiority to L-AmB for the primary ﬁve-part composite endpoint (26.0% versus 30.6%) and is not indicated for this use. In this study, voriconazole was associated with fewer breakthrough IFIs ( p = 0.02) (41). Posaconazole (NOXAFIL®) was approved in 2006 in the United States and European Union for the treatment of oropharyngeal candidiasis and for the prevention of invasive Aspergillus and Candida infections in high-risk patients (42) (Table 2). Efﬁcacy of posaconazole was comparable to that of ﬂuconazole (92% versus 93%) in a randomized study of 350 HIV-infected patients with oropharyngeal candidiasis (43). Two phase III studies of posaconazole as antifungal prophylaxis have been reported. In a randomized double-blind

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study of 600 patients with graft-versus-host disease, posaconazole was as effective as ﬂuconazole in preventing all IFIs (5.3% versus 9.0%) and was superior to ﬂuconazole in preventing IA (2.3% versus 7.0%; p = 0.006) (44). In the second study, 602 patients with neutropenia due to chemotherapy received posaconazole or either ﬂuconazole or itraconazole as prophylaxis (45). Seven patients (2%) in the posaconazole group experienced IFI during the treatment period versus 25 (8%) in the ﬂuconazole/itraconazole group ( p < 0.001). Walsh and colleagues studied oral posaconazole as salvage therapy in 107 patients with IA, using external control cases as a reference group (46). The overall success rate was 42% for posaconazole versus 26% for controls ( p = 0.006). In a compassionateuse study of posaconazole (47), a favorable response was observed in 60% of 91 patients with zygomycosis refractory to or intolerant of prior antifungal therapy. Posaconazole is not currently approved for these indications. Azole in vitro susceptibility testing and resistance. In vitro susceptibility testing for azoles should adhere to the standard approved by the Clinical and Laboratory Standards Institute (CLSI) for testing yeasts, M27A2. CLSI, formerly the National Committee on Clinical Laboratory Standards (NCCLS), has deﬁned the following breakpoints for voriconazole: ≤1 μg/ml as susceptible, 2μg/ml as susceptible dose-dependent, and ≥4 μg/ml as resistant. There are currently no approved breakpoints for posaconazole. Resistance to azoles has been investigated most extensively for ﬂuconazole and has been reported to occur by three mechanisms: (a) efﬂux pumps, (b) alteration in target enzyme, lanesterol 14 α demethylase, a product of the ERG11 gene, or upregulation of ERG11, and (c) change in the ergosterol biosynthetic pathway (primarily ERG3), which prevents accumulation of toxic metabolites in the presence of drug (48–50). Efﬂux is mediated by ATP binding cassette (ABC) transporters CDR1

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and CDR2, and by MDR1, a major facilitator superfamily transporter. The ABC transporters affect most azoles, but MDR1 is speciﬁc for ﬂuconazole. Similar mechanisms of resistance due to upregulation of ABC transporters have been identiﬁed for C. glabrata, C. dubliensis, and C. tropicalis (49, 51). Crossresistance among azoles is common (52). Posaconazole and voriconazole have better activity than ﬂuconazole against C. krusei, but decreased susceptibility of C. glabrata is still a problem (49). Safety profiles. The most common adverse events associated with voriconazole are visual disturbances, fever, chills, headache, tachycardia, nausea, vomiting, abnormal liver function tests, hallucinations, and rash. In clinical trials, 21% of patients reported abnormal vision, color vision change, and/or photophobia; 12% had clinically signiﬁcant transaminase abnormalities, and 7% had treatmentrelated rash (35). The most common adverse events reported with posaconazole are fever, headache, anemia, diarrhea, nausea, vomiting, abdominal pain, hypokalemia, headache, and thrombocytopenia (42). Use in children. Voriconazole is approved in the European Union for use in children at least two years of age. On the basis of milligrams per kilogram body weight, children require higher doses of voriconazole than adults (53). In a compassionate-use study of voriconazole in 58 children with IFI refractory to or intolerant of conventional antifungal therapy, a favorable response was achieved in 18 (43%) of 42 patients with IA, 2 (50%) of 4 with IC, and 5 (63%) of 8 with scedosporiosis (54). Posaconazole plasma levels were studied in 12 children (aged 8–17 years) and 194 adults (aged 18–64 years) who participated in an open-label phase III study of posaconazole 800 mg/day as salvage therapy for proven or probable IFI; posaconazole plasma levels were similar in juvenile and adult patients, and the overall success rates and adverse event proﬁles were comparable between the age groups (55).

Antifungal Therapies in Development Despite considerable progress in the past decade, the morbidity and mortality of IFI are still unacceptably high. There is a need for agents with new mechanisms of action that have a broad spectrum of activity (including resistant pathogens) and can be administered both intravenously and orally. Agents with these characteristics plus a favorable safety proﬁle and few drug interactions would be attractive to evaluate as components of combination therapy regimens for infections that are difﬁcult to treat. There are a number of therapies in development. Nikkomycin Z is a peptide nucleoside inhibitor of chitin synthase in the cell wall of fungi. It has activity against Candida, Histoplasma capsulatum, Blastomycosis dermatidis, and Coccidioides immitis but limited activity against Aspergillus (56). Sodarins block elongation factor 2, an essential part of protein synthesis in fungi but not in humans. Most of the sodarin derivatives evaluated have had a narrow spectrum of activity that did not include all Candida species. Neither Nikkomycin Z nor the sodarins appears to be progressing in development. Several groups of peptides have shown activity against bacteria and fungi, including defensins, protegrins, lactoferrin-derived peptides, and histatins. These peptides are often cationic and amphipathic, characteristics that allow interaction with bacterial membranes and insertion into lipid bilayers, which results in leakage from the cell and eventual death. Some peptides may also have intracellular targets and affect host cellular processes of inﬂammation or innate immunity. Concerns about peptides have included toxicity and the potential for development of resistance (57). Some of these agents have been evaluated in early clinical trials, but no efﬁcacy data are available. Immunotherapy to improve the host response has been evaluated extensively by studies of granulocyte infusions, administration of cytokine growth factors (GCSF, GMCSF), www.annualreviews.org • Advances in Antifungal Therapy

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and administration of proinﬂammatory cytokines (IL-12 and IFNγ). However, all of these studies have limitations, and at this point data regarding the utility of immunotherapy in the treatment of IFI are inconclusive (58). Administration of cytokine growth factors may help patients with protracted neutropenia and IFI by shortening the duration of neutropenia, but no study has demonstrated a reduction in infection-related mortality, and routine use is not recommended (59, 60). Mycograb is a speciﬁc antibody to hsp90, a molecular chaperone for a variety of cellular proteins. It consists of the antigen binding variable domains of the heavy and light chains linked together to create a recombinant protein. Hsp90 is immunodominant and has been identiﬁed in Candida and Aspergillus fumigatus as well as in other fungi (61). Mycograb has been evaluated in a double-blind randomized study of 139 patients with IC (62). Patients were randomized to either Abelcet or Ambisome, alone or in combination with mycograb 1 mg/kg b.i.d. for 5 days. A complete response at day 10 (primary endpoint) was seen in 48% of patients who received amphotericin B alone versus 84% of those who received amphotericin B plus mycograb ( p < 0.001). In addition, patients treated with amphotericin B plus mycograb had lower Candidaattributable mortality than those treated with amphotericin B alone at day 33 (18% versus 4%). Although the results are promising, the study is not deﬁnitive, and questions have been raised about its methodology (63). Based on the results of this trial, mycograb was ﬁled for regulatory approval in the European Union in 2006 and is under regulatory review. Although some of the preliminary data are encouraging, it is uncertain whether any of these approaches will have a signiﬁcant impact on the morbidity and mortality of IFI.

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GM EIA: galactomannan enzyme immunoassay

NEW TREATMENT PARADIGMS Approaches to Early Therapy Because morbidity and mortality are so high once an IFI is established, a number of strate370

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gies for early intervention have been evaluated. It is standard to initiate antifungal prophylaxis in patients who are undergoing chemotherapy that will result in neutropenia and in those undergoing allogeneic HSCT. This prophylactic approach has signiﬁcantly reduced the incidence of Candida infections (64). Empirical antifungal therapy is recommended for neutropenic patients when they have persistent fever despite antibacterial therapy (59, 65), but multiple studies have demonstrated that only ∼5% of these patients have fungal infections. There has been interest in more precisely deﬁning the subset of patients most likely to have IFIs and reserving antifungal therapy for these patients. This approach has been termed pre-emptive therapy. There is no consensus on the deﬁnition of pre-emptive therapy, nor compelling evidence that this approach provides better outcomes than empirical therapy. Pre-emptive therapy. Maertens et al. (66) evaluated a combination of galactomannan enzyme immunoassay (GM EIA) plus highresolution computed tomography (CT) scanning as a trigger for pre-emptive therapy in 88 patients receiving ﬂuconazole prophylaxis and undergoing chemotherapy for acute leukemia or myelodysplastic syndrome, or myeloablative allogeneic HSCT. Pre-emptive therapy was initiated in 9 episodes; 41 qualiﬁed for empirical therapy. Ten other patients received pre-emptive therapy because of positive test results while they were afebrile (and would not have received empirical therapy). This approach reduced the administration of antifungal therapy to 7.7% of patients from 35% if empirical therapy had been used. However, GM EIA is speciﬁc to Aspergillus; infections due to Candida spp. or other molds would not be detected. One prospective open-label randomized comparative trial has evaluated empirical versus pre-emptive therapy. This study enrolled 293 adults with hematologic malignancies receiving chemotherapy or autologous HSCT (67). Patients randomized to empirical

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therapy received a polyene antifungal for persistent neutropenic fever, whereas those randomized to pre-emptive therapy received polyene therapy only if they developed clinical evidence of fungal infection, Aspergillus colonization, or positive GM EIA. Patients in the pre-emptive therapy arm received signiﬁcantly less antifungal therapy (46% versus 66%; p < 0.001) but experienced more IFIs ( p < 0.02). Between the two groups, there was no difference in survival [–2.9% (95% CI –6.4% to 0.6%)] or mortality related to IFI ( p = 0.12). Economic analysis showed no difference between empirical and pre-emptive therapy for total medication costs or antifungal therapy costs (68). Although reducing unnecessary antifungal therapy is an important goal, there are currently insufﬁcient data to justify an approach of pre-emptive therapy.

Combination Therapy The introduction of new antifungal agents with different mechanisms of action has heightened interest in combination therapy for IFI. Potential advantages of combination therapy include a broader spectrum of activity, synergy, and prevention of resistance. Many antifungal combinations have been studied in vitro and in animal models against various Candida spp. (69, 70), but these results do not always predict clinical outcomes. In a randomized double-blind study of ﬂuconazole plus amphotericin B versus ﬂuconazole alone in patients with candidemia, combination therapy was associated with a higher overall success rate (69% versus 56%; p = 0.043) and fewer patients remaining candidemic (6% versus 17%; p = 0.02). However, success rates as measured by Kaplan-Meier time-to-failure analysis were not signiﬁcantly different between the groups ( p = 0.08) (71). Because effective monotherapy is available for candidiasis, recent interest in combination therapy has focused on Aspergillus infections. Studies of antifungal combinations in vitro against Aspergillus spp. and in animal mod-

els of aspergillosis have produced variable results, depending on study methodology and strains evaluated (69, 70). Studies of combination therapy with an echinocandin and amphotericin B (72, 73) or an echinocandin and an azole (74) in patients with IA suggest a potential beneﬁt associated with combination therapy. The ﬁrst prospective open-label noncomparative study of combination therapy with an echinocandin was recently reported (75). In this study, 53 patients received caspofungin in combination with voriconazole, itraconazole, or amphotericin B as salvage therapy for IA. A favorable response was observed in 29 (55%) of 53 patients overall: 8 (50%) of 16 who received caspofungin plus a polyene and 21 (57%) of 37 who received caspofungin plus a triazole. (The study was not designed to test equivalency.) Although using combinations of agents with different mechanisms of action is appealing, the available data are difﬁcult to interpret. Unanswered questions include which combination would be optimal, what endpoint is appropriate, how much beneﬁt would have to be seen to justify the adoption of combination therapy, and which populations are likely to beneﬁt. A well-designed prospective randomized comparative trial of monotherapy versus combination therapy is needed to determine if the potential beneﬁt of any combination regimen outweighs the increased cost and potential increased toxicity of this approach.

DIAGNOSTIC METHODS The gold standard for diagnosis of IFI remains culture and histopathology, but these methods lack sensitivity and speciﬁcity and require invasive procedures that are often impossible in patients at highest risk for IFI. The introduction of high-resolution CT scans has improved detection of pulmonary abnormalities, but the most common abnormalities are nonspeciﬁc, and even ﬁndings considered characteristic of IFI may be seen with other diseases. A major focus in the past decade has been the development of noninvasive tests to www.annualreviews.org • Advances in Antifungal Therapy

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provide a rapid and reliable diagnosis of IFI. The most promising diagnostic tests are detection of fungal antigen [galactomannan and (1,3)-β-D glucan] or DNA (PCR).

EORTC/MSG: European Organization for Research and Treatment of Cancer/Mycosis Studies Group

Galactomannan GM is a polysaccharide component of the Aspergillus cell wall that is released into serum during hyphal growth. The GM EIA (Platelia Aspergillus EIA, Bio-Rad Laboratories, Inc., Redmond, WA) was originally approved in the European Union, with a positive test deﬁned as an optical density index (OD) of at least 1.5. There has been considerable debate about the appropriate cutoff, and the GM EIA was approved by the U.S. Food and Drug Administration (FDA) in 2003 with a threshold OD index of 0.5. A positive test is deﬁned as an OD ≥ 0.5 on two consecutive samples to minimize the impact of false positives. The data submitted to the FDA included 1890 blood samples from 170 patients, including 31 with IA. In this study, the GM EIA demonstrated a sensitivity of 80.7% and speciﬁcity of 89.2%. In other studies, sensitivity and speciﬁcity have varied (<50%–90% and 85%–99%, respectively) and have depended on the population studied, frequency of monitoring, and the cutoff used (76–80). The cutoff OD index of 0.5 has now been adopted worldwide (81) and has been reported to result in a diagnosis of IA up to 9 days earlier than radiographic abnormalities (78, 79, 82). The GM EIA is one of the diagnostic criteria for probable IA in the EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycosis Studies Group) deﬁnitions of IFI (83). The test is approved only for use on serum (84), but has been used on cerebrospinal ﬂuid, urine, and bronchoalveolar lavage (BAL) ﬂuid. Data on the utility of GM EIA in these other ﬂuids are limited. The GM EIA has been used for surveillance in patients at high risk and as a diagnostic adjunct in high-risk patients with ﬁndings

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compatible with IA. Pfeiffer et al. (85) conducted a meta-analysis of studies from 1966 to 2005 that used GM EIA for surveillance in high-risk patients. In the 27 studies identiﬁed, the test had a sensitivity of 71% and speciﬁcity of 89% for proven IA. The performance differed by patient population (better for hematological malignancies and HSCT) and the reference standard used. The GM EIA has also been used to follow the course of IA during treatment, but its utility in this setting is still debated. Most of the data on GM EIA have been obtained in patients with hematological malignancies and those who have undergone HSCT. There have been only two small studies in patients with solid-organ transplants, where results have been complicated by false positives and a low percentage of documented cases of IA (86, 87). Caution should be exercised in interpreting results in other patient populations until additional data are available. The GM EIA may be inﬂuenced by a number of factors (81, 82, 88, 89). Antifungal therapy may signiﬁcantly reduce the sensitivity of the assay. False positive results have been reported with use of beta-lactam antibiotics (e.g., piperacillin-tazobactam, ampicillin, amoxicillin, and amoxicillin-clavulanate), the presence of other fungi, cross-reactivity with lipoglycan of biﬁdobacteria in the gastrointestinal tract, or galactomannan translocation from foods in patients with disrupted gastrointestinal epithelium. False positive results occur more frequently in infants and children and in recipients of allogeneic HSCT (90).

(1, 3)-β-D-Glucan (1, 3)-β-D-glucan is an essential component of the cell wall of many pathogenic fungi, including Candida and Aspergillus spp. Fungitell (Glucatell, Associates of Cape Cod, Falmouth, MA) uses a modiﬁed limulus amebocyte lysate assay to detect only the factor G part of the pathway. It does not detect

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zygomycetes or Cryptococcus. The manufacturer indicates that the assay has a sensitivity of ∼80% for IA and IC (91). In other studies, sensitivity and speciﬁcity have varied depending on the population studied and the certainty of diagnosis of the IFI (64%– 100% and 92%–100%, respectively, when a positive cutoff of 80 pg/ml is used) (92, 93). The assay appears most useful in patients with acute myelogenous leukemia and myelodysplastic syndrome and is often positive before clinical or microbiological diagnosis of infection. There is little information in other populations. A single positive test is considered indicative of infection (93). Detection of βglucan is now included in the EORTC/MSG criteria for diagnosis of probable IFI (83). False positive results are common. They have been reported in patients undergoing hemodialysis with cellulose membranes or ﬁlters (94), after administration of speciﬁc immunoglobulin products, with cotton gauze and sponges used in surgery, and with some drugs (92). Hemolyzed and lipemic samples may interfere with the assay and should not be tested. The occurrence of false positives in a signiﬁcant percentage of patients (75%) who have gram-positive bacteremia (95) severely limits the utility of the test. The effect of antifungal therapy on assay performance has not been evaluated.

Polymerase Chain Reaction (PCR) PCR has been evaluated extensively as a diagnostic tool for fungal infections. Panfungal PCR as well as fungus-speciﬁc PCR have been evaluated. Although PCR testing appears promising, there is no standardized method for it, in contrast to galactomannan and β-glucan testing. The sensitivity of PCR ranges from 45% to >90% and its speciﬁcity is >90% (96–99). Introduction of realtime PCR has signiﬁcantly reduced the risk of false positives, and results are available within a few hours (98, 99). However, results are highly dependent on the specimen (whole

blood, serum, or BAL), primer(s), DNA extraction method, population, and type of disease. The risk of cross-contamination is high, and testing requires a specialized laboratory and trained personnel. An additional concern is that PCR testing of BAL cannot differentiate colonization from infection. Comparison to GM EIA has produced varied results (96, 98). Some authors suggest use of both PCR and GM EIA (96, 97), but there are not sufﬁcient data to support this approach. PCR is considered an investigational method in the EORTC/MSG diagnostic criteria for IFI (83). PCR cannot be recommended routinely for diagnosis of IFI until there is a standardized testing methodology that can be evaluated in prospective studies.

SUMMARY There have been signiﬁcant advances in antifungal therapy in the past decade. Several new antifungal agents have received regulatory approval in the United States and European Union. Caspofungin was the ﬁrst echinocandin, introduced in 2001 as salvage therapy for patients with IA. The echinocandins have demonstrated excellent efﬁcacy against Candida with a favorable safety proﬁle and few drug interactions. They are, however, limited by lack of oral formulations. Voriconazole was the ﬁrst antifungal to demonstrate a decrease in mortality in a comparative trial versus amphotericin B for the ﬁrst-line treatment of IA. However, it is associated with signiﬁcant drug interactions. There have also been signiﬁcant improvements in diagnostics with the introduction of the Aspergillus GM EIA and the β-glucan test. Both tests have been incorporated into the EORTC/MSG criteria for diagnosis of IFI but have limitations. Strategies for management of serious IFI continue to be evaluated. Several studies have investigated alternative approaches to treatment, including preemptive therapy and combination therapy, but no deﬁnitive answer is yet available. www.annualreviews.org • Advances in Antifungal Therapy

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FUTURE ISSUES 1. Role of combination therapy. 2. Deﬁnition of pre-emptive therapy and role in management. 3. Role of immunotherapy. 4. Identiﬁcation of new agents with unique mechanisms of action and activity against resistant pathogens. 5. Role of PCR and other new, rapid diagnostic tests.

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DISCLOSURE STATEMENT The authors are current or former employees of Merck & Co., Inc., the manufacturer of CANCIDAS, and may own stock and/or stock options in the company.

LITERATURE CITED 1. Marr K, Carter R, Crippa F, et al. 2002. Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients. Clin. Infect. Dis. 34:909–17 2. Cappelletty D, Eiselstein-McKitrick K. 2007. The echinocandins. Pharmacotherapy 27:369–88 3. Wagner C, Graninger W, Presterl E, et al. 2006. The echinocandins: comparison of their pharmacokinetics, pharmacodynamics and clinical applications. Pharmacology 78:161–77 4. Morrisson VA. 2006. Echinocandin antifungals: review and update. Expert Rev. Anti-Infect. Ther. 4:325–42 5. CANCIDAS® (caspofungin acetate) for injection. Prescribing Information, Feb. 2005. Merck & Co., Inc., Whitehouse Station, NJ. Available at http://www.merck. com/product/usa/pi circulars/c/cancidas/cancidas pi.pdf 6. Villaneuva A, Gotuzzo E, Arathoon EG, et al. 2002. A randomized double-blind study of caspofungin versus ﬂuconazole for the treatment of esophageal candidiasis. Am. J. Med. 113:294–99 7. Mora-Duarte J, Betts R, Rotstein C, et al. 2002. Comparison of caspofungin and amphotericin B for invasive candidiasis. N. Engl. J. Med. 347:2020–29 8. Cornely O, Lasso M, Betts R, et al. 2007. Caspofungin for the treatment of less common forms of invasive candidiasis. J. Antimicrob. Chemother. 60:363–69 9. Maertens J, Raad I, Petrikkos G, et al. 2004. Efﬁcacy and safety of caspofungin for treatment of invasive aspergillosis in patients refractory to or intolerant of conventional antifungal therapy. Clin. Infect. Dis. 39:1563–71 10. Kartsonis NA, Saah AJ, Lipka CJ, et al. 2005. Salvage therapy with caspofungin for invasive aspergillosis: results from the caspofungin compassionate use study. J. Infect. 50:196–205 11. Walsh TJ, Teppler H, Donowitz GR, et al. 2004. Caspofungin versus liposomal amphotericin B for empirical antifngal therapy in patients with persistent fever and neutropenia. N. Engl. J. Med. 351:1391–402 12. MYCAMINE® (micafungin sodium) for injection. Prescribing Information, June 2006. Astellas Pharma US, Inc., Deerﬁeld, IL. Available at http://www.astellas.us/ docs/mycamine.pdf 374

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13. de Wet N, Bester AJ, Viljoen JJ, et al. 2005. A randomized, double blind, comparative trial of micafungin (FK463) vs ﬂuconazole for the treatment of oesophageal candidiasis. Aliment. Pharmacol. Ther. 21:899–907 14. van Burik JA, Ratanatharathorn V, Stepan DE, et al. 2004. Micafungin versus ﬂuconazole for prophylaxis against invasive fungal infections during neutropenia in patients undergoing hematopoietic stem cell transplantation. Clin. Infect. Dis. 39:1407–16 15. Pappas PG, Rotstein CM, Betts RF, et al. 2007. Micafungin versus caspofungin for treatment of candidemia and other forms of invasive candidiasis. Clin. Infect. Dis. 45:883–93 16. Denning DW, Marr KA, Lau WM, et al. 2006. Micafungin (FK463), alone or in combination with other systemic antifungal agents, for the treatment of acute invasive aspergillosis. J. Infect. 53:337–49 17. ERAXISTM (anidulafungin) for injection. Prescribing Information, Mar. 2006. Pﬁzer Inc., New York, NY. Available at http://media.pfizer.com/files/products/uspi eraxis.pdf 18. Krause DS, Simjee AE, van Rensburg C, et al. 2004. A randomized, double-blind trial of anidulafungin versus ﬂuconazole for the treatment of esophageal candidiasis. Clin. Infect. Dis. 39:770–75 19. Reboli AC, Rotstein C, Pappas PG, et al. 2007. Anidulafungin versus ﬂuconazole for invasive candidiasis. N. Engl. J. Med. 356:2472–82 20. Katiyar S, Pfaller M, Edlind T. 2006. Candida albicans and Candida glabrata clinical isolates exhibiting reduced echinocandin susceptibility. Antimicrob. Agents Chemother. 50(8):2892– 94 21. Balashov S, Park S, Perlin D. 2006. Assessing resistance to the echinocandin antifungal drug caspofungin in Candida albicans by proﬁling mutations in FKS1. Antimicrob. Agents Chemother. 50(6):2058–63 22. Park S, Kely R, Kahn N, et al. 2005. Speciﬁc substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates. Antimicrob. Agents Chemother. 49(8):3264–73 23. Niimi K, Maki K, Ikeda F, et al. 2006. Overexpression of Candida albicans CDR1, CDR2 or MDR1 does not produce signiﬁgant changes in echinocandin susceptibility. Antimicrob. Agents Chemother. 50(4):1148–55 24. Walsh TJ, Adamson PC, Seibel NL, et al. 2005. Pharmacokinetics, safety, and tolerability of caspofungin in children and adolescents. Antimicrob. Agents Chemother. 49:4536–45 25. Jafri H, Zaoutis T, Keller N, et al. 2006. Prospective, multicenter study of caspofungin (CAS) for treatment (Rx) of documented fungal infections in pediatric patients (ped pts). Program Abstr. 46th Intersci. Conf. Antimicrob. Agents and Chemother., Abstr. G-873a. Washington, DC: Am. Soc. Microbiol. 26. Seibel NL, Schwart C, Arrieta A, et al. 2005. Safety, tolerability, and pharmacokinetics of micafungin (FK463) in febrile neutropenic pediatric patients. Antimicrob. Agents Chemother. 49:3317–24 27. Heresi GP, Gerstmann DR, Reed MD, et al. 2006. The pharmacokinetics and safety of micafungin, a novel echinocandin, in premature infants. Pediatr. Infect. Dis. J. 25:1110–15 28. Arrieta AC, Telles Filho F, Berezin E, et al. 2006. A randomized, double-blind trial comparing micafungin (MCFG) and liposomal amphotericin B (L-AMB) in pediatric patients with invasive candidiasis (IC). Program Abstr. 46th Intersci. Conf. Antimicrob. Agents and Chemother., Abstr. M-1308b. Washington, DC: Am. Soc. Microbiol. 29. Flynn PM, Seibel N, Arrieta A, et al. 2006. Treatment of invasive aspergillosis (IA) in pediatric patients (pts) with micafungin (MICA) alone or in combination with other systemic antifungal agents. Program Abstr. 46th Intersci. Conf. Antimicrob. Agents and Chemother., Abstract M-891. Washington, DC: Am. Soc. Microbiol. www.annualreviews.org • Advances in Antifungal Therapy

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30. Benjamin DK, Driscoll T, Seibel NL, et al. 2006. Safety and pharmacokinetics of intravenous anidulafungin in children with neutropenia at high risk for invasive fungal infections. Antimicrob. Agents Chemother. 50:632–38 31. Scott LJ, Simpson D. 2007. Voriconazole: a review of its use in the management of invasive fungal infections. Drugs 67:269–98 32. Koﬂa G, Ruhnke M. 2005. Voriconazole: review of a broad spectrum triazole antifungal agent. Expert Opin. Pharmacother. 6:1215–29 33. Groll AH, Walsh TJ. 2005. Posaconazole: clinical pharmacology and potential for management of fungal infections. Expert Rev. Anti Infect. Ther. 3:467–87 34. Keating GM. 2005. Posaconazole. Drugs 65:1553–67 35. VFEND® (voriconazole) I.V., tablets, and oral suspension. Prescribing Information, Mar. 2006. Pﬁzer Inc., New York, NY. Available at http://media.pfizer.com/files/ products/uspi vfend.pdf 36. Ally R, Schurmann D, Kreisel W, et al. 2001. A randomized, double-blind, double-dummy, multicenter trial of voriconazole and ﬂuconazole in the treatment of esophageal candidiasis in immunocompromised patients. Clin. Infect. Dis. 33:1447–54 37. Kullberg BJ, Sobel JD, Ruhnke M, et al. 2005. Voriconazole versus a regimen of amphotericin B followed by ﬂuconazole for candidaemia in non-neutropenic patients: a randomised noninferiority trial. Lancet 366:1435–42 38. Herbrecht R, Denning DW, Patterson TF, et al. 2002. Voriconazole versus amphotericin B for primary therapy in invasive aspergillosis. N. Engl. J. Med. 347:408–15 39. Schwartz S, Ruhnke M, Ribaud P, et al. 2005. Improved outcome in central nervous system aspergillosis, using voriconazole treatment. Blood 106:2641–45 40. Mouas H, Lutsar I, Dupont B, et al. 2005. Voriconazole for invasive bone aspergillosis: a worldwide experience of 20 cases. Clin. Infect. Dis. 40:1141–47 41. Walsh TJ, Papas P, Winston DJ, et al. 2002. Voriconazole compared with liposomal amphotericin B for empirical antifungal therapy in patients with neutropenia and persistent fever. N. Engl. J. Med. 346:225–34 42. NOXAFIL® (posaconazole) oral suspension. Prescribing Information, Oct. 2006. Schering Corp., Kenilworth, NJ. Available at http://www.spfiles.com/pinoxafil.pdf 43. Vazquez JA, Skiest DJ, Nieto L, et al. 2006. A multicenter randomized trial evaluating posaconazole versus ﬂuconazole for the treatment of oropharyngeal candidiasis in subjects with HIV/AIDS. Clin. Infect. Dis. 42:1179–86 44. Ullmann AJ, Lipton JH, Vesole DH, et al. 2007. Posaconazole or ﬂuconazole for prophylaxis in severe graft-versus-host disease. N. Engl. J. Med. 356:335–47 45. Cornely OA, Maertens J, Winston DJ, et al. 2007. Posaconazole vs ﬂuconazole or itraconazole prophylaxis in patients with neutropenia. N. Engl. J. Med. 356:348–59 46. Walsh TJ, Raad I, Patterson TF, et al. 2007. Treatment of invasive aspergillosis with posaconazole in patients who are refractory to or intolerant of conventional therapy: an externally controlled trial. Clin. Infect. Dis. 44:2–12 47. van Burik JH, Hare RS, Solomon HF, et al. 2006. Posaconazole is effective as salvage therapy in zygomycosis: a retrospective summary of 91 cases. Clin. Infect. Dis. 42:e61–65 48. Anderson J. 2005. Evolution of antifungal drug resistance: mechanisms and pathogen ﬁtness. Microbiology 3:547–56 49. Sanguinetti M, Posteraro B, Fiori B, et al. 2005. Mechanisms of azole resistance in clinical isolates of Candida glabrata collected during a hospital survey of antifungal resistance. Antimicrob. Agents Chemother. 49:668–79 50. Sanglard D. 2002. Resistance of human fungal pathogens to antifungal drugs. Curr. Opin. Microbiol. 5:79–85

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70. Baddley JW, Pappas PG. 2005. Antifungal combination therapy: clinical potential. Drugs 65:1461–80 71. Rex JH, Pappas PG, Karchmer AW, et al. 2003. A randomized and blinded multicenter trial of high-dose ﬂuconazole plus placebo versus ﬂuconazole plus amphotericin B as therapy for candidemia and its consequences in nonneutropenic subjects. Clin. Infect. Dis. 36:1221–28 72. Aliff TB, Maslak PG, Jurcic JG, et al. 2003. Refractory aspergillus pneumonia in patients with acute leukemia: successful therapy with combination caspofungin and liposomal amphotericin. Cancer 97:1025–32 73. Kontoyiannis DP, Hachem R, Lewis RE, et al. 2003. Efﬁcacy and toxicity of caspofungin in combination with liposomal amphotericin B as primary or salvage treatment of invasive aspergillosis in patients with hematologic malignancies. Cancer 98:292–99 74. Marr KA, Boeckh M, Carter RA, et al. 2004. Combination antifungal therapy for invasive aspergillosis. Clin. Infect. Dis. 39:797–802 75. Maertens J, Glasmacher A, Herbrecht R, et al. 2006. Multicenter, noncomparative study of caspofungin in combination with other antifungals as salvage therapy in adults with invasive aspergillosis. Cancer 107:2888–97 76. Pinel C, Fricker-Hidalgo H, Lebeau B, et al. 2003. Detection of circulating Aspergillus fumigatus galactomannan: value and limits of the Platelia test for diagnosing invasive aspergillosis. J. Clin. Microbiol. 41:2184–86 77. Herbrecht R, Letscher-Bru V, Oprea C, et al. 2002. Aspergillus galactomannan detection in the diagnosis of invasive aspergillosis in cancer patients. J. Clin. Oncol. 20:1898–906 78. Sulahian A, Boutboul F, Ribaud P, et al. 2001. Value of antigen detection using an enzyme immunoassay in the diagnosis and prediction of invasive aspergillosis in two adult and pediatric hematology units during a 4-year prospective study. Cancer 91:311–18 79. Maertens J, Van Eldere J, Verhaegen J, et al. 2002. Use of circulating galactomannan screening for early diagnosis of invasive aspergillosis in allogeneic stem cell transplant recipients. J. Infect. Dis. 186:1297–306 80. Wheat L. 2003. Rapid diagnosis of invasive aspergillosis by antigen detection. Transpl. Infect. Dis. 5:158–66 81. Verweij P, Mennink-Kersten M. 2006. Issues with galactomannan testing. Med. Mycol. 44:S179–83 82. Marr K, Balajee A, McLaughlin L, et al. 2004. Detection of galactomannan antigenemia by enzyme immunoassay for the diagnosis of invasive aspergillosis: variables that affect performance. J. Infect. Dis. 190:641–49 83. de Pauw BE, Patterson TF. 2005. Should the consensus guidelines’ speciﬁc criteria for the diagnosis of invasive fungal infection be changed? Clin. Infect. Dis. 41:S377–80 84. Platelia Aspergillus EIA Product Sheet, Bio-Rad Laboratories, Inc., Redmond, WA. Available at http://www.bio-rad.com/cmc upload/Literature/57083/J-115 Asperfillus.pdf 85. Pfeiffer C, Fine J, Safdar N. 2006. Diagnosis of invasive aspergillosis using a galactomannan assay: a meta-analysis. Clin. Infect. Dis. 42:1417–27 86. Kwak E, Husain S, Obman A, et al. 2004. Efﬁcacy of galactomannan antigen in the platelia Aspergillus enzyme immunoassay for diagnosis of invasive aspergillosis in liver transplant recipients. J. Clin. Microbiol. 42:435–38 87. Husain S, Kwak E, Obman A, et al. 2004. Prospective assessment of PlateliaTM Aspergillus galactomannan antigen for the diagnosis of invasive aspergillosis in lung transplant recipients. Am. J. Transpl. 4:796–802 88. Mennink-Kersten M, Donnelly J, Verweij P. 2004. Detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis. Lancet 4:349–57

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89. Aubry A, Porcher R, Bottero J, et al. 2006. Occurrence and kinetics of false-positive Aspergillus galactomannan test results following treatment with β-lactam antibiotics in patients with hematological disorders. J. Clin. Microbiol. 44:389–94 90. Herbrecht R, Letscher-Bru V, Oprea C, et al. 2002. Aspergillus galactomannan detection in the diagnosis of invasive aspergillosis in cancer patients. J. Clin. Oncol. 20:1898–906 91. FUNGITELLTM instructions for use. 2004. Associates of Cape Cod, Inc., East Falmouth, MA. Available at http://www.acciusa.com/pdfs/fungitell insert.pdf 92. Ostrosky-Zeichner L, Alexander B, Kett D, et al. 2005. Multicenter clinical evaluation of the (1→3) β-D-glucan assay as an aid to diagnosis of fungal infections in humans. Clin. Infect. Dis. 41:654–59 93. Odabasi Z, Mattiuzzi G, Estey E, et al. 2004. β-D-Glucan as a diagnostic adjunct for invasive fungal infections: validation, cutoff development, and performance in patients with acute myelogenous leukemia and myelodysplastic syndrome. Clin. Infect. Dis. 39:199– 205 94. Kato A, Takita T, Furuhashi M, et al. 2001. Elevation of blood (1→3)-beta-D-glucan concentrations in hemodialysis patients. Nephron 89:15–19 95. Digby J, Kalbﬂeisch J, Glenn A, et al. 2003. Serum glucan levels are not speciﬁc for presence of fungal infections in intensive care unit patients. Clin. Diag. Lab. Immunol. 10:882–85 96. White P, Archer A, Barnes R. 2005. Comparison of non-culture-based methods for detection of systemic fungal infections, with an emphasis on invasive Candida infections. J. Clin. Microbiol. 43:2181–87 97. White P, Linton C, Perry M, et al. 2006. The evolution and evaluation of a whole blood polymerase chain reaction for the detection of invasive aspergillosis in hematology patients in a routine clinical setting. Clin. Infect. Dis. 42:479–86 98. Costa C, Costa JM, Desterke C, et al. 2002. Real-time PCR coupled with automated DNA extraction and detection of galactomannan antigen in serum by enzyme-linked immunosorbent assay for diagnosis of invasive aspergillosis. J. Clin. Microbiol. 40:2224–27 99. Schabereiter-Gurtner C, Selitsch B, Rotter M, et al. 2007. Development of novel real-time PCR assays for detection and differentiation of eleven medically important Aspergillus and Candida species in clinical specimens. J. Clin. Microbiol. 45:906–14

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:361-379. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

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Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle1,2,3,4,5 and Lawrence Corey1,2,4 1

Department of Medicine, 2 Department of Laboratory Medicine, 3 Interdisciplinary Graduate Program in Pathobiology, University of Washington, Seattle, Washington; 4 Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, Seattle, Washington; 5 Benaroya Research Institute, Seattle, Washington; email: [email protected], [email protected]

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Key Words

The Annual Review of Medicine is online at http://med.annualreviews.org

herpes simplex virus, vaccine, lymphocyte, human immunodeﬁciency virus

This article’s doi: 10.1146/annurev.med.59.061606.095540 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0381$20.00

Abstract Herpes simplex viruses are evolutionarily ancient and ubiquitous. In the past 20 years, there has been increasing recognition of a worldwide pandemic of HSV-2 infection. Moreover, HSV-2 prevalence has increased despite fairly widespread use of antiviral drugs for HSV. The success of HSV-1 and HSV-2 stems from latency within longlived neurons and frequent mucocutaneous shedding. The generally mild medical consequences of HSV infection reﬂect a functional equilibrium between host and microbe in most immunocompetent persons. However, signiﬁcant gaps in our knowledge of the correlates of disease severity and HSV immune evasion are limiting rational advances in these areas. Human genetic studies are gradually outlining important innate responses, while recent imaging and biopsy studies have begun to show that the temporal and spatial anatomic interplay between virus reactivation and host immune response may be important in reactivations and disease expression.

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INTRODUCTION

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HSV: herpes simplex virus Reactivation: HSV DNA in sensory ganglia neurons directs synthesis of mRNA molecules that encode proteins required for HSV DNA replication and assembly of daughter virions. Viral components move anterograde toward epithelia, and infectious virus is released without neuronal death

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Herpes simplex viruses types 1 and 2 (HSV-1, HSV-2) are ubiquitous human pathogens. A balanced household survey (1) from 1999 to 2004 estimated that 57% of US adults are infected with HSV-1 and 17% with HSV-2. The accessibility of animal models and clinical populations, the ease of virus propagation, the medically serious aspects of infection, and fascination with the relapsing pattern of disease have attracted the attention of many investigators. Every advanced technique in molecular medicine has been applied to HSV (2). Nevertheless, a vaccine has remained elusive because of the subtle and successful adaptation of the HSV to its human hosts, as well as our lack of understanding of the virologic and innate and acquired immune correlates of infection severity. The recurrent mucocutaneous infections associated with HSV are painful and socially concerning (3), and account for the bulk of health care utilization. However, they are not the most serious manifestations of infection. Primary HSV infections can be devastating in newborns (4) or immune compromised hosts (5). Because HSV infection is so prevalent, rare complications of recurrent HSV have a considerable medical burden in both immune competent and immune compromised persons. These complications include encephalitis, hepatitis, pneumonia, esophagitis, and keratitis. In rare cases, summarized below, speciﬁc or generalized immune deﬁciencies or dysregulation have been implicated in these diseases. For most patients, however, the host or viral factors associated with these unusual, severe infections remain unknown. The use of sensitive diagnostic tests, particularly sensitive PCR assays of cerebrospinal ﬂuid or serum, and safe, systemic antiviral therapy may limit further damage. The licensure of the nucleoside analog acyclovir in 1982 was a landmark in the development of safe, effective antiviral therapy (6). Detailed discussions of diagnosis and treatment (7, 8) are beyond the scope of this review. Instead, we emphasize how molecular, Koelle

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cellular, animal model, and human research can help explain the clinical manifestations of HSV infection and disease and provide context for current and future interventions.

INTERACTIONS BETWEEN HSV AND HIV-1 INFECTIONS Increasing evidence of signiﬁcant interplay between HSV and HIV-1 has renewed interest in HSV-2 control. HSV-2 infection is signiﬁcantly related to HIV-1 transmission (recently reviewed in References 9–11). Coinfection with HSV-2 is very common in HIV-1-infected persons worldwide. HSV-2 is now appreciated as a major cause of genital ulcer disease worldwide (12–14). HSV-2 reactivation is associated with increasing amounts of HIV-1 RNA and infectious virus in the genital tract (15). In coinfected West African women not receiving antiretroviral therapy (ART), daily anti-HSV therapy reduced the frequency and amount of genital HIV-1 RNA shedding (9, 16). Among coinfected women receiving efavarenz-based highly active antiretroviral therapy (HAART) who had residual cervicovaginal HIV-1 shedding at baseline, daily suppressive anti-HSV therapy further reduced the frequency and titer of genital HIV-1 RNA shedding (17). An interventional study (18) in which coinfected potential HIV-1 transmitters receive anti-HSV therapy is under way to determine if this reduced genital tract HIV-1 shedding translates into reduced HIV-1 transmission to susceptible partners. A recent study of East African women showed that previously or recently acquired HSV-2 infection was signiﬁcantly associated with HIV-1 acquisition (19). A second interventional study (HPTN 039) is evaluating the efﬁcacy of daily anti-HSV therapy in preventing acquisition of HIV-1 among HSV-2 infected persons (18). The within-person impact of HSV infection on HIV-1 pathogenesis may vary with HIV-1 disease status and treatment. Untreated coinfected persons have somewhat (0.3–1 log10 ) higher HIV-1 plasma viral loads

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than do HSV-uninfected persons (10, 20). Providing anti-HSV therapy for three months to coinfected persons not on ART lowered the mean plasma HIV-1 RNA level by 0.53 log10 (16). These HIV-1 viral load differences are clinically meaningful (21). It is possible that an inexpensive and safe intervention—daily anti-HSV therapy, costing as little as $30 per year in the developing world—could delay the need for ART. Longer-term data and measures of CD4 T cells and immune activation are needed to explore this possibility.

HSV-2 TRANSMISSION AND ITS PREVENTION Classically, primary HSV infection in the absence of pre-existing immunity is symptomatic and prolonged. It has been surprising, therefore, to learn that the majority of HSV-2 seroconversion events are asymptomatic. These data come from the placebo arms of prospective vaccine trials (22). Some individuals experience typical recurrences, but others remain permanently asymptomatic. Prospective studies have also shown that most horizontal HSV-2 transmission events occur during asymptomatic shedding (23). Patient education coupled with intensive pathogen detection can move the dividing line between symptomatic and asymptomatic shedding (24, 25), but avoidance of contact during obvious recurrences is not enough. HSV-2 shedding is quite variable between individuals, and no biomarker for frequent reactivation exists. Among immunocompetent, HSV-2-seropositive persons, PCR analysis of swabs of the anogenital region, collected on a daily basis, show that 95% of persons shed HSV-2. The median shedding rate averages 25% of days but varies from 2% to 75%, with more than one third of patients shedding on >40% of days. This high frequency of reactivation is a major factor in transmission to others. Absent a vaccine, how can we limit transmission? Retrospective analyses indicate that regular condom use is associated with lower

sexual HSV-2 transmission (26). Unfortunately, intensive safer-sex counseling did not prevent HSV-2 acquisition in a prospective study of men who have sex with men (27). Daily antiviral therapy can reduce HSV-2 mucocutaneous shedding by 60%–80% depending on detection method, and titers are lower during residual sheds (28). During a one-year observation period, transmission of HSV-2 within a group of largely heterosexual couples was reduced by 48% if the potentially transmitting partner took valacyclovir daily (29). The US Food and Drug Administration has approved valacyclovir for prevention of HSV-2 transmission. Natural history data suggest additional measures to limit HSV infections, although clinical trial data are lacking. For vertical transmission, these include avoidance of unprotected sex by HSV-seronegative pregnant women, use of antiviral therapy for recurrences in late pregnancy, and possibly prophylactic treatment of newborns known to have been exposed during delivery. Patients should be counseled to avoid sex when they have lesions or prodromes. The higher rates of asymptomatic and total HSV-2 shedding in the ﬁrst year after primary genital herpes (GH) infection (30) suggest that advising patients to reduce exposures during this time is also rational. Up to 50% of ﬁrst-episode GH is due to HSV-1 in some clinical settings (31). Given the much lower rate of HSV-1 reactivation in the genital tract compared to HSV-2, typing of genital isolates (HSV-1 versus HSV-2) can rank GH patients for their risk of transmitting HSV genitally and allow individualized, stratiﬁed counseling.

Asymptomatic/ unrecognized shedding: HSV detection with no signs or symptoms of infection GH: genital herpes

INNATE IMMUNE RESPONSE TO HSV Herpesviruses are evolutionarily ancient and infect hosts as simple as mollusks. Innate virus sensor and effector mechanisms, predating the acquisition of rearranging antigen receptors on T and B lymphocytes, remain important in initiating and modulating www.annualreviews.org • Herpes Simplex

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“classical” immune responses. We have known for several decades that interferon (IFN)-α and -β, secreted by blood cells and ﬁbroblasts, respectively, have potent anti-HSV activity. Siegal and colleagues characterized the “natural” IFN-α producing cells in blood as plasmacytoid dendritic cells (pDCs) (32). pDC numbers decline signiﬁcantly in untreated HIV-1 infection (33), and even if present, pDCs can be nonresponsive to HSV (34). More recently, non-HIV-infected subjects with defective pDCs and severe HSV infection have been described (34, 35). pDCs seem to function mainly as viral sensors. They are known to express toll-like receptors 7 and 9, and HSV DNA can trigger responses via TLR9 (36). Mouse studies of HSV-1 skin infection and HSV-2 vaginal infection indicate that pDCs are not the proximal antigenpresenting cells (APCs) for T cell priming (37, 38) but are more involved in innate resistance (39). While DCs typically inﬂuence Th1/Th2 balance via IL-12/IL-10, the role of the pDC subset at this innate/acquired interface is controversial. The importance of IFN-α and -β (type 1 IFN) in the host response to HSV is highlighted by the enhanced virulence of HSV in IFN-receptor-deﬁcient mice, and by the speciﬁc type 1 IFN evasion functions mediated by several HSV-encoded proteins (40). The HSV γ34.5 protein allows HSV to evade type I IFN activity by activating a cellular phosphatase that activates eukaryotic translational initiation factor 2 alpha (EIF2α). This negates the IFN-induced effect of protein kinase R, an IFN-stimulated gene (ISG), and permits resumption of viral protein synthesis. An important early event after innate sensing of viruses is the translocation of interferon response factor (IRF)-3 to the nucleus, where it upregulates IFN-β expression. An HSV protein, ICP0, blocks the type 1 IFN response upstream of IFN-β by inhibiting IRF-3 nuclear translocation (41). Yet another HSV protein, US11, counteracts the antiviral function of another ISG, 2 –5 oligoadenylate synthetase (42). Candidate live attenuated HSV vaccine Koelle

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strains typically contain deletions of these and other virulence factors that act by evading the host innate or acquired immune system. IFN-γ, also known as type 2 or immune interferon, is secreted both by antigen-speciﬁc Tc1 CD8 and Th1 CD4 T cells in response to peptide/major histocompatibility complex (peptide-MHC), and by innate and borderline lymphocytes such as NK, NKT, and γδ T cells. A further blurring of the innate/acquired divide occurs at the level of target cells: Although type 1 and 2 IFNs bind to separate receptors, both trigger transcription factors, termed signal transducers of activated T cells (STAT)-1 and -2, to be phosphorylated, move to the nucleus with or without IRF cofactors, and stimulate expression of ISG mRNAs. The consequences of lesions in the STAT pathway can be severe. For persons with a rare homozygous mutation at an amino acid in STAT-1, HSV infection can be fatal (5). Innate lymphocytes such as NK and γδ T cells accumulate at sites of HSV infection (43, 44) and can lyse HSV-infected cells (45, 46), but as yet, little is known about positive signaling to these cells that might result in IFN-γ release or cell-mediated cytotoxicity. NFκB is another mobile transcription factor that is a convergence point for signaling after pathogen detection by TLR2 and other sensors. HSV signals through TLR2 and causes brisk activation of the NFκB pathway (47, 48). Clinical and laboratory strains of HSV have unexplained differences in their ability to activate cells via TLR2 (48). NFκB movement to the nucleus triggers expression of anti-HSV effectors such as TNF-α. NEMO/IKK-γ is a molecule that regulates NFκB, and hemizygous mutation in NEMO is associated with fatal HSV (49). Another interesting mutation in an innate immunity pathway has recently been associated with fatal HSV encephalitis (HSE) in children. Up to 13% of HSE cases occur in consanguineous families, and HSE patients typically have no evidence of overt immune deﬁciency to other infectious agents. Casanova and colleagues studied whole blood

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from HSE patients in consanguineous families and reported that IFN-α responses to HSV and TLR7 and TLR9 agonists were low to absent (49a). Unique, homozygous, nonsynonymous mutations in the UNC-93B gene were detected in two patients. UNC-93B encodes an endoplasmic reticulum transmembrane involved in TLR signaling. Mutations in other proteins at the innate-acquired crossroads have also been associated with severe HSV infection. These include CD16, an Fc receptor expressed by NK cells and involved in antibody-dependent cellular cytotoxicity, and CD40L, which is required for the licensing of DCs as well as B cell class switching (50, 51). With the exception of a recent study of TLR2 (52), little research has investigated more subtle variations in these innate or crossover immune molecules and the nonmorbid spectrum of HSV severity. The TLR7 and -8 agonist resiquimod is a powerful inducer of IFN-α. Recent phase III trials showed that topical application of resiquimod to GH lesions decreased the subsequent rate of HSV-2 shedding (53). Although clinically apparent recurrences were not delayed at the dose studied, the authors consider the induction of IFN-α via TLRs a promising therapeutic approach for treating mucosal HSV infection in humans. Topical TLR9 agonists are powerful inducers of a local anti-HSV state in the genital tract and have strong adjuvant activity for local vaccines (54). pDCs and their type I IFN product also have favorable effects on the skin-homing characteristics of memory, HSV-speciﬁc T cells (55) (Figure 1), further supporting a role for manipulations of innate immunity in future efforts to reduce the spread and health impact of HSV.

ACQUIRED IMMUNE RESPONSE TO HSV The acquired immune response to HSV includes CD4 and CD8 T cells and antibodies. CD4 T cells are probably important in both antibody and CD8 responses. HSV-speciﬁc

CD8 T cells develop poorly in the absence of CD4 T cell help, and HSV-speciﬁc CD4 T cells “license” DCs to prime HSV-speciﬁc CD8 T cells. The same DCs that present HSV antigen to CD4 T cells, and are licensed in the process, are necessary and sufﬁcient to prime naive CD8 T cells (56). The importance of CD4 cells as pure effector cells remains controversial. In the immune mouse intravaginal HSV-2 rechallenge model, CD4 T cells and IFN-γ are clearly important mucosal effectors (57). In our cross-sectional human study of non-HAART-treated HIV-1/HSV2 coinfected men, low HSV-2-speciﬁc CD8, but not CD4, T cell number correlated with GH lesion severity (58). In these subjects, CD4 T cells may have been available to assist CD8 priming if HSV-infection predated HIV-1 infection. CD4 T cell loss clearly correlates with HSV-2 shedding in non-HAARTtreated HIV-1-infected persons (59). When HAART is administered, GH lesion severity improves while HSV-2 shedding remains high (60). HSV-2-speciﬁc CD4 T cells certainly localize to GH lesions (61) and can interact with infected skin keratinocytes that have been activated by IFN-γ, as has been shown to occur in lesions (62). These data are consistent with a model in which HSV-2-speciﬁc CD4 T cells participate in lesion resolution. CD8 T cells may be important for controlling HSV replication and shedding in the ganglia and skin/mucosa. Vaccines stimulating CD8 responses alone can be protective in animal models (63). During primary infection, inﬁltration of CD8 T cells is associated with control of acute ganglionic infection. In the chronic stage, HSV-speciﬁc CD8 T cells that recognize a structural HSV glycoprotein persistently inﬁltrate the dorsal root ganglia of latently infected mice (64). This implies some level of lytic protein expression sufﬁcient to drive local immune monitoring. These cells have cytolytic and IFN-γ effector functions, and explant studies show the latter are functionally important for HSV suppression. Human autopsy studies (65) show that HSV-1-speciﬁc CD8 T cells with these www.annualreviews.org • Herpes Simplex

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effector functions also localize to trigeminal ganglia in the natural host. Neuron dropout or death is not observed clinically or histologically. Inhibitory molecules capable of modulating T cell activation have been detected in

a

infected ganglia (66). T cells’ effector functions are probably also reduced by the suppressive environment, including the presence of regulatory T cells, in the anterior chamber during ocular infections (67). It will be

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important in future studies to determine how inﬂammation and immune privilege interact in these important sites of infection. In the periphery, HSV-2-speciﬁc CD8 T cells are attracted to GH lesions, where the inﬁltration of local cytotoxic T cells correlates with viral clearance (43). These cells can be detected at low levels in peripheral blood mononuclear cells by ELISPOT or peptideMHC oligomers but are greatly enriched in herpetic skin. Stable, speciﬁc, and high expression of a skin-speciﬁc lymphocyte vascular adressin, cutaneous lymphocyte-associated antigen (CLA), by circulating HSV-2-speciﬁc CD8 T cells may participate in their rapid homing to a site of skin infection (68). Recently, the spatial and temporal relationships between inﬁltrating HSV-2-speciﬁc CD8 T cells and HSV-2 replication were studied in human genital skin biopsies using peptideMHC oligomers (69). When lesions are active, these cells are abundant in the dermis, and they also penetrate the epidermal layer where HSV-2 is replicating. After clinical healing, HSV-2-speciﬁc CD8 T cells persist for up to eight weeks, often adjacent to the sensory nerve endings that are believed to be sites of HSV egress from neurons (Figure 2). These studies were performed with CD8 T cells speciﬁc for proteins in the tegument layer of the HSV virion. Unbiased library screens and clonal dominance studies (70) indicate that tegument-speciﬁc CD8 T cells may be somewhat immunodominant, but re-

search in this area is just beginning. A screen of about half the HSV-2 proteome using synthetic peptides revealed surprising diversity for CD8 target antigens, with some HLA dependence and a median of 11 open reading frames recognized per subject (71). The functional properties and within-epitope diversity of the CD8 response are also important. Mouse experiments show that CD8 T cell avidity and clonotypic complexity are associated with mild disease (72). These newer anatomic data and, by implication, the failure of antibody and CD4-stimulating vaccines to signiﬁcantly impact HSV in preventative or therapeutic modalities argue for a renewed effort to design and test vaccines that can maximally stimulate this arm of the immune response, while carefully monitoring for excessive immune activation in the ganglia. Antibodies are functionally relevant in the prevention of neonatal transmission of HSV-1 or HSV-2 in the setting of chronic infection, a mature IgG response that can cross the placenta, and recurrent cervicovaginal shedding at delivery. The per-delivery risk of neonatal infection is quite low, in contrast to maternal primary infection in which IgG antibody is lacking. This disparity implies that passively transferred antibody is protective (73). Isolated antibody deﬁciency is rarely associated with severe primary or recurrent HSV infection, and elicitation of neutralizing antibodies alone, to levels seen during natural infection, has thus far not correlated with preventative

Peptide-MHC oligomer: a ﬂuorescent probe with multiple functional groups of major histocompatibility complex (MHC) molecule loaded with an antigenic viral peptide. The oligomer binds speciﬁcally to T lymphocytes that recognize the viral peptide. Oligomers of human leukocyte antigen (HLA) class I molecules bind human virus-speciﬁc CD8+ T lymphocoytes; class II oligomers bind CD4+ cells. Murine homologs contain MHC rather than HLA molecules

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 1 Model for selected events in a recurrent cutaneous HSV-2 lesion. Top: Dermal venule and infected epidermal keratinocytes. Circulating HSV-2-speciﬁc CD8 T cells express cutaneous lymphocyte-associated antigen (CLA), allowing them to adhere to E-selectin, which is upregulated on endothelial cells in HSV-2 lesions. Unpublished data and animal gene knockout models support possible roles for CXCR3 and its ligands, IP-10, Mig, and IP-9, in attracting HSV-speciﬁc T cells. HSV-2-speciﬁc CD4 T cell expression of CLA may be assisted by innate plasmacytoid dendritic cell (pDC) secretion of IFN-α and/or IL-12 in response to HSV (H in red hexagons). In the skin, HSV-2-speciﬁc CD8 T cells kill HSV-2-infected keratinocytes (yellow arrows). Expression of human leukocyte antigen (HLA) class I, necessary for this recognition, may be assisted by IFN-γ secreted by HSV-2-speciﬁc CD4 and NK cells. CD4 cells secrete IL-2, supporting CD8 cells. Bottom: Circulating HSV-2-speciﬁc CD8+ T cells, identiﬁed by staining with a speciﬁc ﬂuorescent tetramer reagent (right upper quadrant of left panel ), overexpress the skin-homing marker CLA (upper right) compared to bystander CD8+ T cells (lower right). Data from References 55 and 68. www.annualreviews.org • Herpes Simplex

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H and E Normal skin

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Figure 2 Histologic features of recurrent human genital HSV-2 lesions. Top left and center: Hematoxylin and eosin-stained frozen sections of normal and buttock genital HSV-2 lesional skin, showing dermal inﬁltrate and loss of epidermis. Top right: two-color confocal image of CD8+ cells (green, mostly in dermis) and HSV-2 antigen (red, conﬁned to superﬁcial skin). Bottom left: transverse frozen section of HSV-2 lesion showing occasional CD8+ cells ( green) in epidermis. Nuclei are stained blue with DAPI. Center: Three color stains including visualization of HSV-2-speciﬁc CD8 T cells (red ) using HLA-peptide multimers conjugated to quantum dots. HLA–peptide–quantum dot multimers stain HSV-2-speciﬁc CD8 T cells red. At right is a section parallel to the skin surface, showing CD8 T cells in the dermal rete ridges. Skin was obtained four weeks after resolution of clinical lesions. From Reference 69.

gD2: glycoprotein D of HSV-2 gB2: glycoprotein B of HSV-2 388

vaccine efﬁcacy in humans (74). Functional roles for antibody are implied by the fact that HSV encodes a potent Fc receptor that contributes to pathogenesis in appropriate mouse models (75). The recent elucidation of structures for HSV envelope proteins involved in receptor binding and fusion (76, 77) may allow rational vaccine design to further boost neutralizing antibody levels and revitalize this area of vaccine research. Because HSV in genital lesions and vaginal/cervical secretions appears to exist as “free virus,” it is likely that an optimal vaccine will need to provide high levels of neutralizing antibodies. Prior subunit vaccines containing the major neutralizing proteins gD2 and Koelle

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gB2 have not effectively blocked infection, suggesting that neutralizing antibodies may provide a necessary but insufﬁcient immune response to prevent infection in humans. A vaccine containing truncated gD2 in a novel lipid adjuvant was recently shown to prevent HSV-2 disease in phase III clinical trials (77a). This vaccine elicits antibody and CD4 T cell responses. Clinical activity was observed only in women who were both HSV-1- and HSV2-uninfected. A conﬁrmatory trial is under way in this population. Vaccine regimens that elicit both effector antibodies and adaptive T cell responses appear to offer the greatest hope for developing an effective immunogen. Some of the approaches currently being

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developed in the HIV-1 vaccine ﬁeld, such as the inclusion of fusion intermediates as targets for antibodies and the use of potent viral vectors to elicit CD8 T cell responses, may be applicable to HSV vaccine development.

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UNANSWERED QUESTIONS Many of the most important questions in the immunobiology of HSV infections remain unanswered or unaddressed. As discussed above, there are few data on the correlates of protection in humans and few clear associations between adaptive or innate immune responses and frequencies of reactivation of infection. There are documented, though infrequent, examples of recovery of multiple strains on more than one occasion each, consistent with reinfection of immune hosts (78). It is likely that wild-type infection is partially protective against second-strain infection and disease. Knowledge of the strength of this effect would provide a rational target for vaccine research. We do not know how local HSV-speciﬁc T cells in dorsal root ganglia interact with infected neurons. Secretion of IFN-γ appears to be crucial to limiting viral reactivation (64), implicating T cell activation; but clinically and histologically, infected neurons appear to be protected from cytotoxic at-

tack. Antiapoptotic HSV miRNA expressed during latency (79) and inhibitory T cell receptor/ligand pairings in the ganglia (66) may modulate the destructive potential of the T cell inﬁltrate, but further research is needed. The factors responsible for variation in HSV-2 severity in apparently immunocompetent persons, including severe loss of control of HSV replication in persons with encephalitis or hepatitis, remain unknown. The host polymorphisms described to date (mentioned above) provide a few clues, but most of the spectrum remains mysterious. The role of viral polymorphisms in disease severity remains largely unexplored. Epidemiologic studies suggest a poor correlation between the severity of GH in transmitting and receiving persons (80). However, many HSV genetic markers have been associated with dramatic pathogenesis effects in animal models (81), and little sequencing has been performed on clinical isolates. The tools for such a project are now available. The long coevolution of host and pathogen may limit the power of animal models to address some of these issues. The only certainties are that human behavior guarantees ongoing opportunities for HSV-2 transmission and research, and that HSV is assuredly wilier than we are clever.

Latency: HSV DNA exists extrachromosomally in nuclei of sensory ganglia neurons. Speciﬁc latency-associated RNA transcripts, not proven to encode proteins, accumulate in these neurons. Newer shedding data indicate latency may be less tight than previously believed (see text)

SUMMARY POINTS 1. HSV-2 infection is a signiﬁcant cofactor for HIV-1 transmission. 2. Clinical trials of anti-HSV drugs to prevent HIV-1 acquisition and transmission are nearing completion. Results, if positive, will provide further impetus for medical approaches in prevention as well as vaccine initiatives. 3. HSV-2-speciﬁc CD8 T cells have surveillance functions to limit HSV-2 reactivation in ganglia and skin. 4. A HSV-2 vaccine eliciting antibody and CD4 T cell responses with partial clinical activity in HSV-uninfected women only is being investigated in a conﬁrmatory phase III clinical trial. 5. Asymptomatic shedding accounts for the majority of HSV-2 transmission.

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6. Suppression of viral shedding in the source partner using nucleoside analog drugs decreases HSV-2 transmission. This raises the possibility that immunotherapy could also reduce shedding and transmission, as well as alleviate symptoms. 7. The frequency of asymptomatic genital HSV-2 shedding, averaging 25% of days using multiple samples and sensitive methods, indicates that persistent lytic infection, rather than latency with intermittent reactivation, may more accurately describe HSV-2 in the human sacral ganglia.

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8. Innate immunity defects have emerged as correlates of severe HSV infections. Innate immunity stimulators have powerful anti-HSV effects in animals but have not reached clinical licensure.

FUTURE ISSUES 1. What are the innate and acquired immune correlates of disease severity? 2. Will anti-HSV interventions prevent HIV-1 transmission or acquisition? 3. Will efﬁcacy of the gD2/alum/ASO4 vaccine in HSV-seronegative women be conﬁrmed? 4. Will CD8-directed vaccines ameliorate severity and shedding when used prophylactically? Will they have therapeutic beneﬁt?

DISCLOSURE STATEMENT Dr. Koelle has received grant support from Antigenics, Inc.; 3M, Inc.; and GlaxoSmithKline. He is a coinventor on patents related to HSV-2 vaccines. Dr. Corey is director of the University of Washington Virology Division, which has received grant support from GlaxoSmithKline and Novartis, two companies that make antiviral drugs for the treatment of HSV-2. However, he receives no salary support from these studies. In the past he has received consulting fees from Antigenics, which is developing an HSV-2 vaccine.

LITERATURE CITED 1. Xu F, Sternberg MR, Kottiri BJ, et al. 2006. Trends in herpes simplex virus type 1 and type 2 seroprevalence in the United States. JAMA 296:964–73 2. Roizman B, Knipe DM, Whitley RJ. 2007. Herpes simplex viruses. In Fields Virology, ed. DM Knipe, PM Howley, pp. 2501–602. Philadelphia: Lippincott, Williams, and Wilkins 3. Corey L, Wald A. 1999. Genital herpes. In Sexually Transmitted Diseases, ed. KK Holmes, PF Sparling, PA Mardh, et al., pp. 285–312. New York: McGraw-Hill 4. Kimberlin DW. 2005. Herpes simplex virus infections in neonates and early childhood. Semin. Pediatr. Infect. Dis. 16:271–81 5. Dupuis S, Jouanguy E, Al-Hajjar S, et al. 2003. Impaired response to interferon-alpha/beta and lethal viral disease in human STAT1 deﬁciency. Nat. Genet. 33:388–91 6. Dorsky DI, Crumpacker CS. 1987. Drugs ﬁve years later: acyclovir. Ann. Intern. Med. 107:859–74

2. Reviews the early history of HSV research.

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7. Workowski KA, Berman SM. 2006. Sexually transmitted diseases treatment guidelines, 2006. Morb. Mortal. Wkly. Rep. 55(RR-11):16–22 8. Corey L. 2004. Herpes simplex viruses. In Harrison’s Principles of Internal Medicine, ed. DL Kasper, E Braunwald, AS Fauci, et al., pp. 1035–42. New York: McGraw-Hill 9. Corey L. 2007. Synergistic copathogens—HIV-1 and HSV-2. N. Engl. J. Med. 356:854– 56 10. Corey L. 2007. Herpes simplex virus type 2 and HIV-1: the dialogue between the 2 organisms continues. J. Infect. Dis. 195:1242–44 11. Freeman EE, Weiss HA, Glynn JR, et al. 2006. Herpes simplex virus 2 infection increases HIV acquisition in men and women: systematic review and meta-analysis of longitudinal studies. AIDS 20:73–83 12. Beyrer C, Jitwatcharanan K, Natpratan C, et al. 1998. Molecular methods for the diagnosis of genital ulcer disease in a sexually transmitted disease clinic population in northern Thailand: predominance of herpes simplex virus infection. J. Infect. Dis. 178:243–46 13. Hoyo C, Hoffman I, Moser BK, et al. 2005. Improving the accuracy of syndromic diagnosis of genital ulcer disease in Malawi. Sex. Transm. Dis. 32:231–37 14. Lai W, Chen CY, Morse SA, et al. 2003. Increasing relative prevalence of HSV-2 infection among men with genital ulcers from a mining community in South Africa. Sex. Transm. Infect. 79:202–7 15. Schacker T, Ryncarz AJ, Goddard J, et al. 1998. Frequent recovery of HIV-1 from genital herpes simplex virus lesions in HIV-1-infected men. JAMA 280:61–66 16. Nagot N, Ouedraogo A, Foulongne V, et al. 2007. Reduction of HIV-1 RNA levels with therapy to suppress herpes simplex virus. N. Engl. J. Med. 356:790–99 17. Ouedraogo A, Nagot N, Vergne L, et al. 2006. Impact of suppressive herpes therapy on genital HIV-1 RNA among women taking antiretroviral therapy: a randomized controlled trial. AIDS 20:2305–13 18. Celum CL, Robinson NJ, Cohen MS. 2005. Potential effect of HIV type 1 antiretroviral and herpes simplex virus type 2 antiviral therapy on transmission and acquisition of HIV type 1 infection. J. Infect. Dis. 191(Suppl. 1):S107–14 19. Kapiga SH, Sam NE, Bang H, et al. 2007. The role of herpes simplex virus type 2 and other genital infections in the acquisition of HIV-1 among high-risk women in northern Tanzania. J. Infect. Dis. 195:1260–69 20. Gray RH, Li X, Wawer MJ, et al. 2004. Determinants of HIV-1 load in subjects with early and later HIV infections, in a general-population cohort of Rakai, Uganda. J. Infect. Dis. 189:1209–15 21. Mellors JW, Munoz A, Giorgi JV, et al. 1997. Plasma viral load and CD4+ lymphocytes as prognostic markers of HIV-1 infection. Ann. Intern. Med. 126:946–54 22. Langenberg AGM, Corey L, Ashley RL, et al. 2000. A prospective study of new infections with herpes simplex virus type 1 and type 2. N. Engl. J. Med. 341:1432–38 23. Mertz GJ, Schmidt O, Jourden JL, et al. 1985. Frequency of acquisition of ﬁrst-episode genital infection with herpes simplex virus from asymptomatic and symptomatic source contacts. Sex. Transm. Dis. 12:33–39 24. Langenberg A, Benedetti J, Jenkins J, et al. 1989. Development of clinically recognizable genital lesions among women previously identiﬁed as having asymptomatic herpes simplex virus type 2 infection. Ann. Intern. Med. 110:882–87 25. Frenkel LM, Garratty EM, Shen JP, et al. 1993. Clinical reactivation of herpes simplex virus type 2 infection in seropositive pregnant women with no history of genital herpes. Ann. Intern. Med. 118:414–18 www.annualreviews.org • Herpes Simplex

16. Daily anti-HSV therapy among HIV-infected women not on HAART lowered plasma HIV-1 by 1/2 log10 , as well as genital HIV-shedding.

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26. Wald A, Langenberg AG, Krantz E, et al. 2005. The relationship between condom use and herpes simplex virus acquisition. Ann. Intern. Med. 143:707–13 27. Brown EL, Wald A, Hughes JP, et al. 2006. High risk of human immunodeﬁciency virus in men who have sex with men with herpes simplex virus type 2 in the EXPLORE study. Am. J. Epidemiol. 164:733–41 28. Wald A, Corey L, Cone R, et al. 1997. Frequent genital herpes simplex virus 2 shedding in immunocompetent women: effect of acyclovir treatment. J. Clin. Invest. 99:1092–97 29. Corey L, Wald A, Patel R, et al. 2004. Once-daily valacyclovir to reduce the risk of transmission of genital herpes. N. Engl. J. Med. 350:11–20 30. Koelle DM, Benedetti J, Langenberg A, Corey L. 1992. Asymptomatic reactivation of herpes simplex virus in women after the ﬁrst episode of genital infection. Ann. Intern. Med. 116:433–37 31. Engelberg R, Carrell D, Krantz E, et al. 2003. Natural history of genital herpes simplex virus type 1 infection. Sex. Transm. Dis. 30:174–77 32. Siegal FP, Kadowaki N, Shodell M, et al. 1999. The nature of the principle type 1 interferon-producing cells in human blood. Science 284:1835–37 33. Chehimi J, Campbell DE, Azzoni L, et al. 2002. Persistent decreases in blood plasmacytoid dendritic cell number and function despite effective highly active antiretroviral therapy and increased blood myeloid dendritic cells in HIV-infected individuals. J. Immunol. 168:4796–801 34. Abbo L, Vincek V, Dickinson G, et al. 2007. Selective defect in plasmacytoid dendritic cell function in a patient with AIDS-associated atypical genital herpes simplex vegetans treated with imiquimod. Clin. Infect. Dis. 44:e25–27 35. Dalloul A, Oksenhendler E, Chosidow O, et al. 2004. Severe herpes virus (HSV-2) infection in two patients with myelodysplasia and undetectable NK cells and plasmacytoid dendritic cells in the blood. J. Clin. Virol. 30:329–36 36. Lund J, Sato A, Akira S, et al. 2003. Toll-like receptor 9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells. J. Exp. Med. 198:513–20 37. Zhao X, Deak E, Soderberg K, et al. 2003. Vaginal submucosal dendritic cells, but not Langerhans cells, induce protective Th1 responses to herpes simplex virus-2. J. Exp. Med. 197:153–62 38. Belz GT, Behrens GM, Smith CM, et al. 2002. The CD8alpha+ dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens. J. Exp. Med. 196:1099–104 39. Lund JM, Linehan MM, Iijima N, et al. 2006. Cutting edge: plasmacytoid dendritic cells provide innate immune protection against mucosal viral infection in situ. J. Immunol. 177:7510–14 40. Leib DA. 2002. Counteraction of interferon-induced antiviral responses by herpes simplex viruses. Curr. Top. Microbiol. Immunol. 269:171–85 41. Lin R, Noyce RS, Collins SE, et al. 2004. The herpes simplex virus ICP0 RING ﬁnger domain inhibits IRF3- and IRF7-mediated activation of interferon-stimulated genes. J. Virol. 78:1675–84 42. Sanchez R, Mohr I. 2007. Inhibition of cellular 2 –5 oligoadenylate synthetase by the herpes simplex virus type 1 Us11 protein. J. Virol. 81:3455–64 43. Koelle DM, Posavad CM, Barnum GR, et al. 1998. Clearance of HSV-2 from recurrent genital lesions correlates with inﬁltration of HSV-speciﬁc cytotoxic T lymphocytes. J. Clin. Invest. 101:1500–8

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44. Verjans GM, Roest RW, Van Der Kooi A, et al. 2004. Isopentenyl pyrophosphate-reactive Vgamma9Vdelta 2 T helper 1-like cells are the major gammadelta T cell subset recovered from lesions of patients with genital herpes. J. Infect. Dis. 190:489–93 45. Sciammas R, Johnson RM, Sperling AI, et al. 1994. Unique recognition by a herpesvirusspeciﬁc TCRgd cell. J. Immunol. 152:5392–97 46. Yasukawa M, Zarling JM. 1983. Autologous herpes simplex virus-infected cells are lysed by human natural killer cells. J. Immunol. 131:2011–16 47. Hargett D, Rice S, Bachenheimer SL. 2006. Herpes simplex virus type 1 ICP27dependent activation of NF-kappaB. J. Virol. 80:10565–78 48. Sato A, Linehan MM, Iwasaki A. 2006. Dual recognition of herpes simplex viruses by TLR2 and TLR9 in dendritic cells. Proc. Natl. Acad. Sci. USA 103:17343–48 49. Niehues T, Reichenbach J, Neubert J, et al. 2004. Nuclear factor kappaB essential modulator-deﬁcient child with immunodeﬁciency yet without anhidrotic ectodermal dysplasia. J. Allergy Clin. Immunol. 114:1456–62 49a. Casrouge A, Zhang SY, Eidenschenk C, et al. 2006. Herpes simplex virus encephalitis in human UNC-93B deficiency. Science 314:308–12 50. Jawahar S, Moody C, Chan M, et al. 1996. Natural killer (NK) cell deﬁciency associated with an epitope-deﬁcient Fc receptor type IIIA (CD16-II). Clin. Exp. Immunol. 103:408– 13 51. Garcia-Perez MA, Paz-Artal E, Correll A, et al. 2003. Mutations of CD40L ligand in two patients with hyper-IgM syndrome. Immunobiology 207:285–94 52. Bochud PY, Magaret AS, Koelle DM, et al. 2007. Increased viral shedding and lesional rate in patients with genital HSV-2 infection. J. Infect. Dis. 196:505–9 53. Mark KE, Corey L, Meng T-C, et al. 2007. Topical resiquimod 0.01% gel decreases herpes simplex virus type 2 genital shedding: a randomized, controlled trial. J. Infect. Dis. 195:1324–31 54. Wu JJ, Huang DB, Tyring SK. 2004. Resiquimod: a new immune response modiﬁer with potential as a vaccine adjuvant for Th1 immune responses. Antivir. Res. 64:79–83 55. Koelle DM, Huang J, Hensel MT, et al. 2006. Innate immune responses to herpes simplex virus type 2 inﬂuence skin homing molecule expression by memory CD4+ lymphocytes. J. Virol. 80:2863–72 56. Smith CM, Wilson NS, Waithman J, et al. 2004. Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity. Nat. Immunol. 5:1143–48 57. Milligan GN, Bernstein DI, Bourne N. 1998. T lymphocytes are required for protection of the vaginal mucosae and sensory ganglia of immune mice against reinfection with herpes simplex virus type 2. J. Immunol. 160:6093–100 58. Posavad CM, Koelle DM, Shaughnessy MF, et al. 1997. Severe genital herpes infections in HIV-infected individuals with impaired HSV-speciﬁc CD8+ cytotoxic T lymphocyte responses. Proc. Natl. Acad. Sci. USA 94:10289–94 59. Schacker T, Zeh J, Hu H-L, et al. 1998. Frequency of symptomatic and asymptomatic herpes simplex virus type 2 reactivations among human immunodeﬁciency virus-infected men. J. Infect. Dis. 178:1616–22 60. Posavad CM, Wald A, Kuntz S, et al. 2004. Frequent reactivation of herpes simplex virus among HIV-1-infected patients treated with highly active antiretroviral therapy. J. Infect. Dis. 190:693–96 61. Koelle DM, Abbo H, Peck A, et al. 1994. Direct recovery of HSV-speciﬁc T lymphocyte clones from human recurrent HSV-2 lesions. J. Infect. Dis. 169:956–61 62. Cunningham AL, Turner RR, Miller AC, et al. 1985. Evolution of recurrent herpes simplex lesions: an immunohistologic study. J. Clin. Invest. 75:226–33 www.annualreviews.org • Herpes Simplex

49a. Children with fatal HSV encephalitis but not other infections had separate, homozygous UNC-93B mutations and impaired innate responses to HSV.

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64. Proves that HSV-specific CD8 T cells localize to DRG, in proximity to latently infected neurons. Implies intermittent antigen presentation in the ganglia. 65. HSV-1-infected human trigeminal ganglia are infiltrated by HSV-specific CD8 and CD4 T cells.

69. In situ genital skin studies using HSV-2-specific peptide-HLA stains show virus-specific CD8 T cells adjacent to HSV-infected cells and axonal endings.

77a. Phase III vaccine trials showing activity in HSV-seronegative women only. The reason(s) for the gender difference are unknown.

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63. Orr MT, Orgun NN, Wilson CB, et al. 2007. Cutting edge: recombinant listeria monocytogenes expressing a single immune-dominant peptide confer immunity to herpes simplex virus-1 infection. J. Immunol. 178:4731–35 64. Khanna KM, Bonneau RH, Kinchington PR, et al. 2003. Herpes simplex virusspecific memory CD8+ T cells are selectively activated and retained in latently infected sensory ganglia. Immunity 18:593–603 65. Verjans GMGM, Hintzen RQ, van Dun JM, et al. 2007. Selective retention of herpes simplex virus specific T cells in latently infected human trigeminal ganglia. Proc. Natl. Acad. Sci. USA 104:3496–501 66. Suvas S, Azkur AK, Rouse BT. 2006. Qa-1b and CD94-NKG2a interaction regulate cytolytic activity of herpes simplex virus-speciﬁc memory CD8+ T cells in the latently infected trigeminal ganglia. J. Immunol. 176:1703–11 67. Suvas S, Azkur AK, Kim BS, et al. 2004. CD4+ CD25+ regulatory T cells control the severity of viral immunoinﬂammatory lesions. J. Immunol. 172:4123–32 68. Koelle DM, Liu Z, McClurkan CM, et al. 2002. Expression of cutaneous lymphocyteassociated antigen by CD8+ T-cells speciﬁc for a skin-tropic virus. J. Clin. Invest. 110:537– 48 69. Zhu J, Koelle DM, Cao J, et al. 2007. Peripheral virus-specific CD8+ T cells contiguous to sensory nerve endings limit HSV-2 reactivation in human genital skin. J. Exp. Med. 204:595–603 70. Koelle DM, Liu Z, McClurkan CL, et al. 2003. Immunodominance among herpes simplex virus-speciﬁc CD8 T-cells expressing a tissue-speciﬁc homing receptor. Proc. Natl. Acad. Sci. USA 100:12899–904 71. Hosken N, McGowan P, Meier A, et al. 2006. Diversity of the CD8+ T cell response to herpes simplex virus type 2 proteins among persons with genital herpes. J. Virol. 80:5509– 15 72. Messaoudi I, Guevara Patino JA, Dyall R, et al. 2002. Direct link between MHC polymorphism, T cell avidity, and diversity in immune defense. Science 298:1797–800 73. Brown ZA, Gardella C, Wald A, et al. 2005. Genital herpes complicating pregnancy. Obstet. Gynecol. 106:845–56 74. Corey L, Langenberg AGM, Ashley R, et al. 1999. Two double-blind, placebo-controlled trials of a vaccine containing recombinant gD2 and gB2 antigens in MF59 adjuvant for the prevention of genital HSV-2 acquisition. JAMA 282:331–40 75. Lubinski JM, Jiang M, Hook L, et al. 2002. Herpes simplex virus type 1 evades the effects of antibody and complement in vivo. J. Virol. 76:9232–41 76. Spear PG. 2004. Herpes simplex virus: receptors and ligands for cell entry. Cell Microbiol. 6:401–10 77. Heldwein EE, Lou H, Bender FC, et al. 2006. Crystal structure of glycoprotein B from herpes simplex virus 1. Science 313:217–20 77a. Stanberry LR, Spruance SL, Cunningham AL. 2002. Glycoprotein-D-adjuvant vaccine to prevent genital herpes. GlaxoSmithKline Herpes Vaccine Efficacy Study Group. N. Engl. J. Med. 347:1652–61 78. Sucato G, Wald A, Wakabayashi E, et al. 1998. Evidence of latency and reactivation of both herpes simplex virus (HSV)-1 and HSV-2 in the genital region. J. Infect. Dis. 177:1069–72 79. Gupta A, Gartner JJ, Sethupathy P, et al. 2006. Anti-apoptotic function of a microRNA encoded by the HSV-1 latency-associated transcript. Nature 442:82–85 Koelle

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80. Langenberg AGM, Corey RL, Ashley RL, et al. 1999. Acquisition of symptomatic and asymptomatic HSV-1 and HSV-2 infections: a prospective study of their incidence and clinical spectrum. N. Engl. J. Med. 341:1432–38 81. Brandt CR, Kolb AW, Shah DD, et al. 2003. Multiple determinants contribute to the virulence of HSV ocular and CNS infection and identiﬁcation of serine 34 of the US1 gene as an ocular disease determinant. Invest. Ophthalmol. Vis. Sci. 44:2657–68

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RELATED RESOURCES 1. Morrison LA. 2004. The Toll of herpes simplex virus infection. Trends Microbiol. 12:353–56 2. Dudek T, Knipe DM. 2006. Replication-defective viruses as vaccines and vaccine vectors. Virology 344:230–39 3. Cunningham AL, Diefenbach RJ, Miranda-Saksena M, et al. 2006. The cycle of human herpes simplex virus infection: virus transport and immune control. J. Infect. Dis. 194 (Suppl. 1):S11–18 4. Roizman B, Whitley RJ. 2001. The nine ages of herpes simplex virus. Herpes 8:23–27

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:381-395. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Contents

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Medical Management of Inﬂuenza Infection Annu. Rev. Med. 2008.59:397-413. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Anne Moscona Department of Pediatrics, Weill Cornell Medical College, New York, New York 10021; email: [email protected]

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Key Words

First published online as a Review in Advance on October 16, 2007

antiviral, neuraminidase inhibitor, prophylaxis, drug resistance

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.061506.213121 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0397$20.00

Abstract Antiviral drugs are important in the management of seasonal inﬂuenza and critical to pandemic planning. Although several other classes of anti-inﬂuenza compounds exist, the neuraminidase (NA) inhibitors are currently the only option in most clinical settings. These drugs, zanamivir and oseltamivir, prevent the release of newly replicated inﬂuenza virions from infected cells. They are highly effective when used for treatment of seasonal inﬂuenza early in the course of infection, or for prevention when given soon after exposure. Treatment strategies for avian inﬂuenza infections in humans are still provisional owing to inadequate clinical data. As predicted by molecular studies, resistance to the NA inhibitors is now emerging, although at a level less signiﬁcant than adamantane resistance. NA inhibitor resistance is a cause for concern if indeed some mutant strains of avian inﬂuenza are transmissible and pathogenic. In the near term, appropriate use of the available NA inhibitors will be of major beneﬁt in lessening morbidity and mortality due to inﬂuenza infection. Priorities include developing appropriate formulations and guidelines for use of these drugs in children and people infected with avian inﬂuenza, study of the mechanisms and clinical signiﬁcance of drug resistance, and identiﬁcation of new antiviral therapies that target different points in the viral life cycle in order to limit the effect of emerging drug resistance.

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INTRODUCTION

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Once the inﬂuenza virus successfully infects a host and initiates the disease cascade, our ability to cure the disease is limited. Therefore, prevention is an important goal. Intensive efforts are under way to ﬁnd universal vaccines that will protect regardless of the inﬂuenza virus’ antigenic drift and shift in the future. At present, however, many situations require medical treatment of inﬂuenza disease, and effective antiviral agents are critical. A current goal is the application of the available antiviral agents to prevent and treat inﬂuenza disease, both in seasonal epidemic inﬂuenza and as part of a pandemic plan. A future goal is the design of new antiviral drugs that are targeted to various stages in the inﬂuenza viral life cycle and in the pathogenesis of disease, which would avoid eliciting drug resistance.

AVAILABLE CLASSES OF INFLUENZA ANTIVIRALS: OVERVIEW Two classes of drugs are currently available for the treatment and/or prophylaxis of inﬂuenza infections: the adamantanes or M2 inhibitors and the neuraminidase (NA) inhibitors. The emergence of resistance to both classes of drugs is encouraged by the use of single drugs that target a single stage in the viral life cycle, to treat a rapidly evolving pathogen. At the time of this writing, NA inhibitors are the only reliable antiviral option for treatment of seasonal inﬂuenza infection; the utility of the adamantanes (amantadine and rimantadine) has been virtually eliminated by the development of resistance (1).

ADAMANTANES The adamantanes (amantadine and rimantadine) block inﬂux of H+ ions through the M2 (inﬂuenza matrix protein 2) ion channel, preventing the acid-triggered fusion reaction mediated by the inﬂuenza hemagglutinin and thus interfering with viral uncoating inside 398

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the cell. These compounds are effective only against inﬂuenza A and are associated with several toxicities, particularly of the central nervous system, and with rapid emergence of drug-resistant variants. Adamantane-resistant isolates of inﬂuenza A are genetically stable, are as transmissible and as pathogenic as wildtype isolates in susceptible contacts, and can be shed for prolonged periods in immunocompromised patients. The rapidity with which the adamantanes elicit viral resistance (30% of treated patients shed resistant viruses within three days of treatment) severely limits their utility for treatment of seasonal inﬂuenza. In terms of pandemic planning, future pandemic strains may retain sensitivity, and the use of adamantanes in combination with NA inhibitors is at least a theoretical consideration for prophylaxis (2) (discussed below). However, recently, the vast majority of circulating seasonal inﬂuenza strains have been adamantane-resistant (3, 4). More than 98% of these resistant variants bear the same S31N substitution in the M2 protein, which renders this class of drugs virtually useless. In fact, in the 2005–2006 inﬂuenza season, the U.S. Centers for Disease Control instructed physicians not to prescribe adamantanes for prevention or treatment of inﬂuenza after ﬁnding 91% of samples of the dominant circulating strain of human inﬂuenza to be adamantaneresistant (5). Because resistance seems to exist in a relatively homogeneous strain of H3N2 (with the S31N-modiﬁed M2 protein), curtailing the use of these drugs may allow adamantine-susceptible strains to re-emerge (6). Widespread inappropriate use of adamantanes may have contributed to the current situation (6) and needs to be controlled if there is any hope for restoring the utility of these compounds in the future. In addition to the licensed adamantanes, several new adamantine derivatives (7) and more distantly structurally related compounds (8) have in vitro activity against inﬂuenza A, and it is possible that these may be useful against resistant variants. However,

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Figure 1 Mechanism of action of the neuraminidase inhibitors (1). (A) Neuraminidase is essential for the continued replication of inﬂuenza virions. (B) Replication is blocked by the neuraminidase inhibitors, which prevent the release of new virions from the infected cell surface. (From Reference 1 with permission.)

these compounds remain to be studied in animal models and in humans.

NEURAMINIDASE INHIBITORS The NA inhibitors interfere with the release of progeny inﬂuenza viruses from infected host cells in the respiratory tract, preventing infection of new host cells and halting the spread of infection (see Figure 1). Compared to the adamantanes, the two licensed NA inhibitors zanamivir (Relenza) and oseltamivir (Tamiﬂu) are associated with very little toxicity and are less prone to selecting for resistant inﬂuenza viruses (1, 9). The NA inhibitors are effective against all NA subtypes and, therefore against all strains of inﬂuenza, providing another important advantage over the adamantanes, which are only effective against sensitive strains of inﬂuenza A. The two inﬂuenza virus surface glycoproteins, hemagglutinin (HA) and NA, are the antigens that deﬁne the particular inﬂuenza strain (HXNY). The variation of these molecules over time, either through the gradual process of antigenic drift or through acquisition of a totally new HA or NA by means of reassortment, permits the virus to

evade human immune responses. HA binds to cellular molecules bearing sialic acid (Nacetylneuraminic acid) to allow entry of the virus into the target cell, then mediates fusion of viral with cellular membranes in the endosome of the host cell, delivering the viral genome into the cell’s cytoplasm. NA— the target molecule of the NA inhibitor compounds—cleaves cellular receptor sialic acid residues that bind the newly formed virions to the cell and to each other (Figure 1), enabling the spread of infection to new host cells and establishment of ongoing infection. The NA inhibitor molecules mimic the natural substrate of NA, sialic acid (Nacetylneuraminic acid). These small mimics bind to the NA active site, preventing the enzyme from cleaving the sialic acid residues on host cell receptors and thus preventing the release of new viruses. A milestone in rational drug development was reached when the three-dimensional structure of inﬂuenza NA (10) disclosed the location and structure of the catalytic site; this permitted the design of highly effective inhibitors. Zanamivir mimics the natural substrate closely, ﬁtting smoothly into the active site pocket (11–13). Oral inhalation delivers drug directly to the www.annualreviews.org • Medical Management of Influenza

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respiratory tract. Shortly after zanamivir, oseltamivir was developed through modiﬁcations to the sialic acid analog framework, including addition of a lipophilic side chain, that make the drug orally available (14). The NA inhibitors are effective for both treatment and prophylaxis of inﬂuenza A and B infection in humans, and they are effective in vitro and in animal models for treatment of avian inﬂuenza infection. Although the data are inadequate for rigorous assessment of efﬁcacy in the treatment of avian inﬂuenza infection in humans, these drugs are expected to be valuable in the event of an avian inﬂuenza pandemic (15, 16). Structure-based design has made the NA inhibitors highly effective antiviral compounds, but evolution under selective pressure inevitably produces resistant variant viruses. The NA inhibitors are far less likely to promote drug resistance than the adamantanes, and the development of resistance to NA inhibitors had seemed to be of little concern; however, this picture may be changing.

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Efficacy and Timing of Treatment for Seasonal Influenza Zanamivir and oseltamivir are effective for both prophylaxis and treatment of inﬂuenza A and B infection. Several large treatment trials in widely diverse geographical locations showed that when otherwise healthy adults with inﬂuenza were treated with zanamivir or oseltamivir within 36–48 h after onset of illness, a one- to two-day decrease in symptomatic illness occurred (1). Treatment also decreases the number and severity of lower respiratory tract complications and reduces the use of antibiotics (17). However, the positive effects of treatment are greatly enhanced if treatment is started early. Because replication of inﬂuenza virus in the respiratory tract peaks between 24 and 72 h after onset of illness, the earlier the administration of the drug, the shorter the duration of the fever and the faster the return to baseline health (18). Two important studies, in 2003 for inﬂuenza 400

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A (18) and in 2005 for inﬂuenza B (19), directly investigated the relationship between the time to starting oseltamivir therapy and the duration of illness and other efﬁcacy measures. Treatment started within 12 h after onset of fever shortened the illness more than three days compared to treatment started at 48 h, and initiating treatment at intermediate times shortened the illness proportionately (18). Because early administration (0–12 h after onset of symptoms) signiﬁcantly increases the effectiveness of oseltamivir therapy for both inﬂuenza A and B, prompt identiﬁcation of illness and initiation of treatment as early as possible will be essential for producing the best outcomes. In children, both zanamivir and oseltamivir shorten the duration and severity of inﬂuenza symptoms by ∼1.5 days if started within 48 h after onset of illness (20, 21), and presumably would be even more effective if initiated earlier. Oseltamivir may currently be used to treat inﬂuenza in children aged one year and older, whereas the use of zanamivir is reserved for children ﬁve years and older because of the need for an inhaler device. Given the lack of therapeutic options for infants, the evaluation of the pharmacokinetics and tolerance of oseltamivir in infants less than one year old is of critical importance. Of note, however, especially in young children, oseltamivir is much less effective as a treatment for inﬂuenza B than for inﬂuenza A virus infection (22, 23). Zanamivir binds more strongly than oseltamivir in the active site of the inﬂuenza B virus NA (24, 25), and this afﬁnity difference may affect clinical efﬁcacy. The use of oseltamivir against inﬂuenza B infection in young children may need to be reconsidered, and the options of either increasing the dosage of oseltamivir for inﬂuenza B or using zanamivir are worth considering (22, 26).

Prophylaxis for Seasonal Influenza Zanamivir and oseltamivir are both 70%– 90% effective in preventing clinical inﬂuenza in healthy adults, when used either

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as postexposure prophylaxis for close contacts such as household members or as seasonal prophylaxis in the community (1). Oseltamivir may be used for protection of elderly persons in residential institutions, where a 92% reduction in the incidence of inﬂuenza was observed even though most of the elderly residents had been appropriately vaccinated (27). Zanamivir has not been approved for prophylaxis in the nursing home setting as of this writing. NA inhibitors are effective for postexposure prophylaxis in children as young as one year (28), although the inhaler device limits the use of zanamivir to children aged ﬁve years and older. A prospective randomized study of the efﬁcacy of postexposure prophylaxis along with treatment of index cases with oseltamivir (28) showed a reduction in incidence of inﬂuenza disease in children as young as one year of 80% compared to the incidence of inﬂuenza disease if only index cases were treated. These data highlight the importance of recognizing an exposure before viral replication has begun in the pediatric contact. Though not yet licensed for use, the NA inhibitor peramivir (29) is being developed in intravenous and intramuscular formulations. When used orally in humans, the drug was poorly protective, probably owing to poor oral bioavailability (30); however, when given intramuscularly or intravenously in mice, the compound is highly protective against inﬂuenza A infection—both H1N1 (i.e., seasonal) (31) and H5N1 (32). The availability of an intravenous NA inhibitor could be important for patients hospitalized with severe inﬂuenza, and for cases where neither oral nor inhaled routes are an option. In 1999, an intravenous formulation of zanamivir was shown to be safe and effective in prophylaxis for seasonal inﬂuenza A and B (33, 34). Availability of such a formulation of zanamivir would be valuable in the future, especially to deal with issues of oseltamivir resistance in ill patients (see further discussion below). Dimeric derivatives of zanamivir are 100-fold more potent inhibitors of inﬂuenza virus replication in vitro than zanamivir is, and they persist for

long periods in the lungs (35). The development of long-acting NA inhibitors (LANI), now under way, offers the possibility that a drug could be used in a single dose for treatment, or given once a week for prevention of infection (35).

Safety Zanamivir is well tolerated. Equal numbers of patients in drug and placebo groups report adverse effects, primarily minor transient upper respiratory and gastrointestinal complaints. However, in response to postlicensure reports that zanamivir may cause cough, bronchospasm, and a reversible decrease in pulmonary function in some patients (36), it is recommended that if patients with pulmonary dysfunction receive zanamivir they have a fastacting bronchodilator available and discontinue the drug if respiratory difﬁculty develops. However, some convincing data suggest that the recommended dosages of zanamivir do not adversely affect pulmonary function in patients with respiratory disorders (37). Oseltamivir is also well tolerated (28), the most frequent side effects being transient nausea, vomiting, and abdominal pain. These occur in ∼5%–10% of patients, generally only once, soon after initiation of therapy, and resolve spontaneously within 1–2 days (38). Food taken with oseltamivir may reduce nausea and vomiting. Recent reports have suggested an association between oseltamivir use and abnormal behavioral episodes in children and teenagers in Japan (39). In Japan, the use of oseltamivir for children aged 10– 19 years was halted in March 2007 (40), and the government of Korea has also suspended oseltamivir use in teenagers (41). The apparent association is under investigation at the time of this writing, and parents must be informed about this potential issue.

AVIAN INFLUENZA Inﬂuenza pandemics arise when a virus strain emerges with the following characteristics: www.annualreviews.org • Medical Management of Influenza

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it is efﬁciently transmitted between humans, it causes human disease, and most humans have not been previously exposed to it. Although the next pandemic strain may yet surface from an unanticipated source, widespread transmission of H5N1 avian inﬂuenza has focused global attention on the pandemic potential of avian inﬂuenza viruses. Despite several probable incidences of human-to-human transmission (42), as of this writing there is no documented sustained transmission between people. However, the ongoing sporadic human infection rate has steadily increased; in 2006 there were 115 human cases with 79 deaths (43). This situation, along with the virus’ endemicity in birds (44) and the expanding mammalian host range (45), provides the H5N1 virus with many opportunities to adapt to human hosts via mutation in the avian genes, reassortment with mammalian inﬂuenza viruses, or both. Infection with H5N1 in humans causes severe, often fatal disease, with rapid progression to respiratory failure and manifestations of acute respiratory distress syndrome (ARDS) (15). The rapid development of pneumonia is consistent with the ﬁnding that whereas human inﬂuenza viruses preferentially attach initially to host cell sialic acid receptors in the nose and throat, the avian viruses target receptors in the lungs (15). Antiviral drugs will be essential for dealing with a pandemic caused by a new inﬂuenza virus of any origin and will remain important as new vaccination approaches are developed. In the 1968 and 1977 pandemics, adamantanes showed a protective efﬁcacy of ∼70%, only slightly lower than during the interpandemic period (46). The protective efﬁcacy of the NA inhibitors during a pandemic should be at least as high as that of the adamantanes. At present, clade 1 H5N1 viruses are resistant in vitro to the adamantanes but are sensitive to zanamivir and oseltamivir (47, 48). Only some clade 2 H5N1 viruses (primarily Indonesian) are resistant to the adamantanes (49), leaving open the possibility of adamantane use in the future (see comments on com-

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bination therapy below). However, given the rapidity with which adamantine-resistant inﬂuenza A viruses emerge on treatment, the beneﬁt is likely to be short-lived. The NA inhibitors are also effective against NA from the virus that caused the 1918 pandemic (50) and the avian viruses that caused outbreaks in 1997–1999 (51, 52), bolstering their theoretical utility. Because of their likelihood of efﬁcacy and lower risk of resistance, zanamivir and oseltamivir are the drugs of choice for treatment and prevention of avian inﬂuenza. Unfortunately, the use of these drugs for avian inﬂuenza has been anecdotal, and therefore efﬁcacy cannot be accurately estimated. However, oseltamivir protected ferrets (an excellent animal model for human inﬂuenza disease) against fatal H5N1 infection (53). Infection in mice with the highly pathogenic A/Vietnam/1203/04 strain was also effectively treated with oseltamivir, but it required prolonged treatment at a higher dose (54). Zanamivir has also been shown to be effective against highly pathogenic H5N1 in mice (51, 55). The World Health Organization (WHO) has recently developed guidelines for the pharmacological management (treatment and prevention) of avian inﬂuenza A H5N1 virus infections in humans (16). The guidelines highlight the complexity of planning for the treatment and chemoprophylaxis of H5N1 inﬂuenza, given the paucity of clinical data and the urgency of clinical investigation of treatment strategies in affected countries. There are virtually no data to use in developing recommendations for prophylaxis of H5N1 infection in a pandemic setting; thus far, virtually all the human infections have been sporadic and not related to human transmission. Obtaining data on the virologic course, host immune response, and efﬁcacy of antiviral medications in human H5N1 infections is critical. The WHO guidelines recommend that individuals who are perceived to be at risk for serious infection receive chemoprophylaxis with either oseltamivir or zanamivir in the event

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of exposure to H5N1 inﬂuenza. The panel recommends oseltamivir more strongly but acknowledges that there was little evidence on which to base recommendations for either drug. Direct data on oseltamivir treatment were available for 37 cases of human H5N1 infection, and there was no clinical experience with zanamivir in H5N1 infection. Another factor that contributed to the stronger recommendation for oseltamivir is the possibility of extrapulmonary viral replication in humans, and the consideration that systemically administered oseltamivir might have an advantage over inhaled zanamivir. It is unknown whether the presence of extrapulmonary viral genetic material in human H5N1 infection represents ongoing extrapulmonary replication or results from the known high level of pulmonary viral replication. However, the initial site of H5N1 infection and the primary site of replication is the lung mucosa, and zanamivir inhalation provides high levels of drug to the lungs within minutes (56, 57). In addition, once available, the intravenous formulations of zanamivir (33, 34) or peramivir (32) mentioned above could be valuable. Given the emergence of oseltamivirresistant H5N1 viruses that are sensitive to zanamivir (58, 59), the current recommendations may be reconsidered in the future (see comments on resistance below).

RESISTANCE TO NEURAMINIDASE INHIBITORS IN SEASONAL AND AVIAN INFLUENZA Overview of Resistance A strength of the NA inhibitors oseltamivir and zanamivir over the adamantanes is that they are less prone to selecting for resistant inﬂuenza viruses (3). Little spontaneous resistance to NA inhibitors has been documented, no spontaneously resistant inﬂuenza viruses were identiﬁed prior to the introduction of the drugs, and no virus resistant to zanamivir has yet been isolated after the treat-

ment of immunocompetent people. The recent emergence of oseltamivir-resistant variants is therefore a matter of great concern. Beginning in 2004, resistance to oseltamivir in strains of inﬂuenza A has been gradually growing more common (9, 60). Because the clinical relevance and transmissibility of the resistant variants have been unknown, planning for epidemic and pandemic inﬂuenza thus far has discounted the issue of oseltamivir resistance. However, recently, inﬂuenza B viruses with decreased sensitivity to oseltamivir and zanamivir were isolated from individuals in Japan who had not been treated with antiviral medications. The pattern of virus isolation strongly suggested that person-to-person transmission of these resistant viruses had occurred, either within families or in the community (26, 61). The evidence that some NA inhibitor–resistant inﬂuenza virus variants are indeed transmissible and are vigorous pathogens may require a change in treatment guidelines.

Molecular Basis for Resistance Both NA and HA mutations can confer resistance in vitro (62). Whereas resistance due to NA mutations can be detected by phenotypic enzyme inhibition assays, there is as yet no validated assay for identifying HA mutations that confer resistance in humans. Hence, only NA inhibitor–resistant inﬂuenza viruses from humans with mutations in the NA molecule can be identiﬁed. Structural analysis of inﬂuenza NA several years ago predicted that resistance to oseltamivir would be more likely to arise than resistance to zanamivir under the selective pressure of drug treatment (9, 63), and this prediction is now being validated at an escalating pace by clinical data (9, 60, 61). The mechanism of development of resistance is illustrated in Figure 2. As discussed above, the inhibitor molecules mimic NA’s natural substrate and bind to the active site, preventing NA from cleaving host cell receptors and releasing new virus. A rearrangement of amino acids in www.annualreviews.org • Medical Management of Influenza

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Figure 2 Mechanism of resistance to oseltamivir (9). (A) Rearrangement of amino acids in the neuraminidase active site is necessary to accommodate oseltamivir’s hydrophobic side chain. (B) Mutations that prevent this rearrangement may lead to resistance to oseltamivir. The oseltamivir-resistant virus can still bind to the host cell sialic acid receptor and to zanamivir. (C) Analysis of the involved residues of the active site shows that E276 has to rotate and bond with R224 to form a pocket for the larger side chain of oseltamivir, and that the mutations R292K, N294S, and H274Y inhibit this rotation and prevent pocket formation, leading to oseltamivir resistance. None of these mutations prevent the binding of zanamivir or of the natural sialic acid substrate. (From Reference 9 with permission.)

the active site is necessary to accommodate oseltamivir’s hydrophobic side chain (panel A); mutations that prevent this rearrangement may lead to resistance to oseltamivir (panel B). Zanamivir is more structurally similar to the natural substrate of NA and ﬁts directly into the active site, and thus the mutations that prevent the rearrangement would not bring about resistance to zanamivir (panel B). Detailed analysis of the involved residues of the active site (panel C) shows that E276 has to rotate and bond with R224 to form a pocket for the larger side chain of oseltamivir, and that the mutations R292K, N294S, and H274Y inhibit this rotation and prevent pocket formation, leading to 404

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oseltamivir resistance. It has been proposed that oseltamivir-resistance mutations, which do not affect the catalytic residues, are less likely than zanamivir-resistance mutations to have a severe impact on viral ﬁtness (65), and in fact these mutations allow the binding of natural sialic acid substrate (panel B), so that mutated virus can survive and propagate. In contrast, the binding of zanamivir does not require any reorientation of amino acids, so the mutated viruses remain sensitive to that drug. Peramivir, like oseltamivir, requires reorientation of amino acids in order to bind the NA active site (66), and therefore many mutations that confer resistance to oseltamivir or zanamivir also confer resistance to peramivir.

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Resistance in Clinical Isolates The mutations identiﬁed in the resistant inﬂuenza A viruses isolated from humans infected with seasonal or avian inﬂuenza are in the amino acids predicted: R292K, H274Y, and N294S, which prevent the active site pocket formation, and E119V, which interferes with drug binding. A 2004 study of 50 children with H3N2 inﬂuenza treated with oseltamivir in Japan found that nine (18%) harbored viruses with drug-resistance mutations in the NA gene; these mutations were R292K, N294S, and E119V (reviewed in Reference 1). A 2000–2001 Japanese study also identiﬁed resistant inﬂuenza A (H1N1) viruses with the H274Y mutation in 7 of 43 oseltamivir-treated children (16%) (15). Seasonal inﬂuenza virus strains resistant to zanamivir have not been detected in immunocompetent people who were treated with zanamivir, and laboratorygenerated zanamivir-resistant H3N2 strains (with E119G, E119A, or R152K NA mutations) have inefﬁcient NAs that limit their replication efﬁciency and ﬁtness in vitro (65). The majority of the mutations observed in the resistant inﬂuenza B viruses isolated in Japan (61) have been shown to arise in response to the selective pressure of oseltamivir (9) and have led to oseltamivirresistant viruses. However, a separate virus with a novel S250G mutation in the NA and a 15-fold decrease in sensitivity to zanamivir (but still fully sensitive to oseltamivir) was also isolated. More than 90% of all NA inhibitor prescriptions in Japan are for oseltamivir, suggesting that this mutation may have been spontaneous rather than a result of selective pressure imposed by drug treatment. However, because zanamivir is more effective than oseltamivir against inﬂuenza B viruses, the clinical exposure levels of zanamivir would overcome this in vitro efﬁcacy decrease and be well within the range of therapeutic efﬁcacy, even for infections with viruses containing the NA mutations identiﬁed.

The H274Y mutation in NA has now been reported in several H5N1 viruses isolated from infected patients treated with oseltamivir (58, 59). The potentially serious consequences of development of oseltamivirresistant H5N1 inﬂuenza were evident in the report of an ill patient whose clinical course worsened dramatically on treatment, once the resistant variant virus emerged (59). The H274Y mutation does not alter inhibition by zanamivir, and ferrets infected with this variant responded to treatment with zanamivir (66). In December 2006, WHO reported isolation of a strain of avian inﬂuenza from two family members in Egypt with a N294S mutation in the NA gene that conferred moderate resistance to oseltamivir (67) but did not affect sensitivity to zanamivir. The N294S mutation had previously arisen also in a 14-year-old Vietnamese girl exposed to prophylactic doses of oseltamivir (58).

Transmissibility and Medical Relevance of Resistant Isolates The clinical relevance of these resistance mutations and the pathogenicity and transmissibility of these resistant variant viruses are urgent topics for further study. The uncertainty about the importance of NA inhibitor– resistant inﬂuenza has resulted in part from experiments in vitro and in animal models that have generally found NA inhibitor–resistant inﬂuenza viruses to be compromised in infectivity and transmissibility. Such ﬁndings led to the idea that signiﬁcant transmission is unlikely among humans. However, experimental transmission of the virus with a H274Y NA mutation in ferrets showed that the resistant variants grew well in both the index ferret and contact animals, and they were readily transmitted (68). In these particular animal transmission experiments, more variant than wildtype virus was required to infect the animals, and spread to the contact animals was slower, but the virus ultimately grew to the same titers in the contact animals.

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In light of the recent evidence for transmissibility of NA inhibitor–resistant inﬂuenza B (26, 61), the issue of resistance needs to be reevaluated. Resistant strains may have an impact on epidemic or pandemic inﬂuenza. One mathematical model of the spread of resistant viruses in a pandemic concluded that the decreases in biological ﬁtness and transmissibility of NA inhibitor–resistant variants that had been observed at that time would limit community spread of those variants (69), but a more recent modeling study reaches a different conclusion (70). In this model, although antiviral use could signiﬁcantly slow or stop pandemic virus transmission in the absence of drug resistance, the emergence of resistance could severely reduce this beneﬁt. The impact of drug resistance would depend on the degree of reduction in ﬁtness of the speciﬁc resistant virus. If the resistance mutations led to only a modest biological ﬁtness cost and the virus remained highly transmissible, the effectiveness of antiviral use would drop dramatically. Even if resistant strains emerge at extremely low frequencies during antiviral treatment, these strains could contribute signiﬁcantly in an epidemic or pandemic setting. The transmission of resistant inﬂuenza B variants, if indeed it occurred, took longer than that of wild-type inﬂuenza viruses (26). These resistant viruses may thus be somewhat less infectious and require more time to replicate in the host; nonetheless, they eventually cause disease equal in severity to the parent strain.

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DRUG COMBINATIONS AND INVESTIGATIONAL ANTIVIRAL TARGETS Combination Therapy The rapid, nimble evolution of inﬂuenza viruses means that, over time, these viruses may develop resistance to any single antiviral agent, as a result of selective pressure. Treatment with two or more compounds that act at different stages of the viral life cycle would be 406

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more effective and make it less likely for single mutations in the viral genome to confer drug resistance. Current options for combination therapy are few, but this strategy may prove useful as new antivirals are developed. At present, consideration should be given to combinations of the adamantanes with the NA inhibitors for avian inﬂuenza. In vitro, rimantadine acts synergistically with the NA inhibitors to reduce replication of inﬂuenza A (2), and the combination also provides enhanced protection aginst lethality in H5N1infected mice (71). While most clade 1 H5N1 strains are adamantane-resistant, only some clade 2 H5N1 viruses are resistant, and therefore this option may still prove useful.

Sialidase Fusion Protein A recombinant fusion protein consisting of the NA (sialidase) catalytic domain of Actinomyces viscosus fused with a cell surface– anchoring sequence is a promising broadspectrum inhibitor of inﬂuenza virus infection (72). The NA fusion protein is inhaled to remove sialic acid–containing receptors for inﬂuenza from the airway epithelium. FludaseTM DAS181, a sialidase fusion construct now under development, cleaves sialic acid receptors used by both human and avian inﬂuenza viruses, and was highly effective in vitro against a panel of laboratory strains and clinical isolates of inﬂuenza A and inﬂuenza B (72).

HA Inhibitors: Cyanovirin-N The HA surface protein provides an excellent target for antiviral development with the goal of blocking viral entry. The protein cyanovirin-N has been shown to interact with inﬂuenza HA and demonstrates highly potent antiviral activity in vitro against almost all strains of inﬂuenza A and B virus, including clinical isolates and an NA inhibitor–resistant strain (73). HA inhibitors should be pursued further.

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siRNAs Short interfering RNAs (siRNAs) that are speciﬁc for conserved regions of inﬂuenza virus genes, given intravenously to mice in complexes with a polycation carrier, reduced viral replication in the lungs (74). Similar inﬂuenza-speciﬁc siRNAs protected mice from lethal virus challenge with highly pathogenic H5 and H7 avian inﬂuenza A viruses (75). The siRNA strategy holds promise for inﬂuenza as well as for other lethal pathogens, although their mode of delivery in humans has yet to be determined.

Influenza Replication Inhibitors: T-705 and Ribavirin T-705, a substituted pyrazine with potent and selective inhibitory activity against inﬂuenza virus in vitro and in vivo, is active against an NA inhibitor–resistant virus and amantadineresistant viruses (76). It may act by inhibiting the virus RNA polymerase. Ribavirin is a broad-spectrum antiviral agent with activity against inﬂuenza, and it does not seem to generate resistant strains, probably because its target—the cellular enzyme IMP dehydrogenase—is important in the biosynthesis of GTP and viral RNA. This mechanism may also account for ribavirin’s mutagenic potential, which limits its broad use. Viramidine (the prodrug of ribavirin) is active against human and H5N1 avian inﬂuenza (77) and can be administered by intravenous, aerosol, or oral routes; the intravenous and aerosol routes demonstrate efﬁcacy for seasonal inﬂuenza (78) and may be useful in settings of severe disease. Several other promising antiviral targets are under investigation, including signal transduction inhibitors (79, 80), interferon (IFN) inducers (81), and molecules that target the interaction between the inﬂuenza NS1A protein and the 30-kDa subunit of cleavage and polyadenylation speciﬁcity factor (CPSF30). Binding of CPSF30 to NS1A is required for viral replication (82). With the addition of antiviral compounds that target an

array of different stages of the viral life cycle, we will be better equipped to design regimens that avoid emergence of resistant strains of inﬂuenza.

MODULATION OF IMMUNOPATHOLOGIC HOST RESPONSES The primary focus thus far in treatment of inﬂuenza infection has been on the mechanisms of viral infection and replication. Less attention has been directed toward the mechanisms of the inﬂammatory reactions in the lungs that may ultimately lead to severe lung damage and death. Pulmonary inﬂammation is a major feature of the immune response to inﬂuenza virus infection. It promotes the production of proinﬂammatory cytokines and recruitment of macrophages, with production of iNOS and resulting nitric oxide (NO) synthesis following cytokine release. Mouse model experiments have shown that overproduction of free radicals including NO contribute to the development of pneumonia during inﬂuenza virus infection (83, 84). Excess NO generation in response to inﬂuenza virus infection could lead to the observed pulmonary damage in humans as well. It is plausible that therapeutic strategies that inhibit iNOS expression may ameliorate the severe effects of infection in the lungs. In fact, it has been suggested that the NA inhibitors suppress NO production in response to IFN-γ stimulation or inﬂuenza infection in cultured macrophages, and that the NA inhibitors possess antiinﬂammatory effects (suppression of NO production) that are distinct from their suppression of viral replication (85). Intriguing recent data on the use of statins suggest that their anti-inﬂammatory and immunomodulatory effects may reduce pathogenesis in diseases associated with unchecked immune response to infections (86), and may in fact lower the risk of death from seasonal inﬂuenza (87), although speciﬁc evidence for a direct effect of statins on inﬂuenza mortality is lacking. www.annualreviews.org • Medical Management of Influenza

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During the inﬂuenza pandemic of 1918, more than half the deaths occurred among largely healthy people between 18 and 40 years of age and are thought to have been caused by a virus-induced cytokine storm that led to the acute respiratory distress syndrome (ARDS) (88, 89). The key element in generating the storm has been proposed to be an uncontrolled immune response to the virus, accompanied by an outpouring of proinﬂammatory cytokines and chemoattractants (89). Recent experiments in macaques have shown that atypical expression of the innate immune antiviral response is a critical determinant of the severity of disease caused by the 1918 inﬂuenza virus (90). In human H5N1 inﬂuenza infection, high chemokine and cytokine levels, along with high pharyngeal viral loads, have recently been found to correlate with fatal outcomes, suggesting that the high viral load and consequent intense inﬂammatory

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responses contribute to the pathogenesis of severe H5N1 disease (59). The rapid escalation in the inﬂammatory response occurred before antiviral therapy could provide protection, supporting the notion that early initiation of antiviral therapy for avian inﬂuenza is essential (59). It is timely to study the application of immunomodulatory or anti-inﬂammatory agents to medical management of inﬂuenza disease, in order to prevent the intense cytokine response and the oxidative/nitrative stress that appear to contribute directly to fatal outcome. These modalities may then be applied in the future in conjunction with antiviral drugs that target several different stages of the viral life cycle and avoid promoting the development of resistance. We may prevail by addressing both the viral and the host components of disease pathogenesis in the treatment of inﬂuenza.

DISCLOSURE STATEMENT The author is a consultant and/or advisor to Roche Laboratories Inc., GlaxoSmithKline, and Medimmune.

ACKNOWLEDGMENTS I thank Jennifer McKimm-Breschkin, Matteo Porotto, and Michael Sporn for helpful discussions.

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:397-413. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Bacterial and Fungal Bioﬁlm Infections Annu. Rev. Med. 2008.59:415-428. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

A. Simon Lynch and Gregory T. Robertson Cumbre Pharmaceuticals Inc., Dallas, Texas 75235-2304; email: [email protected], [email protected]

Annu. Rev. Med. 2008. 59:415–28

Key Words

First published online as a Review in Advance on October 15, 2007

antibiotic, indwelling medical device, antibiotic tolerance, persistent infection, staphylococcal infections, antibiotic resistance

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.110106.132000 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0415$20.00

Abstract Bioﬁlms are communal structures of microorganisms encased in an exopolymeric coat that form on both natural and abiotic surfaces and have been associated with a variety of persistent infections that respond poorly to conventional antibiotic chemotherapy. Bioﬁlm infections of certain indwelling medical devices by common pathogens such as staphylococci are not only associated with increased morbidity and mortality but are also signiﬁcant contributors to the emergence and dissemination of antibiotic resistance traits in the nosocomial setting. Current treatment paradigms for bioﬁlm-associated infections of semipermanent indwelling devices typically involve surgical replacement of the device combined with long-term antibiotic therapy and incur high health care costs. This review summarizes the existing data relating to the nature, prevalence, and treatment of bioﬁlm-associated infections and highlights experimental approaches and therapies that are being pursued toward more effective treatments.

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INTRODUCTION IMD: indwelling medical device

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FBI: foreign body infection

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In their natural environments, most bacterial and fungal species alternate between planktonic (free-living) and sessile states in response to environmental stimuli such as the availability of essential nutrients. Transition to growth in a sessile state frequently occurs in response to nutrient limitation and is thought to reﬂect a developmental switch to a perseverance mode wherein a biopolymer-encased, structured community of cells—a bioﬁlm— forms on a biotic or abiotic surface. Because the availability of nutrients often parallels the abundance of competing organisms, the developmental switch to the bioﬁlm state is commonly regulated by inter- and intraspecies quorum-sensing mechanisms. The formation and maturation of a bioﬁlm involve a number of transitional states that span the initial attachment phase and the terminal dispersal phase (1) and have been characterized through genome-wide expression proﬁling and proteomic analysis. Such efforts have identiﬁed a number of proteins that play specialized roles in the bioﬁlm phase and have revealed more comprehensive changes in gene expression that reﬂect a downshift in overall metabolic activity consistent with enhanced perseverance (2). At the phenotypic level, a microbial bioﬁlm can also be characterized as a communal population of cells that exhibits tolerance (or insensitivity) to antimicrobial agents that are active on the cells of the bioﬁlm once dispersed into their planktonic state. In situations where the planktonic susceptibility of a pathogen to an antibiotic is known, this type of deﬁnition may be informative in predicting whether an ongoing infection may involve a bioﬁlm component. In addition to the phenotypic response of bioﬁlms to antibiotic challenge, these multicellular structures also exhibit a genotypic response that is of clinical signiﬁcance in the development and spread of antibiotic resistance traits in nosocomial pathogens. This genotypic response can include the de novo

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emergence of antibiotic resistance through elevated mutation rates, as well as an enhanced propensity of cells in bioﬁlms to exchange genetic elements bearing antibiotic resistance traits through intra- and interspecies transfer mechanisms (3, 4).

BIOFILM-ASSOCIATED INFECTIONS: DISEASE STATES, CAUSATIVE PATHOGENS, AND IMPACT ON HEALTH CARE COSTS Deﬁnitive studies to satisfy Koch’s postulates regarding the essentiality of bioﬁlms as speciﬁc etiological agents of disease are lacking (2). In light of the inherent difﬁculties in establishing an unambiguous role for bioﬁlminduced pathogenesis, a minimal set of criteria was recently proposed for determining a bioﬁlm etiology of an infection. These criteria include the presence of substratum-adherent, matrix-encased clusters of bacteria that exhibit phenotypic resistance to antibiotics active on the composite planktonic cells (5). Overall, the most persuasive existing evidence for a speciﬁc role for bioﬁlms in human disease pathogenesis comes from direct observational studies wherein microscopic methods have been employed to identify bioﬁlm structures either at infection sites in situ or on infected tissues or devices freshly recovered from patients.

Foreign Body Infections Infections that are thought to involve bioﬁlm foci are most frequently associated with microbial colonization of the abiotic surface(s) of indwelling medical devices (IMDs) and other materials and are commonly referred to as foreign body infections (FBIs). Microscopic examination of IMDs extracted from patients, including those with FBIs refractory to antibiotic treatment, has revealed the presence of bioﬁlm-like structures formed by bacteria and fungi in a number of studies (2). Although

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clinical bioﬁlms are typically of lower cell density than those grown in vitro, their overall architecture as revealed by electron microscopic methods is similar. These studies have also provided evidence for interactions of the bioﬁlm and host tissues. In the United States, device infections associated with low attributable mortality have been estimated to have initial rates of infection that vary from 1%–3% for mammary implants, penile implants, and joint prosthesis to 10%–30% for urinary catheters (2, 6, 7). IMDs associated with infections of moderate attributable mortality include vascular grafts (1%–5%), cardiac pacemakers (1%–7%), and central venous catheters (CVCs) (3%–8%). Infections associated with cardiac assist devices at the time of placement arise in 25%–50% of cases and attributable mortality can exceed 25% (6). However, these estimates are derived from cases of infections documented by appropriate diagnostic cultures and are probably conservative, partly owing to the limited sensitivity of available diagnostic and clinical microbiology techniques (7). Further, IMDs represent a risk factor for the hematogenous seeding of infections throughout the lifetime of the device in place, and the use of prophylactic antibiotic therapy is routine in high-risk patients prior to invasive dental or surgical procedures. In most instances, infection is diagnosed by clinical symptoms and/or bacteremia, or through other nonsurgical cultures of local body ﬂuids; however, such methods often yield false negative results (7). Sonication and polymerase chain reaction (PCR) ampliﬁcation methods have been experimentally shown to improve the detection of bioﬁlms on explanted orthopedic implants (8), but widespread use of such approaches would require specialized equipment and techniques not common in clinical laboratories. Other nonmicrobiologic diagnostic options, such as advanced imaging techniques, have been used in the medical diagnosis of probable bioﬁlm infections of surgical IMDs. For example, transesophageal echocardiography has been

employed to detect bioﬁlms associated with endocarditis on prosthetic heart valves (2), while ultrasonography, computed tomography (CT), and magnetic resonance imaging (MRI) have been applied with varying success to the diagnosis of deep arterial prosthetic infections (9). Although multiple radiologic techniques, particularly radioisotopic labeling with indium and technetium, have been shown to aid in the diagnosis of prosthetic joint infections and other forms of osteomyelitis, they can give false positive results more than a year after IMD implantation (10). Overall, much work remains in overcoming diagnostic challenges associated with bioﬁlm infections of surgical implants. For nonsurgical IMDs, such as CVCs and urinary catheters, bioﬁlm colonization may originate either from the skin at the point of insertion, or by migration of the organism(s) through or around the catheter once implanted (2). For surgical IMDs, tissue damage and clot formation associated with surgical implantation are correlated with enhanced rates of microbial bioﬁlm colonization (2). Delayed or late-onset orthopedic IMD infections tend to be caused by low-virulence pathogens, such as coagulase-negative staphylococci (CoNS) and Propionibacterium acnes, and may result from hematogenous seeding of the implant following other invasive procedures or infections (11). Table 1 lists the principal IMDs whose function can be compromised by bioﬁlm colonization and the prevalent causative pathogens associated with the resulting FBIs. In many instances, colonization precedes the clinical manifestation of infection and is often sufﬁcient to adversely affect the mechanical function of the device. Prominent pathogens most often associated with bioﬁlm infections of IMDs are either normal commensal ﬂora or are nosocomial in origin. Prevalent Grampositive cocci often include CoNS, reﬂecting both their ubiquity as skin ﬂora and their adherence to IMD surfaces, host tissues, and ﬁbronectin (2, 12), and Staphylococcus aureus, which exists both as nasopharyngeal www.annualreviews.org • Biofilm Infections of Humans

CVC: central venous catheter CoNS: coagulase-negative staphylococci

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Table 1

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Indwelling medical devices commonly associated with biofilm infections Prevalent causative pathogens

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Device

Principal

Secondary

central venous catheters

CoNS

S. aureus, enterococci, Candida spp., K. pneumoniae, P. aeruginosa

urethral catheters

E. coli

Candida spp., CoNS, E. faecalis, P. mirabilis

mechanical heart valves

CoNS

S. aureus, Streptococcus spp., GNB, enterococci, diptheroids

ventricular assist devices

CoNS

S. aureus, Candida spp., P. aeruginosa

coronary stents

S. aureus

CoNS, P. aeruginosa, Candida spp.

neurosurgical ventricular shunts

Staphylococci

Streptococcus spp, Corynebacterium, GNB

peritoneal dialysis catheters

S. aureus

P. aeruginosa, other Gram-negative spp., Candida spp.

orthopedic prostheses

Staphylococci

S. pneumoniae, Streptococcus spp., P. acnes

fracture-ﬁxation devices

CoNS

S. aureus, Propionibacterium spp., Corynebacterium, Streptococcus spp.

endotracheal tubes

Enteric GNB

P. aeruginosa, Streptococcus spp., Staphylococcus spp.

inﬂatable penile implants

CoNS

S. aureus, enteric GNB, P. aeruginosa, Serratia spp., fungi

breast implants

Staphylococci

E. coli, peptostreptococci, C. perfringens, P. acnes

cochlear implants

S. aureus

P. aeruginosa, Streptococcus spp., N. meningitidis, fungi

Abbreviations: CoNS, coagulase-negative staphylococci; GNB, Gram-negative bacilli.

MSCRAMM: microbial surface components recognizing adhesive matrix molecules CRBSI: catheter-related bloodstream infection

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ﬂora and as a nosocomial pathogen. Owing to their ability to produce multiple toxins and degradative enzymes, S. aureus bioﬁlm infections of IMDs tend to be more acute, promoting signiﬁcant tissue damage and host immune response. In contrast, CoNS infections tend to follow a more chronic disease progression (2, 11). Two stages of bioﬁlm colonization have been proposed for the staphylococci: (a) the attachment phase, in which cells ﬁrst associate with the IMD or with host-derived products such as ﬁbronectin or ﬁbrinogen that initially coat the device; and (b) the accumulation phase, in which cell-cell adhesion occurs, and the hallmark encasement of the nascent bioﬁlm in an extracellular polymeric substance ensues (12). These two phases are genetically separable, and multiple gene products have been identiﬁed in laboratory strains as contributors to robust bioﬁlm formation in vitro, including ﬁbrinogen and ﬁbronectin binding proteins (MSCRAMM family adhesins), capsular polysaccharide adhesion factors, autolysin, teichoic acids, and polysaccharide intercellular adhesin (12). In contrast, knowledge of factors involved in bioﬁlm-speciﬁc disease Lynch

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progression in clinical isolates is generally lacking. Among fungal pathogens, Candida albicans, a commensal mucosal organism and opportunistic pathogen of the immunocompromised, is most commonly associated with bioﬁlm colonization of IMDs, and the resultant candidiasis is associated with high mortality (13–16). Other opportunistic bioﬁlm-forming Candida species also associated with catheter-related bloodstream infections (CRBSIs) include C. parapsilosis, C. tropicalis, C. lusitaniae, C. krusei, and C. glabrata (15, 17). Bioﬁlm colonization of ventricular shunt catheters, peritoneal dialysis ﬁstulas, and cardiac valves by Crytococcus neoformans has also been reported and is of particular concern in light of the growing use of ventriculo-peritoneal shunts to manage intracranial hypertension associated with cryptococcal meningoencephalitis in immunocompromised patients. Recurrent meningitis in patients with ventriculo-peritoneal shunts has also been associated with bioﬁlm colonization by Coccidioides immitis. Endocarditis associated with infections of prosthetic valves and other cardiac devices by Aspergillus species is a growing concern in immunocompromised

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patients, although a deﬁnitive role of bioﬁlms has yet to be established. Overall, although Candida and Aspergillus species are the etiologic agents in only ∼8% of implant infections, they are emerging as formidable pathogens with a patient survival rate in some settings as low as 50% (18). The isolation of multiple discrete species from bioﬁlm-colonized implants derived from patients provides the most compelling evidence of the medical importance of polymicrobic (or heterotypic) bioﬁlms (19). Studies of polymicrobic bioﬁlms formed by C. albicans and S. epidermidis indicate that the exopolymeric matrix produced by the fungal species may protect the bacteria against antibiotics, while the bacterial matrix protects the fungi from antifungal action. Other studies of polymicrobic bioﬁlms have provided evidence of enhanced interspecies transfer of antimicrobial resistance traits, symbiotic interactions, and sequential colonization patterns. These observations raise the intriguing possibility that virulence traits associated with bioﬁlm commensalism may be coselected through enhanced persistence and antimicrobial tolerance of such polymicrobic bioﬁlms (20, 21).

Native Tissue Infections Although bioﬁlm-related infections are most often associated with colonization of abiotic surfaces of IMDs, there is a growing body of evidence that bioﬁlm colonization of natural surfaces may also be an obligatory component of some infections that involve no foreign body. These include urinary tract infections caused by uropathogenic Escherichia coli (22) and middle ear infections mediated by Haemophilus inﬂuenzae, Streptococcus pneumoniae, and Moraxella catarrhalis, including chronic otitis media (23), as well as more persistent and chemotherapeutically challenging infections including chronic rhinosinusitis, cystic ﬁbrosis mediated by P. aeruginosa (24), and native valve endocarditis caused by Staphylococcus lugdunensis, Enterococcus durans,

or Viridans group Streptococci. In such infections, antimicrobial therapy will continue to be driven by empiric observations. However, increasing acceptance of the potential role for bioﬁlms in chronic infections with no known foreign body component, particularly those that fail to respond to conventional therapy, should promote studies to identify antimicrobial therapies with improved bioﬁlm efﬁcacy in these settings.

Impact on Health Care Costs The overall health care costs attributed to the treatment of bioﬁlm-related infections have been systematically assessed in two recent reviews (6, 25). For infections of some uncomplicated nonsurgical IMDs (e.g., urinary catheters), the economic impact is quite low owing to the ease of implant extraction or replacement, although stress on the patient and the burden on the hospital staff are important considerations. Bacteremia resulting from CRBSIs in hemodialysis-dependent adults, however, is a much more serious problem, with a signiﬁcant attributed cost (i.e., $17,000 and $32,000 for uncomplicated and complicated bacteremia, respectively) and a high probability of secondary complications including infective endocarditis, osteomyelitis, septic arthritis, septic emboli, and stroke (26, 27). Similarly, costs associated with infection of surgical implants range from $15,000 per event for orthopedic fracture-ﬁxation devices to more than $50,000 to replace an infected mechanical heart valve (25). Clearly, we need new treatment options for the management of bioﬁlm-associated infections that will simplify surgical practices and/or shorten typical hospital stay requirements.

MECHANISMS UNDERLYING THE ANTIMICROBIAL TOLERANCE OF BIOFILMS Inadequate exposure of antimicrobial agents at all relevant sites of infection is one of the principal factors underlying www.annualreviews.org • Biofilm Infections of Humans

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chemotherapeutic failure and presumably limits the efﬁcacy of some agents in deep-seated bioﬁlm-associated infections. Assuming adequate exposure at sites of bioﬁlm infections, subsequent trapping of antimicrobial agents in the exopolymeric matrix of bioﬁlms is often cited as a universal mechanism underlying the insensitivity of bioﬁlms to antimicrobial action. It is true that the penetration of experimental bioﬁlms by some antibiotics, including vancomycin and ampicillin, is limited; however, other agents (e.g., tobramycin and ciproﬂoxacin, rifampin, and a variety of antifungal agents) exhibit good apparent diffusion through bioﬁlms grown in vitro. In some cases, effective diffusion may be mediated by water-ﬁlled channels that are an architectural feature of bioﬁlms. However, studies to assess the actual intracellular penetration of antimicrobials into cells within the bioﬁlm matrix are lacking. Overall, the existing evidence does not suggest that exopolymeric matrix materials form a universal barrier that limits antimicrobial penetration to cells residing within a bioﬁlm. For bacteriostatic classes of antibiotics, which typically work in concert with host immune defenses, the location of bacteria within the exopolymeric coating of the bioﬁlm structure is thought to effectively sequester cells from host immune cells and factors. This may in part explain their poor efﬁcacy in this setting (28). However, no deﬁnitive studies have addressed whether there is a systematic difference between the efﬁcacies of bacteriostatic and bactericidal agents in bioﬁlm-associated infections in vivo. Interpretation of such a study would need to take into account differences in the exposure of different agents at the bioﬁlm infection site(s). The elevated expression of efﬂux pumps is another mechanism whereby cells can exhibit decreased susceptibility to antibiotics. The speciﬁc upregulation of genes encoding antibiotic transporters (or their regulators) has been observed in studies of bioﬁlms formed by Pseudomonas aeruginosa (29), uropathogenic

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E. coli (30), and Candida albicans (31, 32). However, studies of global changes in gene expression associated with bioﬁlm adaptation suggest that upregulation of antibiotic transporters is not a universal theme. Of course, this mechanism is restricted to antimicrobial agents that are substrates for efﬂux transporters. Adaptation to survival in the bioﬁlm state requires changes in metabolic and catabolic pathways that can alter the intrinsic activity of antimicrobial agents in light of their mode of action (2). For example, experimental bioﬁlms formed by CoNS are highly resistant to antibiotics that target cell wall biosynthesis while remaining susceptible to antibiotics that target RNA and protein synthesis (33). Such a response is consistent with a diminished role for cell wall biosynthesis in the bioﬁlm population and reﬂects an ongoing role for transcription and translation in bioﬁlm establishment, maturation, and propagation. It is interesting that so-called small colony variants (SCVs) of bacteria, characterized by a reduced in vitro growth rate owing to genotypic changes in metabolic pathways (34), have been isolated from both experimental bioﬁlms and patients with persistent infections that are probably bioﬁlm-associated. These include P. aeruginosa SCVs isolated from bioﬁlms grown in vitro and from cystic ﬁbrosis patients (35), an E. coli SCV isolated from a chronic prosthetic hip infection (36), and S. aureus SCVs from patients with cystic ﬁbrosis, osteomyelitis, and device-related infections (37). Hence, genotypic adaptations that alter metabolic capacity and decrease growth rate may in some instances contribute to both antibiotic tolerance and the persistence of some bioﬁlmrelated infections. Physiologic heterogeneity is another feature that distinguishes bacteria cultivated in a bioﬁlm state from their planktonic counterparts. This phenomenon affects rates of growth and metabolism and is thought to reﬂect a combination of interbacterial quorumsensing signals, accumulation of toxic byproducts, and dissimilar local microenvironments

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(e.g., different nutrient or oxygen gradients) that arise in the three-dimensional bioﬁlm community. Such phenotypic heterogeneity has been linked to the decreased susceptibility of a subset of cells in the bioﬁlm to speciﬁc antimicrobials. These so-called persister cells are not resistant to the antimicrobial per se but instead appear to escape killing through what is hypothesized to be a transient dormant state (38). The molecular events that lead to the antibiotic tolerance of such persister variants are still poorly understood, but speciﬁc genetic loci have been identiﬁed that appear to affect their rate of formation. In summary, although the antibiotic tolerance of both medical and experimental bioﬁlms is a well-established phenotype, the molecular mechanisms that govern this phenomenon are complex and appear to vary across species, antibiotic classes, and settings.

EXISTING THERAPIES Prophylaxis Prophylactic use of antibiotics and microbicides can reduce the incidence of bioﬁlmassociated infections of IMDs. Strategies for prophylaxis include device coatings, device immersion, surgical site irrigation, antibioticloaded cements, and antibiotic lock therapy. In the latter, a concentrated antibiotic solution is instilled into a catheter in a volume adequate to ﬁll the lumen. The catheter is then “locked” into place for an extended period while the catheter is not in use, with the goal of preventing a line from becoming colonized and thereby reducing the risk of infection. Although the use of antibiotic prophylaxis is controversial because of its potential to increase antimicrobial resistance, it is increasingly common in high-risk patient groups. With regard to device coatings, a recent and comprehensive meta-analysis of randomized controlled trials of rifampin-impregnated CVCs suggests they are both safe and effective in reducing the rate of catheter colonization and CRBSIs (39). Similarly, in six inde-

pendent studies of the efﬁcacy of antibiotic lock therapy in the prevention of CRBSIs in hemodialysis patients, an overall reduction of 64%–100% in CRBSIs was apparent (40).

Treatment: Surgical Intervention Combined with Antimicrobial Therapy Replacement or removal of an infected IMD, combined with systemic antibiotic and/or antifungal therapy, is the most effective treatment in most settings. Standard practice involves either a one-stage or a two-stage procedure (25, 41, 42). For managing IMD infections in nonsurgery patients, long-term antimicrobial suppressive therapy remains the only option, and current salvage rates are highest with early diagnosis (43). Recommendations of antibiotic therapies for the management of bioﬁlm-associated infections have been driven largely by empiric observations and typically involve the use of combination regimens over extended periods (25, 41, 42). Recent reviews summarize current recommended practices for the treatment of infections of prosthetic joints (10, 41, 44), arterial prostheses (9), vascular catheters (45), prosthetic heart valves (46, 47), central nervous system shunts (48), pacemakers and deﬁbrillators (49), endotracheal and tracheostomy tubes (50), and hemodialysis and peritoneal hardware (51), as well as treatment of FBIs of the urinary tract (52). In most settings, the preferred treatment option(s) have arisen through cross-study comparisons of the cure rates or other clinical outcomes achieved for particular treatment courses. Relatively few clinical studies have directly compared alternate therapeutictreatment regimens. In the clinical evaluation of antibiotic regimens, draft guidelines for the design and conduct of efﬁcacy studies in the United States are available only for the CRBSI setting, and direct comparator studies have rarely been undertaken in any IMD infection setting. However, of all agents studied, the utility of rifampin combination therapies for www.annualreviews.org • Biofilm Infections of Humans

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staphylococcal IMD infections has perhaps been most thoroughly evaluated in clinical trials. In these studies, rifampin was combined with quinolones (53–57), beta-lactams (53) or fusidic acid (58), and rifampin-containing regimens are now established as standard therapies for a range of device-associated infections (10, 11, 41, 43, 46, 48). Fungal bioﬁlm-associated infections are notoriously difﬁcult to treat systemically with antifungal agents. This poor in vivo efﬁcacy is not surprising, given the in vitro insensitivity of bioﬁlms formed by C. albicans and other species to various classes of antifungal agents (13, 15, 16). However, agents of the echinocandin class exhibit signiﬁcantly superior in vitro activity in killing fungal cells in established bioﬁlms (14) and in suppressing bioﬁlm colonization of materials (59). Echinocandins have also demonstrated activity in a rabbit model of an intravascular catheter infection (60). These activities may reﬂect a specialized role for glucan synthesis, the cellular pathway targeted by echinocandins, in the bioﬁlm state or the resulting increased osmotic instability of echinocandintreated cells. Lipid-based formulations of amphotericin B have also proven effective in in vitro assays of bioﬁlm activity. The management of fungal bioﬁlm-associated IMD infections involves exchange of the infected device, where possible, combined with systemic antifungal therapy (16). Data from in vitro studies and animal studies of bioﬁlm efﬁcacy, combined with the observed clinical success in management of disseminated candidiasis, suggest that antifungals of the echinocandin class (caspofungin, micafungin, anildafungin) and amphotericin B lipid formulations represent the best available options for the management of bioﬁlm-associated infections with a known or suspected fungal involvement.

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IMDs are listed in Table 2. These include a wide range of modiﬁed devices and surgical materials, most of which are intended for prophylaxis applications. They utilize new materials that resist colonization by microbial pathogens and novel device-coating approaches wherein antibiotics, microbicides, or quorum-sensing inhibitors are either covalently bound to the device or locally eluted from it. In the latter category are the so-called intelligent implants, which are designed to locally release agents when they detect microbial colonization (61). Finally, IMDs that emit low-energy surface acoustic waves (62), electric currents (63), or pulsed ultrasound (64) have been reported to either reduce device colonization or enhance the release and/or effectiveness of locally applied antibiotics. In addition to these device modiﬁcations, new studies have evaluated the effectiveness of new antibiotic or microbicide immersion practices with IMDs to suppress surgical-site infections. Table 3 lists new investigational and recently approved antibiotics that have been evaluated for antibioﬁlm activity through in vitro studies and/or animal models to assess their clinical potential in both prophylaxis and treatment. In the prophylaxis setting, agents of the lipopeptide and lipoglycopeptide classes show promise as potential new antibiotic lock therapies, which may reﬂect their common mechanistic feature as rapidly bactericidal agents (65). In the treatment setting, only dalbavancin has been evaluated in a speciﬁc human comparator trial, where it was found to exhibit efﬁcacy superior to that of vancomycin in treating CRBSIs (66). In studies of refractory Gram-positive infections in orthopedic patients, quinupristin-dalfopristin (67) and linezolid (68) have been reported to have clinical cure rates of 78% and 55%, respectively, although long-term use of these agents has been associated with signiﬁcant adverse events. In light of the paucity of effective agents for the treatment of bioﬁlm-associated infections, systematic studies have been undertaken to

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Experimental approaches for the treatment or prophylaxis of biofilm-associated infections

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Approach

Mode of action

Key reference

pilicides

inhibits bacterial pilus biogenesis and surface attachment

71

RNA III inhibiting peptide (RIP)

disrupts quorum-sensing pathways in staphylococci

72

acyl-homoserine lactone mimetics

disrupts quorum-sensing pathways

73

furanones

disrupts quorum-sensing pathways

74

omigard (omiganan cationic peptide)

topical gel for prophylaxis settings including CRBSIs

http://www.cadencepharm.com

aganocides

hypochlorous acid–based compounds

http://www.novacal.com

ceragenins

depolarizes membrane potential; device coatings

http://www.ceragenix.com

lysostaphin

prevents or disrupts staphylococcal bioﬁlms

http://www.biosynexus.com

device coatings

controlled release of antimicrobials from device surfaces

http://www.surmodics.com

hydrogel coatings

controlled release of silver compounds

http://www.bardmedical.com

surface acoustic waves

disrupts device adhesion and colonization

62

pulsed ultrasound

enhances local release of antibiotic from cements

64

electric direct current

prevents or disrupts bioﬁlm colonization

63

intelligent implants

MEMS-based release of antimicrobial(s) from reservoir

61

gallium compounds

antimicrobial potentiator via disruption of iron metabolism

75

http://www.bacterin.com

Abbreviations: CRBSI, catheter-related bloodstream infection; MEMS, microelectromechanical systems.

identify the most promising antibiotic combinations in both in vitro assays and animal models of bioﬁlm efﬁcacy (69). Of all agents tested, rifampin was again the most common constituent of combinations active against Table 3

staphylococcal bioﬁlms, reﬂecting this agent’s excellent efﬁcacy against slow-growing and adherent staphylococci and its excellent tissue penetration. However, because of the signiﬁcant resistance liability associated with

Biofilm-related activity of approved and investigational antimicrobial agents

Antimicrobial

Description

Biofilm-related activity

Key reference

dalbavancin

lipoglycopeptide

CRBSI treatment

66

daptomycin

lipopeptide

bioﬁlm reduction as lock solution in CVC model

65

right-sided endocarditis

77

poor in vitro activity on adherent staphylococci

78

linezolid

oxazolidinone

effective in combination with rifampin

76

quinupristin-dalfopristin

streptogramins

bone and joint infections

67

bioﬁlm reduction as lock solution in CVC model

79

telavancin

lipoglycopeptide

bioﬁlm reduction in sorbarod model

80

tigecycline

glycylcycline

bioﬁlm reduction in silicone disk model

76

effective in combination with rifampin

81

optimized for bioﬁlm activity

70

CBR-2092

rifamycin-quinolone hybrid

Abbreviations: CRBSI, catheter-related bloodstream infection; CVC, central venous catheter. www.annualreviews.org • Biofilm Infections of Humans

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all approved or investigational rifamycins, these agents are limited to use in combination with an appropriate partner (11, 70). Most recently, a series of stable rifamycinquinolone hybrid antibiotic agents have been reported (70) that were optimized for antimicrobial activity and resistance prevention properties against staphylococci in the bioﬁlm setting.

CONCLUSION Annu. Rev. Med. 2008.59:415-428. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Microbial bioﬁlms have gained recognition as the etiologic agents of many chronic and persistent infections of native tissues and IMDs. Owing in part to their adherent nature and slow growth, such infections are difﬁcult to diagnose and treat with conventional approaches. As the use of IMDs increases, the incidence of bioﬁlm-related infections represents a current and growing unmet medical need. With current technologies and treatment options, prophylaxis to prevent or limit bioﬁlm colonization of IMDs appears to be the single most effective approach. A number of national bodies have adopted more stringent barrier control policies to limit bacterial contamination of IMDs at the time of insertion. Routine use of antimicrobials in this setting remains controversial because compelling evidence of their effectiveness is lacking and their application is likely to further exacerbate the overall problem of antibiotic resistance in the nosocomial setting. Therefore, the prophylactic use of existing antibiotics for treatment of sys-

temic infections will probably continue to be discouraged. In the immediate future, the application of noninvasive imaging technologies, combined with a wider recognition of the role of bioﬁlms in IMD infections, is likely to lead to more rapid diagnosis of bioﬁlm-related infections and consequent earlier adoption of the best available treatment options. For the longer term, systematic studies to discover or optimize antimicrobial agents that either exhibit improved killing of organisms in the bioﬁlm state or potentiate the bioﬁlm activity of existing antimicrobial agents hold promise. However, the existing regulatory climate does not provide a clear path toward the design and implementation of clinical trials to evaluate the efﬁcacy of antibiotics (or antibiotic potentiators) in bioﬁlm-related infection settings, and there is limited current evidence of the pursuit of this approach in the pharmaceutical industry. Industry efforts in this area may be encouraged by revision of the existing clinical study guidelines to address inclusion and exclusion criteria and efﬁcacy endpoints for bioﬁlm-related infections such as CRBSIs. With regard to efﬁcacy endpoints, the use of biomarkers and/or advanced imaging technologies may be considered once they are appropriately validated in animal models of bioﬁlm-related disease. Finally, a better understanding of host-pathogen relationships and further elucidation of basic bioﬁlm biology may reveal new biomarkers with utility in diagnostic settings and new bioﬁlm-speciﬁc targets amenable to chemotherapeutic or immune intervention approaches.

DISCLOSURE STATEMENT The authors are employees of Cumbre Pharmaceuticals Inc., which has a rifamycin-quinolone hybrid antibiotic agent (CBR-2092) in clinical development that is intended for the treatment of bioﬁlm infections.

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66. Raad I, Darouiche R, Vazquez J, et al. 2005. Efﬁcacy and safety of weekly dalbavancin therapy for catheter-related bloodstream infections caused by gram-positive pathogens. Clin. Infect. Dis. 40:374–80 67. Drew RH, Perfect JR, Srinath L, et al. 2000. Treatment of methicillin-resistant Staphylococcus aureus infections with quinupristin-dalfopristin in patients intolerant of or failing prior therapy. J. Antimicrob. Chemother. 46:775–84 68. Razonable RR, Osmon DR, Steckelberg JM. 2004. Linezolid therapy for orthopedic infections. Mayo Clin. Proc. 79:1137–44 69. Saginur R, Stdenis M, Ferris W, et al. 2006. Multiple combination bactericidal testing of staphylococcal bioﬁlms from implant-associated infections. Antimicrob. Agents Chemother. 50:55–61 70. Combrink KD, Lynch AS. 2007. New rifamycins for the treatment of bacterial infections. Expert Opin. Ther. Patents 17:475–85 71. Pinkner JS, Remaut H, Buelens F, et al. 2006. Rationally designed small compounds inhibit pilus biogenesis in uropathogenic bacteria. Proc. Natl. Acad. Sci. USA 103:17897–902 72. Anguita-Alonso P, Giacometti A, Cirioni O, et al. 2007. RNAIII-inhibiting peptide-loaded polymethylmethacrylate prevents in vivo Staphylococcus aureus bioﬁlm formation. Antimicrob. Agents Chemother. 51:2594–96 73. Muh U, Hare BJ, Duerkop BA, et al. 2006. A structurally unrelated mimic of a Pseudomonas aeruginosa acyl-homoserine lactone quorum-sensing signal. Proc. Natl. Acad. Sci. USA 103:16948–52 74. Hume EB, Baveja J, Muir B, et al. 2004. The control of Staphylococcus epidermidis bioﬁlm formation and in vivo infection rates by covalently bound furanones. Biomaterials 25:5023– 30 75. Kaneko Y, Thoendel M, Olakanmi O, et al. 2007. The transition metal gallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobial and antibioﬁlm activity. J. Clin. Invest. 117:877–88 76. Raad I, Hanna H, Jiang Y, et al. 2007. Comparative activity of daptomycin, linezolid and tigecycline against catheter-related methicillin-resistant Staphylococcus bacteremic isolates embedded in bioﬁlm. Antimicrob. Agents Chemother. 51:1656–60 77. Fowler VG Jr, Boucher HW, Corey GR, et al. 2006. Daptomycin versus standard therapy for bacteremia and endocarditis caused by Staphylococcus aureus. N. Engl. J. Med. 355:653–65 78. Blaser J, Vergeres P, Widmer AF, et al. 1995. In vivo veriﬁcation of in vitro model of antibiotic treatment of device-related infection. Antimicrob. Agents Chemother. 39:1134–39 79. Giacometti A, Cirioni O, Ghiselli R, et al. 2005. Comparative efﬁcacies of quinupristindalfopristin, linezolid, vancomycin, and ciproﬂoxacin in treatment, using the antibioticlock technique, of experimental catheter-related infection due to Staphylococcus aureus. Antimicrob. Agents Chemother. 49:4042–45 80. Gander S, Kinnaird A, Finch R. 2005. Telavancin: in vitro activity against staphylococci in a bioﬁlm model. J. Antimicrob. Chemother. 56:337–43 81. Yin LY, Lazzarini L, Li F, et al. 2005. Comparative evaluation of tigecycline and vancomycin, with and without rifampicin, in the treatment of methicillin-resistant Staphylococcus aureus experimental osteomyelitis in a rabbit model. J. Antimicrob. Chemother. 55:995–1002

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:415-428. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch Massachusetts General Hospital Cancer Center, Boston, Massachusetts 02114; email: [email protected]

Annu. Rev. Med. 2008. 59:429–42

Key Words

First published online as a Review in Advance on August 20, 2007

EGFR mutations, personalized medicine, non–small cell lung cancer, geﬁtinib, erlotinib

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.090506.202405 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0429$20.00

Abstract Drugs that target the epidermal growth factor receptor (EGFR) have had a major impact on the treatment of non–small cell lung cancer (NSCLC). The use of these drugs has also motivated pivotal advances in the understanding of the molecular biology of NSCLC, including the discovery that mutations in EGFR are associated with dramatic and sustained responses to anti-EGFR treatments. This review summarizes the clinical development of EGFR tyrosine kinase inhibitors, the discovery of molecular predictors of response, and the future directions for research in the ﬁeld.

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INTRODUCTION Non–small cell lung cancer (NSCLC) is the leading cause of cancer death in the world. It is one of the most lethal types of cancer; a diagnosis of NSCLC portends only a 15% chance of surviving ﬁve years. Nearly half of patients have advanced-stage, incurable cases at the time of diagnosis. In addition, the majority of those with potentially curable NSCLC at diagnosis will relapse with incurable disease within a few years. Cytotoxic chemotherapy has traditionally been the foundation of treatment for advanced NSCLC, but even the

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TKI: tyrosine kinase inhibitor

EPIDERMAL GROWTH FACTOR RECEPTOR Four transmembrane receptor tyrosine kinases (RTKs) make up the EGFR family, including EGFR (or ErbB-1), HER-2 (or ErbB-2), ErbB-3, and ErbB-4 (reviewed in Reference 3). Each ErbB RTK binds a panel of activating ligands except ErbB2/HER-2, which has no known ligand and acts primarily as a dimerization partner for other ErbB RTKs. Upon ligand binding, ErbB receptors form homo- or heterodimers, promoting their reciprocal activation through trans-phosphorylation of distinct intracellular tyrosine residues. The pattern of phosphorylation creates docking sites for the recruitment of diverse effector proteins, leading to the generation of intracellular signal transduction cascades and a variety of cellular responses, including proliferation and survival (antiapoptosis). ErbB-3 lacks intrinsic TK activity, but nonetheless, it can transmit a potent signal following heterodimerization with ErbB-2/HER-2. Hence, the diversity and redundancy within the ErbB family lends ﬂexibility to the transmission and ampliﬁcation of growth signals. The speciﬁc biologic endpoint of the EGFR signaling cascade depends on the cellular context, including the stimulating ligand, the RTK dimerization partner, and the kinetics of the signaling pathways within the cell. Aberrant EGFR activation can promote abnormal cell proliferation by transmitting mitogenic signals. These may result from activating mutations in the RTK, ampliﬁcation of the RTK, autocrine secretion of ligand by tumor cells, or paracrine production by surrounding stromal cells (4–6). In vitro and mouse model experiments have demonstrated the potent transforming ability of such aberrant EGFR signaling in multiple cell types, including NSCLC (7).

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most aggressive regimens offer only about a 20%–35% chance of tumor response and an improvement in survival by an average of 2– 4 months (1, 2). Needless to say, the treatment of NSCLC is an area of medicine in which there is substantial room for improvement in our therapeutic armamentarium. Since their introduction into clinical trials seven years ago, small molecules that inhibit signaling from the epidermal growth factor receptor (EGFR) have greatly inﬂuenced the treatment of NSCLC. Perhaps more importantly, investigations into their mechanism of action have had a profound impact on our understanding of the biology of NSCLC. EGFR inhibitors were originally investigated as therapeutic agents that might be useful for all patients with NSCLC, but their clinical development led to the identiﬁcation of a novel subpopulation of NSCLC patients, and revolutionized the way the disease is conceptualized. This article reviews the still unfolding story of EGFR tyrosine kinase inhibitors (TKIs) in lung cancer.

CLINICAL DEVELOPMENT The EGFR signaling network plays a central role in the growth and maintenance of epidermal derived tissues, and alterations of this network can lead to malignant transformation (see sidebar). As in other epithelial tumors, EGFR expression is detectable in most NSCLC cases, and it is present in premalignant bronchial epithelium as well as in up to 90% of tumors of the squamous cell subtype of NSCLC and 30%–65% of tumors of the adenocarcinoma subtype (8, 9). Based on its central role in cellular growth and its ubiquitous expression, EGFR was posited as a priority target for the development of anti-NSCLC treatments (10). Small molecules were developed to inhibit EGFR signaling by blocking the intracellular TK domain. The ﬁrst two of these EGFR TKIs, geﬁtinib (Iressa) and erlotinib (Tarceva), made their way into clinical trials in the early 2000s. Both were orally administered

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agents that bound at the catalytic cleft of EGFR in competition with adenosine triphosphate (ATP), causing suppression of receptor phosphorylation and downstream signaling (11–13). Broad-based phase I studies showed responses in NSCLC patients and prompted disease-speciﬁc investigations. Phase II studies in NSCLC patients demonstrated an objective response to single-agent TKI therapy in 10%–18% of chemotherapy-pretreated patients (see IDEAL and Perez-Soler studies in Table 1) (14–16). Side effects from treatment with either geﬁtinib or erlotinib were moderate and well tolerated, consisting mainly of acneiform rash, diarrhea, and fatigue, although rare but serious cases of interstitial pneumonitis were observed (17). The manufacturers of geﬁtinib applied to the US Food and Drug Administration (FDA) for a labeling indication as third-line therapy in NSCLC, after treatment with approved ﬁrst- and secondline chemotherapeutics. Based on its phase II demonstration of activity and the “orphan indication” of third-line treatment for NSCLC (i.e., a setting without any other approved treatments), geﬁtinib obtained FDA approval in 2003. During the FDA approval process, geﬁtinib was available at many academic cancer centers as part of a “compassionate use” program of expanded access. The observations made by the geﬁtinib and erlotinib phase II trialists as well as many groups participating in the geﬁtinib expanded access programs turned out to be quite revealing. Females, those with adenocarcinoma histology (and speciﬁcally having bronchioloalveolar cell features), those with minimal to no history of smoking, those of East Asian origin, and those suffering rash while on treatment were more responsive to EGFR TKI therapy (14–16, 18– 21). These characteristics have turned out to be good clinical or phenotypic markers of a genetically deﬁned subgroup of patients with NSCLC, and their early identiﬁcation as a potentially interesting population enabled the subsequent discovery of molecular markers.

The ﬁrst type of randomized study to be performed with EGFR TKIs in NSCLC tested TKIs in combination with standard ﬁrst-line platinum-based chemotherapy against the same chemotherapy with placebo. There were four such studies performed, two with geﬁtinib and two with erlotinib (INTACT-1 and -2, TRIBUTE, and TALENT; see Table 1) (22–25). Altogether >4000 patients participated in these trials, and unfortunately they were all negative, failing to show any survival beneﬁt with the addition of an EGFR TKI to upfront chemotherapy. Interestingly, in a subgroup analysis from one of these studies, the TRIBUTE trial of carboplatin and paclitaxel with erlotinib or placebo, 116 patients who reported that they had never smoked had an impressive prolongation in survival with erlotinib treatment (22.5 months) compared to placebo treatment (10.1 months, p = 0.01) (24). However, the overall negative outcome of the four large trials raised some doubt and controversy within the ﬁeld as to the best use of scarce patient resources and the most successful strategy for developing promising novel targeted agents in NSCLC. As a discipline, should we strive to deﬁne a responding population early in the development of a novel agent, and test for efﬁcacy in that population speciﬁcally? Or rather, given the heterogeneity observed in NSCLC and the incomplete or even incorrect information obtained from preclinical studies about the mechanism of action of many novel agents, should we continue testing new drugs in a broad population and rely on post hoc subgroup analyses to point out clinical populations that beneﬁt the most from therapy? If any of the large ﬁrst-line randomized trials with chemotherapy and TKIs had been performed in a restricted population of light or never-smokers based on the phase II data that suggested this population may have a higher response rate, would it have demonstrated a survival beneﬁt and would TKI-containing regimens now be considered a standardof-care treatment in the ﬁrst line for select patients? Ultimately, the optimal drug

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Table 1

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Major clinical trials examining gefitinib and erlotinib in NSCLC patients

Study

Phasea

IDEAL-1 (14)

R-II

IDEAL-2 (15)

Study population

n

Treatment

Response rate (%)

One-year overall survival (%)

Median survival (months)

p –

prior platinum

103

geﬁtinib 250 mg/day

18

35

7.6

106

geﬁtinib 500 mg/day

19

29

8.0

R-II

prior platinum

106

geﬁtinib 250 mg/day

12

27

7.0

115

geﬁtinib 500 mg/day

9

24

6.0

Perez-Soler (16)

II

prior platinum

57

erlotinib 150 mg/day

12

40

8.4

–

INTACT-1 (22)

R-III

treatment naive

365

cisplatin, gemcitabine, and geﬁtinib 500 mg/day

50

43

9.9

NSb

365

cisplatin, gemcitabine, and geﬁtinib 250 mg/day

51

41

9.9

363

cisplatin, gemcitabine, and placebo

47

44

10.9

347

carboplatin, paclitaxel, and geﬁtinib 500 mg/day

30

37

8.7

345

carboplatin, paclitaxel, and geﬁtinib 250 mg/day

30

41

9.8

345

carboplatin, paclitaxel, and placebo

29

42

9.9

539

carboplatin, paclitaxel, and erlotinib 150 mg/day

22

47

10.6

540

carboplatin, paclitaxel, and placebo

20

44

10.5

533

cisplatin, gemcitabine, and erlotinib 150 mg/day

32

41

9.9

536

cisplatin, gemcitabine, and placebo

30

42

10.1

prior platinum

1129

geﬁtinib 250 mg/day

8

27

5.6

563

placebo

1

21

5.1

prior platinum

488

erlotinib 150 mg/day

9

31

6.7

243

placebo

1

22

4.7

INTACT-2 (23)

TRIBUTE (24)

TALENT (25)

ISEL (26) BR.21 (27)

a b

R-III

R-III

R-III

R-III R-III

treatment naive

treatment naive

treatment naive

For phase of study, II = phase II, R-II = randomized phase II, R-III = randomized phase III. NS, not signiﬁcant.

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–

NS

NS

NS

NS <0.001

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development algorithms are often more obvious in retrospect. At the current time, upfront treatment of advanced NSCLC with TKI agents plus chemotherapy is not recommended outside the setting of a clinical trial. The second type of randomized study to be performed with EGFR TKIs in NSCLC tested TKI monotherapy against placebo in the second- or third-line setting. Again, this design was duplicated with both geﬁtinib and erlotinib (see ISEL and BR.21 studies in Table 1) (26, 27). For the ﬁrst time, the results of similarly designed trials were divergent between the two drugs. The BR.21 study showed a survival advantage for erlotinib treatment: Median survival was prolonged from 4.7 months on placebo to 6.7 months on erlotinib, and there was a corresponding hazard ratio for death of 0.7 ( p < 0.001). The ISEL study of geﬁtinib, in contrast, did not show a survival advantage, with median survivals of 5.1 months on placebo and 5.6 months on geﬁnitib. It remains unclear why BR.21 demonstrated a survival beneﬁt with erlotinib, whereas ISEL failed to show such beneﬁt with geﬁtinib. One theory is that the drugs were administered at doses with differing levels of biological activity. In the phase I clinical development of geﬁtinib, 150 mg/day was sufﬁcient to suppress EGFR signaling in skin biopsies, and in the phase II experience, doses above 250 mg/day did not appear more effective (14, 15, 28). Hence, the decision was made to apply for FDA registration and conduct further studies of geﬁtinib with a dose of 250 mg/day, although the maximum tolerated dose was between 750 and 1000 mg/day (28). In contrast, erlotinib was studied at its maximum tolerated dose of 150 mg/day in all phase II and III trials (29). This distinction could be relevant because, as discussed below, distinct molecular subtypes of NSCLC may require differing biological doses of EGFR blockade to achieve cytotoxicity. Alternatively, the different outcomes in the BR.21 and ISEL trials might reﬂect divergent populations enrolled in the two trials, with respect to the clinical

and/or molecular characteristics of NSCLC that responds favorably to EGFR TKI therapy. It is also possible that the two drugs actually differ in their second- or third-line efﬁcacy. The manufacturers of erlotinib applied for FDA approval of this agent as a secondor third-line treatment for advanced NSCLC based on the results of the BR.21 study, and erlotinib was approved for this indication in 2004. Around the same time, based on the negative results of ISEL, the FDA restricted the use of geﬁtinib to patients participating in a clinical trial or those who had previously beneﬁted from the drug.

EGFR MUTATIONS Three academic groups were inspired by the observation that a subpopulation of NSCLC patients preferentially beneﬁted from TKI therapy, and they searched for the molecular reasons for this phenomenon. In 2004 they reported, nearly simultaneously, the discovery of a collection of somatic mutations in the kinase domain of EGFR that correlated with a high likelihood of response to EGFR TKIs (30–32). Many of the patients harboring these mutations not only responded favorably to EGFR TKIs but also had clinically remarkable results, with rapid, nearly complete reduction of their cancers that lasted, in some cases, for several years. EGFR mutations were much more commonly found in NSCLC patients already clinically identiﬁed as TKI-responsive, i.e., females, never-smokers, Asians, and those with adenocarcinoma histology. The incidence of EGFR mutations in Asians is ∼25%–50%, in contrast to ∼10% in North American and Western European patients, and the strongest predictor of mutation status appears to be absent or low smoking history (33, 34). Approximately 90% of observed EGFR mutations are one of two types: small, inframe deletions in exon 19 clustered around the catalytic site of the receptor, or the single point mutation L858R, which lies within the TK activation loop in exon 21 (30–32, 35–38).

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Functionally, these mutations appear to preserve the ligand dependence of receptor activation but alter the pattern of downstream signaling. Speciﬁcally, downstream activation of antiapoptotic signals (via Akt) is greatly enhanced in cells harboring mutated EGFR compared to wild type, whereas the mutation has minimal effect on proliferative signals (via MAPK/ERK) (39, 40). NSCLC cells with an EGFR mutation also demonstrate enhanced inhibition of biochemical signaling by smallmolecule TKIs compared to wild-type receptors (30, 39). The location of the mutations within critical residues of the catalytic domain, ﬂanking the ATP-binding site, may lead to altered physical structure and enhanced drug binding (41). This is clinically signiﬁcant because complete abrogation of signaling by mutated EGFR may be achieved at relatively low doses of TKIs, whereas complete suppression of the wild-type receptor may require higher plasma drug levels. Other types of EGFR TK domain mutations have been reported, including exon 20 insertions, exon 18 point mutations, and exon 20 point mutations. It is not yet clear whether these rare mutations are TKI-sensitizing like the more common exon 19 deletions and L858R substitution. On the contrary, at least some of the minor mutations are associated with resistance to TKI agents (42–45). There are at least two hypotheses regarding the mechanism by which EGFR mutations lead to rapid and dramatic clinical responses to EGFR TKI therapy. The “oncogene addiction” hypothesis states that because the cancer developed in a setting in which the mutated receptor was constantly transducing high levels of antiapoptotic, i.e., prosurvival signals, the molecular biology of the tumor cell became solely dependent on this signaling and lost its ﬂexibility to adapt to signaling via other parallel pathways (46, 47). In this model, cells harboring EGFR mutations are “addicted” to EGFR prosurvival signaling, and therefore, sudden interruption of EGFR signaling by TKIs causes massive cell death. The “oncogenic shock” hypothesis, a vari-

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ation on oncogene addiction, states that although prosurvival signals dominate in cells with EGFR mutations, EGFR-generated proapoptotic signals are still present in some quantity (48). When receptor signaling is blocked by TKIs, both types of signals are arrested. However, because prosurvival signals decay much more rapidly than proapoptotic signals, a temporary predominance in proapopotic signaling occurs. This sets in motion the irreversible apoptotic cascade, committing the cell to death. Oncogenic shock implicates an active apoptotic pathway as opposed to a passive process. A large number of retrospective series (reviewed in Reference 33) have conﬁrmed that the response rate to TKI treatment in EGFR mutation–positive patients is ∼75% (range 30%–100%, with most series reporting response rates >60%), compared to 10% in wild-type cases (range 0%–33%). These results are notable, given that the typical response rate to combination chemotherapy in NSCLC is ∼20% (1), and highlight the potential impact of using a targeted therapy in a targeted population. Several studies have also demonstrated that patients with EGFR mutations survive longer with TKI therapy than do wild-type patients, with median survivals of up to 30 months (37, 49–55). Emerging data suggest that different types of EGFR mutations may respond to TKIs differently; the exon 19 deletion seems to be associated with a stronger response and longer survival than the L858R substitution (56, 57). Thus far, four phase II studies have been reported using EGFR mutation screening to prospectively deﬁne a population of patients with newly diagnosed advanced NSCLC for ﬁrst-line EGFR TKI therapy. Inoue and colleagues in Japan screened 75 patients, identiﬁed 25 (33%) with exon 19 deletions or L858R point mutations (hereafter termed “classic mutations”), and treated 16 (64%) of these with upfront geﬁtinib (58). A response rate of 75% and a median progression-free survival (PFS) of 9.7 months were observed. Another Japanese group similarly screened 82

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patients, identiﬁed 20 (24%) with classic mutations, treated 16 (80%) with ﬁrst-line geﬁtinib, and achieved a response rate of 75% and a median PFS of 8.9 months (59). Paz-Ares and colleagues in Spain have presented preliminary ﬁndings from 428 patients screened for classic EGFR mutations (60). They identiﬁed 67 (19%) patients harboring mutations, treated 40 (60%) with ﬁrst-line erlotinib, and observed a response rate of 82% and a PFS of 13.3 months. Our own group recently presented the ﬁndings from the ﬁrst such study to be performed in North American patients (60a). We screened 98 patients in a multicenter study and found 34 (35%) with any type of EGFR mutation (i.e., atypical mutations were also allowed). Thirty-one patients were treated with ﬁrst-line geﬁtinib, and we observed a response rate of 55% and a PFS of 11.4 months. Of note, none of the patients with atypical mutations responded, although some did achieve prolonged stable disease. These studies conﬁrm that treatment of EGFR mutation–positive patients with ﬁrstline EGFR TKI therapy can produce durable responses in a high proportion of patients and represents one of the most promising advances in the ﬁeld of lung cancer in recent years. However, it remains important to validate this approach with randomized trials in a population of patients harboring EGFR mutations, as it appears that EGFR mutations are not only predictive of response to EGFR TKIs, but also prognostic and indicate an overall more indolent natural history (61–63). Several such genotype-directed randomized studies are being planned or are under way.

EGFR GENE AMPLIFICATION AND INCREASED GENE COPY NUMBER A second EGFR biomarker that can predict beneﬁt from EGFR TKIs is EGFR ﬂuorescent in situ hybridization (FISH) status. This composite marker identiﬁes an overall increase in the number of copies of the EGFR

gene. FISH+ status is deﬁned as having classical gene ampliﬁcation (tight gene clusters and a ratio of EGFR gene to chromosome of ≥2 or ≥15 copies per cell) and/or chromosome polysomy (≥4 chromosomes in ≥40% of cells) (50). FISH status was ﬁrst examined by Capuzzo and colleagues in a cohort of Italian patients, in which 33% of cases were noted to be FISH+; these patients achieved a higher response rate to geﬁtinib therapy (36%) than did FISH– patients (3%; p < 0.001) and had a longer median survival (18.7 months versus 7.0 months; p = 0.03). Within the same cohort, 17% of cases were EGFR mutation– positive, which was associated with a response rate of 53% compared to 5% in wild-type cases ( p < 0.001), and with a trend toward improved survival. Since the initial report (50), several studies have compared EGFR gene copy number to EGFR mutation status with respect to their relative predictive abilities (see Table 2) (50, 55, 63–67). Some of these studies have used the previously deﬁned EGFR FISH status to measure gene copy number (GCN), and others have used quantitative real-time polymerase chain reaction (qPCR), which measures the absolute number of copies of EGFR. It is not clear which method is superior or which is more indicative of a biologically relevant alteration. The results of the comparison analyses are varied and therefore somewhat difﬁcult to interpret; this is an area of much controversy within the ﬁeld. Although most studies have found EGFR mutation a reliable predictor of response to EGFR TKI therapy, many studies also show that EGFR GCN can predict response. Two studies have concluded that GCN predicts survival whereas mutations do not, and three studies have concluded that mutations predict survival whereas GCN does not. The ethnicity of the patients could be a factor, as all studies that favor mutations as predictors of survival were performed in Asian patients and those that favor GCN were performed in North American or European patients. The groups that examined tissue samples from prospective large clinical

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Table 2 Studies that compared the biomarkers EGFR mutation status and EGFR gene copy number in predicting clinical benefit from EGFR TKI therapya No. in overall study

No. mutation +/ no. assessed for mutations (%)

RR in mutation + pts (%)

MS in mutation + pts (mo)

Method of assessing GCN

No. GCN+/ no. assessed for GCN (%)

RR in GCN + pts (%)

MS in GCN + pts (mo)

Capuzzo/retro (50)

102

15/89 (17)

53

20.8

FISH

33/102 (32)

36

18.7

Bell/IDEAL (63)

425

14/79 (18)

46

∼8b

qPCR

7/90 (8)

29

∼8b

2130

32/312 (10)

72c

14.6c

qPCR

33/453 (7)

56c

11.5c

Takano/retro (55)

66

39/66 (59)

82

20.4

qPCR

29/66 (44)

72

16.4

Han/retro (64)

69

15/69 (22)

53

not reached

FISH

31/66 (47)

32

12.3

Tsao/BR.21 (65)

731

40/170 (29)

16

∼6b

FISH

56/125 (45)

20

∼10b

Hirsch/ISEL (66)

1692

26/215 (12)

38

–b

FISH

114/370 (31)

–b

∼8b

59

17/59 (29)

59

18.9

FISH

26/54 (48)

31

6.4

Author/study

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Bell/INTACTc (63)

Sone/retro a

Abbreviations: pts, patients; RR, response rate; MS, median survival; mo, months; GCN, gene copy number; retro, retrospective collection of patients; FISH, ﬂuorescent in situ hybridization; qPCR, quantitative real-time polymerase chain reaction. Results in boldface reached statistical signiﬁcance for the biomarker-positive group receiving EGFR TKI therapy compared to the same metric for the biomarker-negative group receiving EGFR TKI therapy (for References 50, 55, 63, 64) or when compared to the same metric for the biomarker-positive group receiving placebo treatment (for References 65, 66). b Not directly reported in manuscript. c These patients received chemotherapy plus EGFR TKI.

trials were very limited in that only a small minority of participating patients had sufﬁcient samples for multiple biomarker analyses. For example, in the BR.21 randomized study of erlotinib versus placebo, 731 patients enrolled in the overarching study, but Tsao and colleagues had access to tissue from only 19 (0.03%) EGFR mutation–positive patients and only 25 (0.03%) FISH+ patients treated with erlotinib (65). Furthermore, the extent of similarity between the populations described by these biomarkers remains unclear, as several groups have reported that mutation status is positively correlated with increased GCN (50, 55, 64, 66).

RESISTANCE Unfortunately, virtually all patients who respond to EGFR TKI treatments will ulti436

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mately develop resistance and suffer a clinical relapse. Understanding the mechanisms of resistance and developing new therapies to overcome them is an area of active research. About half of the cases of acquired TKI resistance are attributed to a secondary mutation in EGFR, the point mutation T790M in exon 20 at the “gatekeeper” threonine residue (42, 43). The gatekeeper threonine residue is highly conserved across the oncogenic receptor TKs EGFR, KIT, and ABL, and mutations at this location often lead to kinase-targeted drug resistance (68). In particular, the T790M mutation in EGFR introduces a bulky methionine residue that blocks the ATP-catalytic pocket and sterically hinders the binding of geﬁtinib or erlotinib. Ampliﬁcation of MET has recently been identiﬁed as a second mechanism of EGFR TKI resistance; it probably offers a parallel pathway by which intracellular

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proliferation signals can be activated, thereby obviating the effect of EGFR TKI-mediated blockade (69). Other proposed mechanisms of developing resistance to TKIs include signaling via parallel redundant pathways, constitutive activation of downstream mediators, altered receptor trafﬁcking, efﬂux of the drug from the cell, and mutation of the drug target itself (70–72). Second-generation EGFR TKIs and additional related compounds are being developed to combat T790M and other mechanisms of acquired resistance to geﬁtinib and erlotinib. The second-generation EGFR TKIs tend to employ at least one of the following two strategies to increase efﬁcacy over ﬁrst-generation compounds. First, they may form covalent, irreversible bonds with EGFR, which may increase effectiveness by prolonging the inhibition of EGFR signaling to the entire lifespan of the drug-bound receptor molecule. In cell culture models, irreversibly binding TKIs can effectively kill cells that have acquired resistance to ﬁrst-generation TKIs (71). Second, new drugs target multiple kinases within the cancer cell. The ErbB family signaling network is vast, with a large degree of built-in cross-talk and redundancy (see sidebar, above). The diversity of possible signal transduction routes affords the cell ﬂexibility but, in the case of cancer cells treated with targeted anticancer agents, also allows the emergence of resistant clones that bypass the inhibited receptor (72). Blocking multiple signaling pathways with either a combination of agents or a single multitargeted drug has been promising in preclinical and early clinical work (reviewed in Reference 73). Secondary targets being combined with EGFR inhibition by novel NSCLC drugs include HER-2 and the vascular endothelial growth factor receptor (VEGFR).

CONCLUSIONS AND FUTURE DIRECTIONS The EGFR TKIs geﬁtinib and erlotinib are important and active oral treatments for

NSCLC. Their clinical development sparked a sequence of fascinating discoveries that broadened our understanding of the molecular biology of NSCLC and of the previously underappreciated biological heterogeneity of the disease. Most importantly, we now know that EGFR mutations deﬁne a subgroup of NSCLC patients whose tumors are addicted to EGFR signaling, and these patients are likely to gain signiﬁcant clinical beneﬁt from treatment with EGFR TKIs. The EGFR story has realized the possibility of personalized medicine in epithelial cancers, that is, developing drugs speciﬁcally for use in a molecularly deﬁned subgroup of patients, emulating prior landmark work in patients with chronic myelogenous leukemia (74). Current clinical practice is to use the ﬁrst-generation EGFR TKIs, geﬁtinib and erlotinib, as second- or third-line therapy in advanced NSCLC. However, their utility is being actively investigated in the upfront setting and as part of adjuvant treatment for earlier stages of disease in clinically and molecularly selected subpopulations of NSCLC patients. The optimal method of selecting patients for early EGFR TKI use has yet to be elucidated; the appropriate criterion may be EGFR mutation status, EGFR gene ampliﬁcation, another biomarker, or a combination of biomarkers. In addition to determining which of these selection methods is most effective in clinical decision making, we need to consider cost and ease of use as practical issues for these and all emerging biomarkers. As discussed above, one of the obstacles in uncovering the true predictive nature of any biomarker is that archived tissue from patients participating in clinical trials is very limited and often represents a small minority of the study cohort. NSCLC is particularly challenging in terms of obtaining tumor tissue in sufﬁcient quantity for multiple molecular analyses because the most common biopsy approach (ﬁne-needle aspiration) yields a very limited amount of tissue, and more substantial biopsies require open, more invasive techniques. It is therefore a priority to design

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clinical trials using targeted agents in a way that ensures the availability of sufﬁcient tissue from most, or ideally all, participating patients for correlative molecular analyses. Mandatory collection of tissue specimens from all patients will ensure that as new discoveries are made, complete cohorts of patients can be evaluated for the presence or absence of critical

biomarkers. In addition, the ability to examine EGFR and other biomarkers in peripheral blood is being evaluated as a minimally invasive alternative to core tumor tissue biopsies. A new era of molecular diagnosis and genotype-driven treatments is dawning, and we have to design our drugs, our clinical trials, and our diagnostic technologies accordingly.

DISCLOSURE STATEMENT Annu. Rev. Med. 2008.59:429-442. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

T.L. has served as a consultant (less than $20,000 per year) to AstraZeneca, Genentech, Wyeth, Boehringer Ingelheim, Genzyme, and Xelexis. L.S. has served as a consultant to Genentech; she has received research funding from Genentech and AstraZeneca.

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:429-442. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

Annu. Rev. Med. 2008.59:429-442. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori1 and Ree Dawson2 1

Department of Health Research and Policy, Stanford University School of Medicine, Stanford, California 94305-5405; email: [email protected]

2

Frontier Science Technology and Research Foundation, Boston, Massachusetts 02115; email: [email protected]

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Key Words

First published online as a Review in Advance on October 3, 2007

treatment policy, dynamic treatment

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.062606.122232 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0443$20.00

Abstract An adaptive treatment strategy (ATS) is a rule for adapting a treatment plan to a patient’s history of previous treatments and the response to those treatments. The ongoing management of chronic disease deﬁnes an ATS, which may be implicit and hidden or explicit and well-speciﬁed. The ATS is characterized by the use of intermediate, early markers of response to dynamically alter treatment decisions, in order to achieve a favorable ultimate outcome. We illustrate the ATS concept and describe how the effect of initial treatment decisions depends on the performance of subsequent decisions at later stages. We show how to compare two or more ATSs, or to determine an optimal ATS, using a sequential multiple assignment randomized (SMAR) trial. Designers of clinical trials might ﬁnd the ATS concept useful in improving the efﬁciency and ecological relevance of clinical trials.

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DEFINITION OF “ADAPTIVE TREATMENT STRATEGY”

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ATS: adaptive treatment strategy

An adaptive treatment strategy (ATS) is a rule for adapting a treatment plan to the changing state of an individual patient, taking into account both the history of previous treatments and the response to those treatments (1–4). For example, the clinical management of HIV infection may begin with a particular combination of antiviral medications, and then as the patient’s viral load and CD4 count change over time, the combination may be changed or other treatments may be instituted. A clinician treating a patient with any chronic disorder for an extended period of time may be following an ATS, which may be more or less explicit. By contrast, a single treatment choice, such as whether to start a selective serotonin reuptake inhibitor (SSRI) in a ﬁrst episode of major depressive disorder, may be viewed as an isolated event when taken out of the context of the future treatment plans that will depend on the patient’s response to this ﬁrst treatment decision. This article is an early review of the state of the art of ATS from the perspective of a methodologist interested in the speciﬁcation, assessment, comparison, and optimization of ATSs.

WHY CONSIDER A STRATEGY AS A WHOLE? The reason for considering an ATS as a whole instead of focusing on the individual treatment decisions that comprise it is that the overall consequences of a current treatment choice may depend on the performance of future treatments that are applied after the results of the current treatment are manifested. The optimal choice of current treatment may need to take those future treatments into account. For example, a highly toxic cancer chemotherapy A may produce higher rates of response than a less toxic version B, but many of the failures that occur on B might yet be reversed by A after B. If so, then the 444

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ATS “ﬁrst B, and then if failure, A” may be chosen over the ATS “ﬁrst A, and then if failure, B,” perhaps after consideration of differential treatment mortality and morbidity. Such tradeoffs pervade clinical decisions, including the choice of frontline antibiotic therapy, initial antidepressant therapy, sequencing of cancer chemotherapy, and many others; ATS formalizes these frequently ad hoc treatment sequences. One very general trend in medical care is the gradual substitution of perhaps somewhat less effective but gentler treatments early in the course of clinical management. This substitution is based on the knowledge or hope that, with better follow-on or “rescue” treatments, one can afford to give up some immediate beneﬁt in order to avoid side effects, or other costs, without sacriﬁcing ultimate beneﬁts. An important feature of any ATS is that the individual decisions at each stage must be based on knowledge available at that stage. It would be ideal to know the results of a blood glucose management decision in terms of the clinical outcomes of blindness, cardiovascular events, and quality-adjusted life-years, but it is necessary to depend on a measurable clinical indicator, such as the area under the glycosylated hemoglobin curve over recent time, or some other summary of the current state of glycemic control. Thus, the ultimate outcome “ﬁgure of merit” might not be purely a function of the states used to drive treatments. For example, at one point in an ATS for treating lymphoma, the decision for hematopoeitic stem cell transplant might depend on risk factors for relapse following complete remission, but the outcome is overall or progression-free survival. Despite the ubiquity of two- or threestage ATSs of the kind described above, the evidence base for comparing different versions seldom comes from direct studies of entire ATSs. We show how to compare two or more ATSs, or more generally, how to determine an optimal ATS if it is possible to do all the necessary clinical studies. Then we consider methods for doing the right clinical

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Table 1

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Notation for an adaptive treatment strategy

S1 , S2 , . . . , SK A1 , A2 , . . . , AK S1 , A1 , S2 , A2 , . . . , SK , AK , Y d1 , . . . , d K d 1 (S1 ) d 2 (S1 , A1 , S2 ) d k (S1 , A1 , . . . , Ak−1 , Sk ) E [Y(d )] E [Y(d )] − E Y(d )

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states used as inputs to the adaptive treatment decisions treatment options at each stage the order of observation of states and treatments; Y is the overall outcome the decision rules at each stage the initial treatment, given the ﬁrst state the second treatment, given the ﬁrst state, ﬁrst treatment, and second state the treatment at stage k after a series of k states and k−1 treatments the average outcome (for a population of patients), if all of them were treated with the ATS d the difference in average outcome of two ATSs, d and d

trials to compare real ATSs and make statistical inferences about their effects. We conclude that clinical trial designers might improve the efﬁciency and general applicability of trials by taking an explicitly adaptive view of study treatment conditions.

COMPARING AND OPTIMIZING ADAPTIVE TREATMENT STRATEGIES To compare ATSs, quantiﬁable outcome criteria, collectively termed Y, must ﬁrst be deﬁned. For example, in managing major depressive illness, Y might indicate the achievement of a full remission or summarize quality of life over an extended period. In cancer chemotherapy, ﬁve-year survival might be the right measure, or if the side-effects burden should be considered, Y might be a measure of survival time without symptoms or toxicity. In the management of chronic pain, Y might represent total days without pain or excessive sedation. Second, the sequence of treatments triggered by various clinical outcomes in an ATS must be delineated. The standard notation (4) for an ATS is presented in Table 1. Table 2 S1 A1 S2 A2 Y

We illustrate the optimization process for the two-stage adaptive scenario described in Table 2 because that contains all the essential features and avoids excessive notation. For patients aged 60 or older with untreated diffuse large B-cell lymphoma (DLBCL), the question arises (5) as to the use of rituximab (R) in combination with the standard induction regimen of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), and also the subsequent use of rituximab in the maintenance of patients who have a response. To simplify the scenario, we suppose that the ﬁrst state is deﬁned by the diagnosis and minimum age of the patients, and is therefore the same for all patients being treated. We also use the catch-all label “standard of care” (SOC) to refer to the treatment received by patients who do not respond to the induction. In this simpliﬁed scenario, there are four possible ATSs, depending on whether CHOP or R-CHOP is used for induction and whether maintenance rituximab (MR) or observation (OBS) is used in patients who respond to the induction. Y might be taken as vital status at two years. More elaborate and complete ATSs might specify what to do with younger

DLBCL: diffuse large B-cell lymphoma SOC: standard of care MR: maintenance rituximab OBS: observation

A two-stage ATS for untreated diffuse large B-cell lymphoma in patients >60 years old constant over patients choice of induction therapy (CHOP or R-CHOP) state of disease after induction therapy (response or not) maintenance therapy in responders (MR or OBS) or “standard of care” in nonresponders vital status at two years after induction therapy

Abbreviations: R-CHOP, rituximab plus CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone); MR, maintenance rituximab; OBS, observation. www.annualreviews.org • Adaptive Treatment Strategies

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Table 3

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The backward induction for the DLBCL ATS

x1 y1 m1 = max(x1 , y1 )

2-year survival under MR in patients who respond to R-CHOP 2-year survival under OBS in patients who respond to R-CHOP 2-year survival under best maintenance option, B, in R-CHOP responders

u1 v1 m1 = max(u 1 , v1 )

2-year survival under MR in patients who respond to CHOP 2-year survival under OBS in patients who respond to CHOP 2-year survival under best maintenance option, B , in CHOP responders

f1 h1 z1 w1 r = f1 m1 + (1 − f1 )z1

probability of response to R-CHOP induction probability of response to CHOP induction 2-year survival under SOC in patients who do not respond to R-CHOP 2-year survival under SOC in patients who do not respond to CHOP 2-year survival in strategy beginning with R-CHOP and continuing with best second-stage strategy in responders to R-CHOP 2-year survival in strategy beginning with CHOP and continuing with best second stage strategy in responders to CHOP choose best induction treatment (compare f1 , h 1 ), then choose maintenance strategy that has the best survival in the responders to that induction treatment

r = h 1 m1 + (1 − h 1 )w1 myopic method

Abbreviations: DLBCL, diffuse large B-cell lymphoma; MR, maintenance rituximab; OBS, observation; R-CHOP, rituximab plus CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone); SOC, standard of care.

patients (deﬁning the ﬁrst state based on age, for example) and those who do not respond (by choosing a speciﬁc set of rules for the SOC). Any particular ATS would produce an average Y across all patients managed with that ATS, and the optimal ATS could be deﬁned as the one with the best average Y (6–8). The construction of an optimal two-stage ATS illustrates that the future performance of the strategy deﬁnes the best choice at each stage. The key point is that optimization starts at the end with the effects of the last treatment and works backward (a process referred to as backward induction). The ATS assigns the last treatment (in our example, this is the second treatment) based on the entire history of previous states (here, the ﬁrst two states), and treatments (here, only the ﬁrst treatment). So in our example, we begin by stratifying the patient population by the pretreatment state, the possible ﬁrst treatment choices, and the possible effects of each choice on the posttreatment state. For each of those strata, we ﬁnd the best choice of second treatment. In our simple example, all patients start in the same pretreatment state, and all patients who 446

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do not respond to induction are treated with SOC, so the possible treatments can vary in only two strata, namely R-CHOP responders and CHOP responders (see Table 3). In R-CHOP responders, x1 or y1 —whichever is larger—determines the best maintenance treatment in that stratum. We pick the best treatment, B, in the stratum; the corresponding best survival, m1 , is known as the “optimal beneﬁt to go” in the literature (7). In CHOP responders, whichever is larger of u 1 or v1 deﬁnes the best maintenance strategy (B ) in that stratum (with survival m1 ), and so forth. Notice that the best choice of maintenance treatment in this stratum of CHOP responders does not have to be the same as the best choice for the R-CHOP responders. Furthermore, even though all strategies call for the same treatments in nonresponders to R-CHOP and CHOP, the respective rates of survival, z1 , w1 , do not have to be the same, since the nonresponders to R-CHOP are not necessarily the same patients as the nonresponders to CHOP. Backing up one step, we have to compare the results of the ﬁrst treatment option in terms of the optimal choices for the second

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step. Thus, we plug in the previous results to ﬁnd that the comparison of outcomes at the ﬁrst stage depends on the various probabilities of survival. The choice of initial treatment comes down to a comparison of r and r and completes the deﬁnition of the optimal ATS, since the subsequent decision has been identiﬁed already. With more stages and more possible variation in treatment choices, the method for ﬁnding the optimal ATS elaborates on the basic technique described above: Start at the end, after K – 1 treatments and K states; ﬁnd the best (ﬁnal) treatment a K as a function of the previous history of states and treatments, based on the optimal beneﬁt to go at the end. Then choose treatment a K −1 within strata deﬁned by previous states and treatments to produce the average optimal beneﬁt to go, averaging over the distribution of states SK produced by the treatment a K −1 . Keep stepping backward until the ﬁrst treatment is chosen within strata deﬁned by the ﬁrst state. An alternative way of deﬁning a “good” ATS would be to start at the beginning and ﬁnd the initial treatment for each initial state that produced the best distribution of second stages, and then for each pair of states resulting from that choice, ﬁnd the best followon treatment in terms of Y. In our example, this would amount to choosing the induction strategy that produces the highest rate of response, and then choosing the maintenance strategy in responders that produces the best two-year survival. It should be obvious from the algebra that this need not produce the best overall ATS. The ﬁrst stage of this method looks ahead only to the next state, so we call this the best “myopic” strategy. The myopic method for ﬁnding good strategies may get trapped away from the optimal ATS at an early stage because the myopic method cannot factor in the way that the subsequent treatments succeed or fail given the states that result from applying the ﬁrst treatment. The key point to bear in mind is that one cannot always deﬁne the best ATS by starting at the beginning, since the best initial choice of treatment may

depend on the results that can be obtained by the best choices of subsequent treatments, in the context of the results of the ﬁrst treatment. However, it seems clear that the kinds of studies that would be required to put an evidence base under the best myopic ATS are much faster and simpler than the ones required for the optimal ATS.

ITT: intent-to-treat SMAR trial: sequential multiple assignment randomized trial EMR: electronic medical record

METHODS FOR COMPARING STRATEGIES The practical utility of an ATS will depend on the ability to estimate the quantities in Table 3, and thus to compare and optimize ATSs. There are, broadly speaking, three modes of inquiry into the relative merits of different ATSs (4, 6, 7): (a) observational studies of the uncontrolled variation in sequential treatment choices that occur in practice (“data mining”); (b) one-time randomized trials of whole ATS, in which each patient is experimentally assigned to an entire ATS at the outset of the study and the average Ys compared across those assignments by the intentto-treat (ITT) principle; and (c) sequential multiple assignment randomized (SMAR) trials, in which at each decision point the patient is randomized among the appropriate options for treatments, within strata deﬁned by the history of prior treatments and responses.

Data Mining One of the beneﬁts promised by the electronic medical record (EMR) is that the patient’s history of treatments and responses will be readily available to the clinician, thus making it easier to implement an ATS. Furthermore, the record left by such extended histories of sequential decision making reveals the extent of variation in ATSs apparently followed by different clinicians. In principle, it seems possible to match each observed pattern of treatments to an explicit ATS and use the outcome data (Y ) to compare different ATSs, and even to ﬁnd the optimal ATS (among all those that are apparently used in the observed practice of clinicians). For example, it seems www.annualreviews.org • Adaptive Treatment Strategies

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No unmeasured confounders (NUC): at each stage, given the history of prior states and treatments, the current treatment assignment does not depend on future unobserved potential outcomes on possible future treatments

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possible to ﬁnd the optimal medication sequencing strategy for managing hypertension from the millions of records of such patients maintained by large HMOs or the Department of Veterans Affairs health system. Setting aside the shortcomings of current EMRs (especially their varying quality and lack of pertinent outcome data), there is a more fundamental problem. The data mining or epidemiologic approach has the same weakness as all nonexperimental methods of measuring treatment effects, namely, the reliance on an untestable assumption that there are no unmeasured confounders (NUC) of treatment assignment (9–11). A confounder is a pretreatment patient characteristic that is related to both the choice of treatment and the outcome. If a confounder is measured, statistical adjustment or stratiﬁcation can deal with the bias created by ignoring the confounding in estimating the treatment effect. But if a confounder is not measured, there may be bias remaining even after adjusting for all measured confounders. This central weakness of observational studies is exacerbated by the multi-stage sequential nature of the ATS, which requires a sequential version of NUC at each stage. That is, the choice of treatment at each stage may depend on all the measured patient characteristics and observed states resulting from previous treatments, but we must assume it does not further depend on unmeasured characteristics or states, particularly the potential future outcomes of possible subsequent decisions. Furthermore, the analytic methods needed to produce unbiased estimates of treatment effects from such sequential data (even assuming NUC) differ from those used by data miners in the context of single-stage treatments, mainly traditional classiﬁcation and regression adjustment methods. Finally, a combinatorial explosion results from the many variations in apparent strategy that occur, most of which have no real claim to optimality. Nevertheless, despite the weaknesses identiﬁed above, there is clearly a role for such data mining efforts. They provide crucial inLavori

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formation on the strategies in current use and the range of outcomes that occur. In designing an experiment, one must not ignore current practice, since an experimental intervention that takes clinicians and patients far off the usual road may fail because of nonadherence. The analysis of naturalistic strategies can also reveal the extent of naturally occurring nonadherence to the apparent ATS, and suggest additions to the ATS that accommodate that reality. In the management of hypertension, depression, type II diabetes, substance abuse, and many other disorders, one of the patient states that must be taken into account is the nonadherence state, in which patients do not take their prescribed medications or follow their diet recommendations; they may not even show up for regular diseasemanagement appointments. Success or failure at dealing with nonadherence in all its forms may be one of the most important determinants of overall success or failure of a treatment policy. To be complete, an ATS needs to specify what will be done if the patient is in any of the nonadherent states. The naturalistic data include valuable information on rates of nonadherence and perhaps even clues for how to augment an ATS to provide options for dealing with it. Conversely, successful data mining for comparing treatments in the context of chronic disease requires attention to the adaptive nature of decision making. Standard methods for the statistical adjustment for observed confounders of ﬁxed treatments fail if applied to the time-varying treatment situation.

One-Time Randomization Among a List of Whole Strategies The one-time (sometimes called “baseline”) randomized ATS trial looks much like a standard trial, and the randomization eliminates unmeasured confounders. By randomizing patients to entire strategies before the ﬁrst treatment, it is possible to compare average outcomes by standard statistical methods for

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comparing treatment groups, since the treatment strategy has been ﬁxed by a single randomization. For example, the VANQWISH trial (12) compared an invasive management strategy (immediate angiography followed by revascularization if indicated) to conservative medical management (followed by revascularization, should the patient exhibit ischemic changes) in patients with non-Q-wave myocardial infarction (NQMI), which we can take as the (constant) initial state. This is really a special case of a two-stage strategy: Initial treatment is either immediate angiography followed by revascularization if indicated, or medical management with stress testing; the second state measures ischemic changes for conservatively managed patients (for invasive management, a second state is not needed to drive treatment), and the second treatment is the (delayed) angiography/revascularization procedure for patients who develop ischemic changes during noninvasive testing. The ultimate outcome is overall survival. The VA Cooperative Study #006 (13) compared inpatient and outpatient geriatric evaluation and management units (GEMs) on overall survival, randomizing patients to GEM/non-GEM inpatient care and to GEM/non-GEM outpatient care. The fourway randomization was done at the outset, but of course the second-stage randomization only had an effect in survivors of the inpatient stay. Because only vital status was used to drive treatment, this is an example in which the states and Y measure the same outcome. The four DLBCL ATSs described above could have been tested in a four-wayrandomized trial in which patients were assigned before induction to the sequence they would follow. One drawback to this approach is that it creates an opportunity for patients to fail to adhere to their assigned ATS after induction but before maintenance. For example, patients who do not tolerate R-CHOP well but do have a response might be more likely to opt for OBS as a maintenance treatment, even if they are assigned to RM at the original randomization. If this happens often,

there may be no satisfactory randomizationbased inference from the study. On efﬁciency grounds, it also seems attractive to randomize in two steps, since the outcomes of the ﬁrst treatment are “prerandomization” with respect to the second treatment assignment and can be used in “forced balance” schemes to improve the balance of patient characteristics across the second randomization. As it happens, the trial (5) of the four ATSs for DLBCL described above did use a two-stage randomization. The generalization of this second randomization leads to the SMAR trial.

The Sequential Multiple Assignment Randomized Trial The four ATSs for DLBCL in our example were compared in a SMAR trial with twoyear failure-free survival (FFS) as the primary outcome. For our purposes here we ignore certain issues with the data, notably due to post-randomization exclusions caused by delayed pathologic ﬁndings. After exclusions, there were 267 R-CHOP and 279 CHOP assignments, and a second randomization in responders, 174 to MR and 178 to OBS. If we use the algebra of the example, but for FFS instead of overall survival, we can simplify the example considerably, because the FFS in the patients who do not achieve response after induction is zero by deﬁnition. The ﬁrst backward induction step reveals that MR provides a higher FFS than does OBS in both the CHOP responders (0.74 versus 0.45) and the R-CHOP responders (0.79 versus 0.77), so the optimal maintenance treatment is always MR, and the “optimal beneﬁt to go” is 0.79 for R-CHOP responders and 0.74 for CHOP responders. Then we can take a step back to the ﬁrst stage and observe that since z1 = w1 = 0 by deﬁnition of FFS, we have r = (0.77)(0.79) = 0.608 and r = (0.76)(0.74) = 0.562—so R-CHOP induction has the best combination of response and optimal beneﬁt to go. Thus, the evidence favors the use of rituximab in both induction and maintenance. The authors of the paper www.annualreviews.org • Adaptive Treatment Strategies

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analyze the results in several different ways that come to the same general conclusion. It is possible to provide statistical uncertainties (standard errors, conﬁdence intervals, p values) for the contrast of these results among all four ATSs; these methods take account of the “data sharing” in the estimates; all the patients who are randomized to R-CHOP and respond contribute data to the estimated results of both ATSs that start with R-CHOP (and similarly for CHOP) (6, 7, 14, 15). The analytic methods for SMAR trials are formally the same as those needed for correct data mining of nonexperimental data; but NUC is guaranteed for SMAR trial data, and excessive multiplicity of strategies can be avoided by deliberate, judicious choice of limited options for the ATSs tested in a trial. This is not generally possible for the data miner, who needs to take her ATSs as she ﬁnds them.

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THE INTENT-TO-TREAT PRINCIPLE The concept of ATS clariﬁes some of the issues surrounding the intent-to-treat (ITT) principle in clinical trials. Under this principle, the outcomes of patients in a trial are to be summarized and contrasted as the original randomization dictates. Subjects are not reclassiﬁed if, for example, they receive treatments that differ from the original assignment or do not adhere to treatment. The principle has been defended as providing inferences that are grounded in the randomization, and proponents refer to the pragmatic requirement to compare the policy of “try to give treatment A” versus “try to give treatment B.” The criticisms of ITT focus on the unreality of the comparison of such treatment policies when a large proportion of patients drop out of assigned treatments or switch into treatments to which they were not assigned by the randomization. In ATS terms, the initial randomization assigns the ﬁrst treatment. From that point on, each patient in the trial has an unspeciﬁed ATS that determines subsequent treatments on the 450

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basis of changes in the patient’s and clinician’s state of equipoise. The ITT comparison averages over these ATSs, and to the extent that a “consumer” of the trial results has a different mix of ATSs in mind, there will be dissatisfaction with the ITT comparison. For example, if all the nonadherence to protocol is due to an unacceptable and unavoidable side effect of the experimental treatment in a fraction of the population (not identiﬁable in advance), and the default second-line treatment is clear to all, then the ITT principle is a sure guide to the true value of the treatment. This is because the underlying ATSs are clear, and there are only two: one that begins with the experimental treatment and then defaults to a standard if the side effect occurs, and another that begins and stays with the standard (control) treatment. As a trial departs from this neat scenario, the underlying ATSs become less clear and more various, so averaging over them makes less and less deﬁnite sense to consumers. For example, the ITT principle in the VANQWISH trial averages over the different individualized and unspeciﬁed postrandomization strategies employed by the treating clinicians, deﬁned by the different ways that clinicians interpreted the results of stress testing in the conservative arm (what level of ischemic change led to angiography), and in both arms, by the different decisions about revascularization following angiography that revealed coronary artery blockage (whether to intervene, what vessels to open, and what method to use). The optimal choice of conservative versus invasive strategy depends on how well the stress testing and delayed intervention rescue patients who turn out to need interventional treatment. In the VANQWISH trial, the contrast between the two randomized strategies turned on how well the implicit ATSs were implemented by the team at the study sites, and this was not under experimental control. In the event, the rescue worked exceedingly well, so the conservative strategy was overall the best one; it kept about two thirds of the patients from being

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catheterized during the index hospitalization, preventing complications from the diagnostic procedure and subsequent intervention. However, one could imagine a situation in which the second stage was not so successful. Thus, any clinical trial that uses the standard ITT method to compare treatments on long-term deﬁnitive outcomes is in effect averaging over the success or failure of the intervening (perhaps uncontrolled) treatments that are applied as the patient’s course dictates during the trial. Thus, even though the comparison among randomized initial treatments (A1 ) is not myopic (it uses Y as the criterion for success or failure), its interpretation depends on the speciﬁc mix of subsequent uncontrolled choices of A2 , and so forth. Consideration of the nature of the implicit ATSs that will be averaged over can help deﬁne the value of the ITT analysis and may lead to making them more explicit, perhaps by standardizing those parts of the ATS that clinicians can agree about. The next step involves taking the parts of the ATS that cannot be standardized (because there is disagreement) and randomizing over them in a SMAR trial. By doing so, one brings subsequent treatment variation under experimental control, allowing surer inference about the primary treatments and also creating the possibility of inference about optimal choices for the subsequent options.

EARLY INDICATORS OF RESPONSE Thall (16) describes the difﬁculties of current designs for early-phase trials that use an “early response indicator.” It is highly risky to use even a good early response indicator to select treatments to take forward, or to make the “go/no-go” decision for a deﬁnitive trial. He points out the connection between more efﬁcient phase II–III trials and the ATS idea. This promising line of research may help to improve the performance of the phase II–phase III transition. One inference from Thall’s work is that the deﬁnitive treatment outcome (here, Y ) and the early indi-

cator (which corresponds to a state) need to be studied together. Current practice (which uses the early indicator to select treatments for deﬁnitive testing and then separately uses the deﬁnitive outcome to conﬁrm efﬁcacy in a large trial) neglects the issue of how the two relate to each other statistically, leading to failed phase III trials.

SUMMARY The idea of personalized medicine has seized the imagination of many medical researchers. Instead of “one pill cures all,” personalized medicine offers a strategy, a list of treatment recommendations that depend on the genetic makeup of the individual patient. Of course, physicians have always practiced personalized medicine in a sense; applying genomic knowledge is just the latest version of the usual adaptation of treatment to diagnosis. But each individual’s germline genome is ﬁxed over her lifetime, while everything else changes, including her state of health or illness and response to treatments. Other than timelimited, acute interventions that cure the patient, the typical treatment decision occurs in a sequence of similar decisions, in the context of long-term management of a chronic condition. As medicine improves its ability to stave off mortality, the result is a growing list of chronic conditions formerly considered acute (HIV infection, diabetes, and many others). There is a growing need for adaptive treatment strategies (ATSs): rules that change the treatment as the patient’s history of illness and response to previous treatment evolves. Other terms for this kind of strategy include dynamic treatment, treatment policy, and empirical treatment. An ATS has three components: a list of states that capture the important current features of the patient’s illness, a list of treatments from which to choose the one to apply at the current time, and a rule for choosing a new treatment on the basis of the history of states and treatments. The iterative application of the rule creates the ATS out of the www.annualreviews.org • Adaptive Treatment Strategies

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available treatments, but the state descriptions are also fundamental to the ATS because they capture the information that is used to drive the decisions. The states and rules work together much the way diagnostic technology and treatment guidelines do, the former deﬁning the categories of patients and their diseases as a basis for the actions prescribed by the latter. To evaluate an ATS, one needs at least one metric of success (or failure), which captures the important features of the overall outcome of the patient. This can be as simple as the span of the patient’s life from the start of the strategy, or as complex as a patient-centered utility based on quality of life integrated over that span. An ATS may be based on early markers of treatment effect, but the intent is to inﬂuence an overall ultimate outcome. A potential advantage of the electronic medical record (EMR) is that the patient’s history of treatments and responses will be

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readily available to the treating clinician, making it convenient to implement a coherent ATS. Presumably, mining the data embedded in the EMR can shed light on the natural variation of ATSs for a given condition and perhaps suggest candidate strategies for testing in experiments. But randomization is still the key to reliable inference about treatment effects, especially in the context of ATSs. Testing the effectiveness of different ATSs and ﬁnding optimal ones can challenge the current methods of evidence-based medicine. Methods for evaluating and comparing ATSs are evolving; experimental and observational paradigms for obtaining evidence, statistical methods for analyzing data relating to ATSs, and issues relating to research ethics are all in ﬂux. The ATS perspective may also help resolve the difﬁculties presented by the intentto-treat (ITT) principle and make it possible to do studies that have greater ecological relevance.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENT This work was supported by a grant from the National Institutes of Health (R01 MH051481) to Stanford University.

LITERATURE CITED 1. Lavori PW, Dawson R, Rush AJ. 2000. Flexible treatment strategies in chronic disease: clinical and research implications. Biol. Psychiatry 48:605–14 2. Lavori PW, Dawson R. 1998. Developing and comparing treatment strategies: an annotated portfolio of designs. Psychopharm. Bull. 34(1):13–18 3. Lavori PW, Dawson R. 2000. A design for testing clinical strategies: biased individually tailored within-subject randomization. J. R. Statist. Soc. Ser. A 163:29–38 4. Murphy SA. 2005. An experimental design for the development of adaptive treatment strategies. Stat. Med. 24:1455–81 5. Habermann TM, Weller EA, Morrison VA, et al. 2006. Rituximab-CHOP vs CHOP alone or with maintenance rituximab in older patients with diffuse large B-cell lymphoma. J. Clin. Oncol. 24:3121–27 452

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6. Lavori PW, Dawson R. 2003. Dynamic treatment regimes: practical design considerations. Clin. Trials 1:9–20 7. Murphy SA. 2003. Optimal dynamic treatment regimes. J. R. Statist. Soc. Ser. B 65:331–66 8. Moodie EM, Richardson TS, Stephens DA. 2007. Demystifying optimal dynamic treatment regimes. Biometrics 63(2):447–55 9. Robins JM. 1989. The control of confounding by intermediate variables. Stat. Med. 8:679– 701 10. Murphy SA, Van Der Laan MJ, Robins JM. 2001. Marginal mean models for dynamic treatment regimes. J. Am. Statist. Assoc. 96:1410–23 11. Robins JM. 2004. Optimal structural nested models for optimal sequential decisions. In Proceedings of the Second Seattle Symposium on Biostatistics, ed. DY Lin, P Heagerty, pp. 189–326. New York: Springer 12. Boden WE, O’Rourke RA, Crawford MH, et al. 1998. Outcomes in patients with acute non-Q-wave myocardial infarction randomly assigned to an invasive as compared with a conservative strategy. N. Engl. J. Med. 338:1785–92 13. Cohen HJ, Feussner JR, Weinberger M, et al. 2002. Effectiveness of geriatric evaluation and management units and clinics. A VA Cooperative Trial. N. Engl. J. Med. 346:905–12 14. Dawson R, Lavori PW. 2007. Sequential causal inference: application to randomized trials of adaptive treatment strategies. Stat. Med. In press 15. Lavori PW, Dawson R. 2007. Improving the efﬁciency of estimation in randomized trials of adaptive treatment strategies. Clin. Trials 4:297–308 16. Thall PF. 2007. A review of Phase 2–3 clinical trial designs. Lifetime Data Anal. doi: 10.1007/s10985-007-9049-x

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:443-453. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse,1 Robin Shattock,2 and John P. Moore1 1

Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021; email: [email protected]

2

St. George’s, University of London, London, United Kingdom

Annu. Rev. Med. 2008. 59:455–71

Key Words

First published online as a Review in Advance on September 24, 2007

topical application, vaginal rings, sustained-release formulations, reverse transcriptase inhibitors, entry inhibitors

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.061206.112737 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0455$20.00

Abstract The development of a vaginal (and perhaps a rectal) microbicide would be of major beneﬁt for slowing the global spread of human immunodeﬁciency virus type 1 (HIV-1). A microbicide is a gel or related device that, when inserted vaginally or rectally, acts to prevent infection of a woman or a man by HIV-1 during sexual intercourse. A practical microbicide must be not only effective, safe, and userfriendly but also economically affordable in the developing world. To date, the performance of microbicide candidates in efﬁcacy trials has been disappointing, but next-generation concepts now in or approaching clinical trials offer improved prospects for efﬁcacy. The most plausible approaches involve topical application of antiretroviral agents with speciﬁc activity against HIV-1, compounds similar to drugs used to treat HIV-1 infection. How these inhibitors are applied may also be critical, with sustained-release formulations and vaginal ring delivery systems now becoming a high priority.

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INTRODUCTION ARV: antiretroviral drug

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Pre-exposure prophylaxis: ARVs taken orally to prevent HIV-1 infection Microbicide: vaginally or rectally applied substance to prevent sexual transmission of HIV-1

Each year, ∼4 million new HIV-1 infections occur, mostly through heterosexual intercourse (1). In the effort to prevent HIV1 transmission, behavioral alterations driven by education are critical, condoms and other barrier methods remain essential, and male circumcision may now also have a role (2). Among biology-based interventions, the most important is a prophylactic vaccine, but an effective one remains elusive (3–5). Other strategies now being emphasized involve using antiretroviral drugs (ARVs) for prevention. In pre-exposure prophylaxis, an ARV is regularly and systemically (i.e., orally) administered at a dose and frequency sufﬁcient to impede the establishment of HIV-1 infection in a newly exposed host (6). The microbicide concept, our focus here, involves the vaginal or rectal application of an inhibitory compound(s) to prevent the very earliest stage

of sexual transmission, the infection of target cells close to the sites of virus deposition in the exposed host (Figure 1). One of the most important rationales for developing a microbicide is to enable a woman to protect herself. Three times more 15- to 24-year-old women in sub-Saharan Africa are now HIV-1-infected than men of the same age (7). Young women at risk need protection in a form they themselves can control. Men often refuse to use a condom, but they may not object (or even know) if their female sex partners have applied a microbicide before intercourse. This may be particularly important in populations where a high value is placed on fertility (8). Here we discuss speciﬁc inhibitors of HIV-1 replication that may soon be in microbicide trials, as well as the properties a practical microbicide should possess. Understanding how HIV-1 is transmitted is important for

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 1 The female genital mucosa: one environment where microbicides must prevent transmission. The schematic represents the female genital tract with lumen (above) and submucosal tissue (below) at the juncture of the vaginal squamous stratiﬁed epithelium (left) and the cervical columnar epithelium (right). In the lumen, both X4 and R5 virions, deposited with the semen, are shown (see inset key). R5 virus is preferentially transmitted or has an advantage in establishing infection soon after transmission; X4 virus has multiple potential disadvantages in crossing the mucosa and in the infection of target cells in deeper tissues. A microbicide must block transmission at one or more critical stages in the following scheme. (a) The endocervix is ﬁlled with a plug of mucus that contains mucin, which may preferentially trap X4 virions because of the strong positive charge of their Env proteins. (b) R5 virions have diffused to the columnar cervical epithelium; this monolayer is a weak barrier against infection. One virion is shown traversing the epithelium by transcytosis, but whether this mechanism of transfer across the epithelium plays a role in vivo has not been conﬁrmed; HIV-1 does not penetrate intact cervical explants (14). (c) The stratiﬁed vaginal epithelium provides stronger impediments to viral penetration. It harbors Langerhans cells with interdigitating dendrites expressing the MCLR langerin. The interaction of R5 or X4 virions with langerin may lead to endocytosis and destruction of the virus, although Langerhans cells express CCR5 and can be infected by R5 viruses. (d ) Virus may only be able to gain access to submucosal tissues through rifts in the stratiﬁed epithelium, caused by genital ulcerative disease and exacerbated by coital abrasions. After diffusion through such rifts, R5 or X4 viruses may infect submucosal T cells. (e) R5 viruses preferentially infect macrophages, which are considered important producers of HIV-1 for further dissemination through the organism. ( f ) Dendritic cells (DCs) can trap HIV-1 via DC-SIGN (or another MCLR) and transmit it to T cells, and DCs can also be infected by R5 viruses. (g–h) DCs may transport HIV-1 via afferent lymphatics to regional lymph nodes. Here, at a later stage, the virus will encounter both neutralizing antibodies and cytotoxic T cells; the latter will efﬁciently kill infected T cells, leaving infected macrophages to sustain HIV-1 production. Further dissemination through the organism to the gut-associated lymphatic tissue as well as to spleen, brain, liver, and lungs will occur through efferent lymphatics. In addition to these events initiated by diffusible virions, infected cells may be deposited with the semen. Microbicides may, therefore, also have to counter the cell-to-cell transfer of virus. (From Reference 32 with permission.) 456

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Dendritic cell Macrophage Afferent lymphatics

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the design of intervention strategies, but the topic is beyond the scope of this article. We therefore recommend several reviews on sexual transmission and how host and virus factors inﬂuence its efﬁciency (7, 9–15). The ﬁrst microbicides tested clinically included detergents, pH modiﬁers, and polyanionic gels (14, 16–19). Three efﬁcacy trials have now failed, and the results of others are pending. Nonoxynol-9 (N9), a detergent present in spermicides and some vaginal lubricants, destroys the HIV-1 lipid membrane and hence inactivates the virus very effectively in vitro, but it was not protective in vivo and actually increased the rate of HIV-1 infection slightly. The problem was that the regular use of N9 damaged the naturally protective barrier of the vaginal epithelium and caused inﬂammation, thereby recruiting target cells to the mucosa. These events rendered the women more vulnerable to infection upon subsequent exposure to HIV-1 (20). The critical importance of preserving the body’s natural defenses now mandates thorough preclinical and early clinical testing of microbicide candidates. Despite the failure of N9, a second surfactant candidate, C31G or Savvy®, entered phase III clinical trials in Ghana and Nigeria. These trials were discontinued in 2005/2006 after an interim review of the data revealed that, although Savvy® appeared safe, the incidence of HIV infection was too low among the trial participants for efﬁcacy ever to be shown. Unfortunately, efﬁcacy trials of the polyanion Ushercell (cellulose sulfate) also had to be terminated prematurely in early 2007, owing to an increased rate of infection at some of the clinical sites. What caused this problem remains to be determined (for a discussion, see Reference 20a); there is no evidence that the mechanism was similar to N9’s, as Ushercell is not a detergent. Three other polyanion-based microbicides remain in phase III trials, without any reports of infection enhancement to date. It will be absolutely essential for future progress that any further safety problems be solved early.

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ATTRIBUTES OF A SUCCESSFUL MICROBICIDE To be useful, a microbicide must be safe, acceptable, efﬁcacious, and affordable (17). As mentioned above, it must not impair natural defensive mechanisms against HIV-1 transmission. The naturally occurring lactobacilli that contribute to the maintenance of a low vaginal pH must also be maintained (10). A product intended for use by women who may become pregnant must not be teratogenic, and carcinogenicity is also an issue for any regularly applied compound. Safety studies must also be carried out on women with genital ulceration and inﬂammation, as well as women who have other sexually transmitted diseases or cervical ectopy, because potential users of a microbicide product may have such complicating conditions. Toxicity within the uterus should be monitored, since a gel may easily penetrate there (21, 22). The rectal use of a microbicide raises other safety concerns, as the rectal cellular environment differs from the vagina and the epithelial cell layers are more fragile (23–25). A microbicide should not induce inﬂammation, because any increase in the frequency or activation status of immune system cells near the site where the virus is deposited could facilitate its transmission (11, 12). Chemokine derivatives or other modiﬁers of innate immunity might affect the activation state of local immune system cells, and the potential of peptideand protein-based microbicides to be immunogenic must also be considered. Of note is that two different Toll-like receptor ligands (CpG oligonucleotides and Imiquimod) may have enhanced the vaginal transmission of simian immunodeﬁciency virus (SIV) to macaques via local inﬂammatory responses (26). Any product for use during sexual intercourse must be acceptable to both partners. Women will reject any microbicide that has an unusual smell or color, is too complex to apply, or is too viscous or too ﬂuid for comfort. Formulation issues are therefore important,

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and although they are not directly relevant to the earliest stages of the microbicide development process, it should not be assumed that every compound can be properly formulated for real-world use. Ideally, a microbicide should remain effective for several hours. A product that must be applied only moments before intercourse takes place would be less desirable than one that could be applied a few hours beforehand, particularly if a woman intends to use the product covertly. The slow release of active compounds from intravaginal devices is one way to provide durable protection. This potentially important approach is discussed further below. A microbicide whose manufacturing cost is prohibitive for users in the developing world would not be useful, nor would a product too fragile to be distributed or stored efﬁciently. Some scientiﬁcally sound concepts may be too sophisticated to translate into practical products. For example, new ways to make peptides and proteins on a large scale might have to be developed if some compounds that have performed outstandingly well in preclinical studies are to fulﬁll their potential. Fermentation technologies and plant-based proteinproduction systems represent potential solutions, provided suitable investments are made to cover the initial development costs.

MICROBICIDES BASED ON REVERSE TRANSCRIPTASE INHIBITORS The next generation of microbicides seems likely to involve the topical application of gels Table 1

containing ARVs that inhibit the viral reverse transcriptase (RT) enzyme, which converts HIV-1 genomic RNA to proviral DNA within the newly infected cell. The RT inhibitors can be divided into two broad categories. The nucleoside RT inhibitors (NRTIs) mimic the natural deoxy-ribonucleosides that are the building blocks of DNA, but, unlike the natural compounds, they cannot be further polymerized to create an extended DNA chain. Instead, the nascent chain is terminated at the point where the NRTI has been incorporated (which is why NRTIs are often referred to as chain-terminating inhibitors). In contrast, the non-nucleoside RT inhibitors (NNRTIs) are classic enzyme inhibitors; they bind to RT at a point distinct from the active site and allosterically inhibit the enzyme’s function (27, 28). One NRTI, Tenofovir, is a serious microbicide candidate, and several NNRTIs are currently in early-stage clinical trials (Tables 1 and 2). All the NNRTIs interfere with HIV-1 replication within the newly infected cell, but some (the “tight-binding” compounds) that associate avidly with RT may also be active against any cell-associated HIV1 present in genital tract secretions and have a prolonged duration of activity (29). Several tight-binding NNRTIs are now at various stages of preclinical or clinical development as experimental microbicides, including thiourea-PETT (phenethylthiazolylthiourea) derivatives (PHI-236, PHI-346, and PHI-443), urea-PETT derivatives (MIV150), oxypyrimidines (S-DABO), thiocarboxanilides (UC-781), and diarylpyrimidines (TMC-120). Their individual properties have been reviewed extensively elsewhere (27, 28).

Reverse transcriptase inhibitors in clinical trialsa

Category of ARV microbicideb

Preclinical trial

Safety trial (phase I)

S-DABO

TMC-120, UC-781, MIV-150

NRTI NNRTI

NNRTI: non-nucleoside reverse transcriptase inhibitor

Efficacy trial (phase IIb/III) PMPA (Tenofovir)

a A more comprehensive list of substances and continual updates of phases of the respective trials can be obtained from the Alliance for Microbicide Development, http://www.microbicide.org. b Abbreviations: NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor.

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PRO 542, other soluble CD4 molecules

BMS-378806, BMS-C

Other proteins/ peptides

Small molecule

Mannose shield

tyrosine-sulfated N-terminal CCR5 peptide

Enfuvirtide, T-1249, C52L defensins

Cyanovirin-N,c defensins, other lectins or lectin-like molecules 5MO30

2F5, 4E10, Z13

2G12

Ectodomain

multiple MAbs/anti-CD4i Fabsb

V3 region/ coreceptor bs

NSC 13778

α-and θ-defensins

Q4120, TNX-355, and others

Domain 1 or 2

CD4 CCR5

CMPD167, vicriviroc, maraviroc

PSC-RANTES, 2-RANTES

PRO 140, 2D7, and others

CXCR4

AMD3100, AMD3465

stromal cell-derived factor-1, β-defensins

12G5 and others

Coreceptor

Cell surface

Candidates shown to be active in SHIV-macaque model are in italics. Abbreviations: bs, binding site; CD4i, CD4-induced epitope on gp120; Fab, antigen-binding antibody fragment; ICAM, intracellular adhesion molecule. b CD4i has poor accessibility for whole IgG antibodies in the CD4-Env complex but binds Fabs well (68). Such antibody fragments or any of the antibodies mentioned may not be feasible as microbicide components but are included to illustrate the principles of entry inhibition. c Cyanovirin-N blocks both infection of lymphocytes and capture by dendritic cells.

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To inactivate RT, the inhibitors must penetrate the cell membrane and have a long intracellular half-life, which is why all the above compounds are highly lipophilic. This raises issues concerning their formulation and delivery to aqueous environments such as the vagina or rectum (27, 28). Such concerns could be signiﬁcant in practice, because the local concentrations of microbicide compounds may need to be several logs greater than those sufﬁcient to inhibit HIV-1 infectivity in vitro (17). Like all microbicide candidates, the NNRTIs must not be inactivated under the acidic conditions inherent to the female genital tract, and they must not damage the vaginal or rectal mucosa or perturb the natural microorganisms normally present at those sites (particularly within the vagina).

ENTRY INHIBITORS AS MICROBICIDES The fusion or entry inhibitors speciﬁcally impede HIV-1 from entering its target cells. They are attractive candidates for microbicide use because stopping the virus from entering cells at or close to the site where it is deposited will automatically prevent infection. The surface of HIV-1 virions is studded with around a dozen trimeric envelope glycoprotein (Env) spikes that mediate virus– cell fusion and entry (30, 31). A trimer consists of three monomers, each containing the gp120 surface glycoprotein noncovalently associated with the transmembrane glycoprotein gp41. The gp120 protein contains the binding sites for cell surface receptors, while gp41 contains a membrane-spanning domain that anchors the gp120-gp41 complex into the HIV-1 membrane. Noncovalent associations between the gp41 subunits hold the trimer together. HIV-1 infection is initiated when a functional Env trimer binds the cell surface CD4 receptor. Ancillary interactions between the virus and the cell, mediated by either the Env complex or host cell proteins incorporated within the virion membrane, can pre-

cede the gp120-CD4 interaction and make it more likely by overcoming electrostatic repulsive forces between the viral and cellular membranes. CD4 binding triggers conformational changes in the Env trimer that enable a secondary interaction between gp120 and a coreceptor, CCR5 or CXCR4 (30, 32, 33). CCR5 is by far the more important coreceptor for HIV-1 sexual transmission; viruses using CXCR4 (X4 variants) account for only ∼1%–5% of new infections, either because they are inefﬁciently transmitted or are outreplicated by CCR5-using (R5) viruses during the acute phase of the new infection. The binding of gp120 to either coreceptor triggers a substantial conformational change in gp41 that brings the virus and cell membranes into close enough proximity for the two to fuse together (34, 35). Each stage of the attachment and fusion process is susceptible to blockade by speciﬁc compounds that bind to either the Env proteins or their cellular receptors. Several entry inhibitors are now under evaluation as microbicides, although none is yet in clinical trials (Tables 1 and 2).

Entry inhibitors: compounds that block the steps in viral replication leading to release of the viral core into the cytoplasm of the target cell Envelope glycoprotein (Env): mature HIV-1 Env consists of trimers of gp120-gp41 heterodimers SHIV: simian/human immunodeﬁciency virus, i.e., SIV bearing an HIV envelope glycoprotein

VIRUS-TARGETING ENTRY INHIBITORS Virus-directed inhibitors are inactive until the moment when the infectious inoculum (i.e., semen) is deposited onto the mucosal surface (Figure 1). They must then act rapidly. When infectious SIV or SHIV is experimentally applied, without trauma, within the macaque vagina, infection still occurs if a virus-inactivating solution (soap or mild acid) is added after a delay of more than 30–60 min (36, 37). Within that time, sufﬁcient infectious virus has migrated beyond the reach of an aqueous solution of an inactivating agent, presumably to a site within the local cells or tissues. The virus-binding compounds must, therefore, be present at the right place, and in sufﬁcient quantities, to encounter the incoming virus and impede infection of the ﬁrst potential target cells it meets. To do this, the compounds must, presumably,

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be able to diffuse into the deposited semen; it follows that semen must not block their actions. Neutralizing monoclonal antibodies (MAb) can protect macaques from vaginal challenge. A notable example is the activity of MAb b12, which recognizes the CD4-binding site on gp120 (37). A tetravalent, recombinant form of CD4 (CD4-IgG2 or PRO 542) has similar activity (14). Both b12 and (more so) CD4-IgG2 are broadly active against multiple HIV-1 strains in vitro. Cyanovirin-N (CV-N) is a small, lectin-like protein derived from a cyanobacterium that binds to mannose moieties on the extensively N-glycosylated gp120 protein. Thus CV-N decorates the gp120 surface and acts, in effect, akin to a neutralizing antibody. It has broad and potent anti-HIV-1 activity in vitro and can protect macaques from both vaginal and rectal SHIV challenge (38, 39). Other lectins have a broadly similar, and sometimes more potent, antiviral effect than CV-N in vitro and are also being considered as microbicides (40). Such compounds are broadly active against divergent HIV-1 strains and prevent the binding of gp120 to various mannose C-type lectin receptors (MCLRs), which could be important for impeding sexual transmission (40). The disadvantages of lectins include their cytotoxic or cell-stimulatory effects because of reactivity with cellular glycans. It is also possible that blocking the interaction of HIV-1 with some MCLRs, such as langerin on Langerhans cells and the macrophage mannose receptor, may divert the virus away from unproductive (or even actively inhibitory) pathways and thereby promote infection (41–43). Moreover, the lectins, b12, and CD4-IgG2 are proteins, raising concerns about their ease and cost of manufacture to clinical standards and in large quantities. Additional possible disadvantages of proteins include their degradation or denaturation within the vagina or rectum (e.g., by host proteases), and perhaps the elicitation of immune responses resulting in inﬂammation and the recruitment of susceptible target cells. Klasse

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Peptides based on gp41 sequences inhibit the fusion of both R5 and X4 viruses by interfering with the conformational changes within gp41 that occur after coreceptor binding, and in a coreceptor-independent manner. Speciﬁcally, they prevent gp41 trimers from forming the six-helix bundle conﬁguration necessary for the juxtaposition of the virus and cell membranes (44). Two different gp41based peptides with overlapping sequences, C52L and T-1249, can protect macaques from vaginal SHIV challenge (45; RS Veazey, PJ Klasse, ML Greenberg, JP Moore, unpublished results). T-1249 has been tested extensively as an injectable antiviral drug; it reduced plasma viral load in HIV-1-infected people by up to 2.0 logs (46, 47). An earlier generation gp41 peptide, T-20 (Enfuvirtide), is licensed for the treatment of HIV-1 infection (44). Small-molecule gp41 ligands that mimic the fusion-blocking peptides might also prove to be useful as microbicides (48) because this type of inhibitor has the theoretical advantage of being easier and cheaper to manufacture than proteins. Another small-molecule inhibitor, BMS378806, binds to the CD4-binding region of gp120 to inhibit CD4 association and subsequent structural changes in gp120 that drive the formation of the coreceptor-binding site. Hence BMS-378806 blocks the attachment of HIV-1 to target cells in a coreceptorindependent manner (49). BMS-378806 can protect macaques from vaginal challenge with an R5 SHIV (45), and a follow-up compound, BMS-C, with improved properties, is now being tested (50).

RECEPTOR LIGANDS AS MICROBICIDES Several cell surface receptors can help capture HIV-1 prior to CD4 binding. The tethered virus can more readily encounter CD4 via a “proximity effect.” Among such ancillary receptors are heparan sulfate proteoglycans, LFA-1 (which is the receptor for any ICAM-1 present in the viral envelope) and

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MCLRs, including but not limited to dendritic cell–speciﬁc ICAM-3-grabbing nonintegrin (DC-SIGN). The MCLRs may be relevant to sexual transmission, if HIV-1 capture onto or within dendritic cells (DCs) is involved (13). MAbs speciﬁc for, e.g., DCSIGN that inhibit HIV-1 capture have been identiﬁed; however, other MCLRs may mediate HIV-1 binding to DCs, so a MAb to only one of them might not be sufﬁcient to prevent transmission. In small-scale studies in the macaque model, mannans that block gp120MCLR interactions were not protective (45). It may be pertinent that HIV-1 uptake into macrophages via the macrophage mannose receptor and into Langerhans cells via langerin can counteract the productive route of infection (41–43). In principle, CD4 ligands might be useful by blocking the gp120-CD4 interaction from the cellular side. For example, an anti-CD4 MAb has been considered as a microbicide (14), and a small-molecule ligand for CD4 that blocks gp120 binding has also been described as having antiviral activity, albeit with fairly low potency (51). Little work has yet been carried out to develop CD4 ligands as microbicides. In contrast, the coreceptors are now major targets for microbicide development, particularly CCR5 inhibitors (32, 33) (Figure 2). Two such compounds can inhibit the vaginal transmission of an R5 SHIV to macaques: the modiﬁed chemokine PSC-RANTES and the small-molecule CMPD167 (45, 45a). Given the stringency of the model (progesterone treatment to thin the vaginal epithelium and a “high-dose” viral challenge), the robust protection that has been observed is encouraging for the prospects of developing a CCR5 inhibitor for human use. Other chemically modiﬁed chemokines are also being evaluated in cell culture systems, although 2-RANTES failed to protect macaques; indeed, the liposome carrier was found to confer better apparent protection (52). Various small-molecule CCR5 ligands have antiviral activities comparable to, or greater than, CMPD167’s; mar-

aviroc from Pﬁzer has recently been approved by the US Food and Drug Administration (FDA) as a therapeutic for HIV-1 infection, and vicriviroc from Schering Plough is in advanced clinical trials (53). In principle, MAbs to CCR5 could also be used as microbicides, given their antiviral effects in vitro and in vivo, although they would be subject to the same practical constraints as other protein-based microbicides (54). X4 viruses can sometimes be identiﬁed in semen, and there are recorded examples of primary infections with X4 viruses. CXCR4using viruses can be vaginally transmitted to macaques. Moreover, infections with X4 viruses are associated with a more rapid rate of progression to AIDS and death (32, 33). CXCR4 therefore remains a legitimate target for microbicide development. Several smallmolecule CXCR4 inhibitors have been tested clinically as ARVs, with antiviral efﬁcacy being reported. One such compound, AMD3465, is now being tested for microbicide activity in the macaque vaginal transmission model (55). The virus-targeting entry inhibitors discussed above are also generally active against CXCR4-using viruses.

ICAM: intracellular adhesion molecule DC-SIGN: dendritic cell–speciﬁc ICAM-3–grabbing nonintegrin, an MCLR

COMBINATION MICROBICIDES: A POTENTIAL SOLUTION TO GLOBAL HIV-1 SEQUENCE DIVERSITY No ARV is now used on its own to treat HIV-1 infection. The rate at which the virus replicates and mutates makes it inevitable that escape mutants will arise rapidly. However, a combination of at least three drugs with different mechanisms of action is harder to evade, since a mutation that confers resistance to one drug still leaves the virus vulnerable to the others. Similar arguments apply to the use of inhibitor combinations to counter HIV-1 transmission, although rather than circumventing resistance development per se, a more important advantage of microbicide combinations is to increase the range of HIV-1 variants that can be countered.

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uated for their ability to protect against one or two challenge viruses, invariably (at present) derived from genetic subtype B. And there have been few tests of the breadth of activity of microbicide candidates even in tissue culture systems. A microbicide would only be useful if it were active against most or all of the different HIV-1 phenotypic variants present in the genital ﬂuids of a potential transmitter. If not, then inhibitor-resistant variants would be transmitted, perhaps at lower efﬁciency, even

Coitally-dependent gel delivery system Coitally-independent semi-solid delivery system

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Vaginal concentration of microbicide

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The global diversity of HIV-1 presents huge obstacles for all HIV-1 prevention programs. A practical microbicide must counter any HIV-1 variant it encounters. Within infected individuals, within cohorts, cities, countries, and continents, diversity increases over time, as viable and transmissible sequence variants accumulate (56, 57). The sequence diversity problems arise on two levels: within an infected individual and within a population. Yet microbicide candidates, if they are tested at all in primates, are only eval-

Local irritancy Systemic effects Maximum effective concentration

Optimal concentration range

Minimum effective concentration

No protection

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Time (days)/frequency of dosing

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if the sensitive ones were blocked. For example, if an NNRTI-based microbicide were used in a population of individuals previously exposed to the same inhibitor (or one with a similar resistance proﬁle) as part of HIV1 therapy, and resistant variants had accumulated within the population, the transmission of such variants would probably become inevitable. Over time, as the global effort to increase ARV access continues, this issue will become less and less theoretical. A second problem is that some naturally occurring HIV-1 variants are resistant to certain inhibitors, even in the absence of any drug-induced selection pressures. For example, some variants are naturally insensitive to the BMS-378806 attachment inhibitor because of sequence variation within both gp120 and gp41 (58). Another example relates to viruses that can use either CCR5 or CXCR4 (either as genuinely dual-tropic, R5X4 clones or, perhaps more commonly, as mixtures of R5 and X4 variants). If both variants were present as transmissible viruses in an infected person, a microbicide directed at only one coreceptor would not be effective. The problems are magniﬁed when considering the natural sequence diversity of HIV-1 within populations of infected individuals. A hypothetical microbicide found to be effective in an American research laboratory against

a few, prototypic, commonly used subtype B viruses, then shown to confer protection against a subtype B SHIV in a primate model, would be of little use in, say, southern Africa if it were ineffective against viruses from subtype C. Such issues must now be considered during preclinical research. Panels of test viruses from different genetic subtypes are being used to gauge the breadth of activity of neutralizing antibodies induced in experimentally vaccinated animals or humans (59), and can be used in microbicide research (17, 50). One solution to the sequence diversity problem is to use combinations of inhibitors with complementary mechanisms of action and hence different resistance proﬁles (for both natural and drug-selected variation). On the level of a single HIV-1-infected person, if 90% of the viruses present in, e.g., semen are sensitive to inhibitor 1, and 90% are independently sensitive to inhibitor 2, then the two inhibitors in combination would counter 99% of the potentially transmissible variants (adding a third would increase the coverage to 99.9%). Similar arguments can be made across populations of infected people. As well as increasing the breadth of coverage against natural variants, using a second (or third) inhibitor in a microbicide could reduce the spread of drug-resistant viruses within a population that

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 2 (a) Fluctuations in local inhibitor concentrations after various modes of microbicide delivery. Three delivery systems are compared: coitally dependent gels, coitally independent semisolids, and reservoir-type vaginal rings. Concentration ranges are divided into three categories according to their effect, as shown on the right. One application of a microbicide gel immediately before sex act A would provide protective vaginal microbicide concentrations through sex act A but not B or C. However, a highly retentive, sustained-release, semisolid microbicide formulation administered once daily could protect through all three sex acts. Likewise, the vaginal application of a single controlled-release formulation, such as a vaginal ring device, could theoretically provide prolonged protection by gradually releasing the active ingredient of the microbicide. (b) Examples of reservoir vaginal rings that are in clinical use for other purposes, such as hormone treatment. The ring diameters are around 4 cm. Provided these devices do not cause inﬂammation or epithelial lesions, they could be used to provide sustained release of protective microbicide concentrations. (c) An x-ray of a female pelvis showing a vaginal ring applied to the fornix around the cervix uteri. Such devices are being developed for the continuous release of protective concentrations of microbicides. Like all commercial silicone rings, this ring contains barium sulfate to allow it to show up under x-rays, so that the presence of the drug-loaded cores in the center of the white opaque rings can be conﬁrmed. All three images are provided courtesy of Dr. Karl Malcolm, Queen’s University, Belfast. www.annualreviews.org • Microbicides to Prevent HIV-1 Sexual Transmission

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has access to ARVs. For example, the sexual transmission of viruses resistant to a particular NNRTI would not be prevented by a microbicide based on the same or a broadly similar drug, but the resistant viruses would be blocked if the product also contained an entry inhibitor unaffected by NNRTI resistance mutations. An additional beneﬁt to combining inhibitors is the potential for synergy, i.e., the activity of a combination can be greater than the sum of activities of the constituents. The combination can thus enhance the degree of inhibition at given doses, or it can lower the dose required for a certain effect, thereby cutting costs. However, most inhibitor combinations are not synergistic; and among those that are, synergy is often modest, enhancing potency less than tenfold (60). An example of synergy between entry inhibitors understood, at least partially, at the molecular level is that the activity of a CCR5 inhibitor can delay the rate of fusion of a CD4-attached Env complex, buying more time for a peptide such as T-20 to gain access to its gp41 binding site. Combinations of the CCR5 inhibitor CMPD167, or the attachment inhibitor BMS-378806, with the fusion-blocking peptide C52L increase the protection of macaques from vaginal SHIV challenge, compared with the use of single inhibitors. The synergy observed between C52L and each of the other two compounds in vitro might account for the outcome of the macaque challenges, although more studies are needed (61). Practical issues, however, hinder the development of inhibitor combinations. As emphasized above, a successful microbicide must be an inexpensive product, and using more than one compound will increase its cost even if dose-sparing effects do result from true synergy. Coformulating multiple compounds may also be problematic, particularly if chemical incompatibilities exist. In theory, new safety concerns could arise from drugdrug interactions if two compounds are used together. Another issue is procedural: the FDA requires that microbicide compounds

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be shown to have efﬁcacy individually before they can be tested as combinations (62). Such a rule means that three individual efﬁcacy trials would have to be conducted before any microbicide that contained two active ingredients could be approved, at least two of the trials being conducted sequentially. This adds enormously to the cost and duration of the product development process; any product with more than two active ingredients would, in effect, be impossible to develop under such restrictions. Some sophisticated and imaginative thinking might be required to adjust the present regulations and increase ﬂexibility.

SUSTAINED-RELEASE DEVICES FOR MICROBICIDE DELIVERY As noted above, a microbicide that conferred long-lasting protection without compromising efﬁcacy would be a major asset. Many different methods can be used to deliver substances to the vagina, but microbicide formulation strategies have tended to focus on simple gel-based systems similar to those used for conventional topical/transdermal drug delivery (63). The vaginal use of conventional gel-based systems has several problems, including (a) the limited retention of the gel within the vagina due to leakage/clearance, (b) the need to administer the gel shortly before every act of intercourse, (c) the limited duration of protection provided by a single application, and (d ) the difﬁculty in using a gel without the knowledge of the male partner (8, 13, 63–65). For all these reasons, the ﬁrst-generation, gel-based microbicide formulations may suffer from limited effectiveness and poor compliance. “Modiﬁed-release,” coitally independent formulations may be able to overcome many of these problems (63–65). Such delivery methods include so-called sustained-release and controlled-release systems. A sustainedrelease formulation releases the drug slowly, at a rate governed by the delivery system, and for a prolonged period (typically <24 h), thereby reducing the dosing frequency. For example, a

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highly retentive, sustained-release, semisolid microbicide formulation administered in the morning might be able to maintain the concentration of an inhibitor within the optimal range for the entire day and night; such a microbicide would be coitally independent. This concept is far from implausible. Even in a simple gel formulation, hydroxymethyl cellulose gel, two different entry inhibitors (CMPD167 and BMS-378806) were shown to protect macaques from the vaginal transmission of a SHIV over a multi-hour period (45). In controlled-release formulations, the substance is released at a constant rate over much longer periods (many days, up to a year or more). The inhibitor concentrations achieved soon after administration of the device do not then vary substantially over time (Figure 2) (63–65). An example of such a system is silicone elastomer rings that are designed for intravaginal use in a way that is broadly analogous to a diaphragm (although they would be doughnut-shaped, rather than cap-like, so as to permit the transit of ﬂuids to and from the uterus). The rings, costing ∼$5– $10, are loaded with ARVs and secrete them gradually without further intervention, generating intravaginal inhibitor concentrations that are constantly maintained within the optimal range for the life of the ring, perhaps as long as 3–6 months (63–65). Vaginal rings are being developed for a range of microbicide candidates, including TMC-120 (63–65) and UC781 (65). Rings loaded with TMC-120 release the inhibitor gradually into solution at concentrations well above those that have anti-HIV-1 activity in cervical tissue explants (29, 64). This kind of technology should be aggressively pursued.

PERSPECTIVES AND CONCLUSIONS The next few years will be crucial for microbicide development. It is unclear whether the failure of the Ushercell cellulose sulfate gel will predict how other polyanion-based prod-

ucts will fare in ongoing clinical trials. However, a series of high-proﬁle failures could discourage donors, governments, and potential trial volunteers, particularly if any enhancement of infection is seen again. For the next generation of microbicide candidates, more emphasis must be placed on the selection of only the most potent compounds prior to commencing a clinical trial process that moves inexorably toward phase III efﬁcacy trials. In this respect, the R5 SHIV vaginal challenge model in nonhuman primates (37, 38, 45, 45a) provides the most relevant and stringent available test of how a product might perform in humans. This model is a vitally important tool when rational choices between candidates and formulations must be made. Had the current generation of microbicide products undergone challenge studies in the primate model, fewer might have failed in efﬁcacy trials in humans (66). Irrespective of how the present generation of compounds performs clinically, the technical requirements for making a successful microbicide, although signiﬁcant, are substantially less demanding than those faced by vaccine developers. Success will require close cooperation between scientists familiar with the properties of ARVs and others experienced in developing practical products for intravaginal use. Extensive safety and efﬁcacy testing in the preclinical and animal-model arenas will also be essential, to avoid the problems seen with N9 and Ushercell. But will a microbicide be sufﬁcient to impede the global spread of HIV-1? Eventually, a combination of different prevention science strategies may be needed, and the most logical such combination involves vaccines and microbicides. Can a vaginally applied entry inhibitor substitute for a mucosal neutralizing antibody and synergize with a vaccine that induces only cellular immune responses? Whether antiviral compounds could be applied both orally and vaginally should also be considered [i.e., preexposure prophylaxis (67) plus microbicides]. To prevent sexual transmission of HIV-1, we will have to think outside the box.

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DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS The support for our microbicide studies from the NIH, grant U19 AI 65413, is gratefully acknowledged. We also thank Dr. Karl Malcolm, Queen’s University, Belfast, for the kind permission to reproduce illustrations.

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:455-471. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas Division of Infectious Diseases, Johns Hopkins School of Medicine, Baltimore, Maryland 21205

Annu. Rev. Med. 2008. 59:473–85

Key Words

The Annual Review of Medicine is online at http://med.annualreviews.org

HCV, coinfection, liver disease, injection drug use

This article’s doi: 10.1146/annurev.med.59.081906.081110

Abstract

c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0473$20.00

Hepatitis C virus (HCV) coinfection occurs in an estimated one quarter of HIV-infected persons in Europe, Australia, and the United States. As use of highly active antiretroviral drugs has markedly reduced opportunistic infections, HCV-related liver disease has emerged as a leading cause of death. HIV infection adversely affects both the natural history and the treatment of hepatitis C. Because there are no experimental models of coinfection and because the pathogenesis of each infection is incompletely understood, how HIV infection alters hepatitis C is not clear. This review considers the epidemiology, natural history, treatment, and pathogenesis of hepatitis C in HIV-infected persons.

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The majority of HIV/HCV-coinfected persons are former or current long-term inIt is estimated that 250,000 people in the jection drug users (IDUs). The basis for the HIV/HCVcoinfected: infected United States are infected with both human strong link between HIV/HCV coinfection with both human immunodeﬁciency virus 1 (HIV) and hepatitis and illicit drug use is the greater transmissiimmunodeﬁciency C virus (HCV). These HIV/HCV-coinfected bility of HCV by this route. In studies from virus and hepatitis C persons account for approximately 25% of all accidental needlestick exposures, HCV is apvirus HIV-infected persons and 8% of those with proximately tenfold more transmissible than Injection drug user hepatitis C (1). Similar proportions of HIV- HIV (4). That, coupled with the greater reser(IDU): person who infected persons are dually infected in Europe voir of HCV among IDUs, explains why peruses illicit drugs by intravenous or and in Australia (2). In sub-Saharan Africa and sons who get HIV from injection drug use ofsubcutaneous Asia, where the majority of HIV-infected per- ten already have been exposed to HCV. In one injection sons live, the frequency of HIV/HCV coinfec- large IDU cohort in Baltimore, the incidence tion varies, and in many instances remains of HCV was eight- to tenfold higher than uncertain. It has been estimated that there that of HIV (5). Likewise, in IDUs in virtually are 10 million HIV/HCV-coinfected persons all Western cities, including Seattle, Chicago, worldwide (http://www.euro.who.int/HEN/ Los Angeles, Amsterdam, Rome, Sydney, and Melbourne, the incidence of HCV infection Syntheses/hepatitisC/20050411 7). Individuals coinfected with HIV and HCV is higher than that of HIV. Consequently, comprise a distinct subset of those infected the typical HIV/HCV-coinfected person has with each virus. Persons who acquired HCV long-standing injection drug use and a season infection through blood transfusions or brief of HIV infection superimposed on decades of injection drug use are not usually dually in- chronic hepatitis C. Transmission of HCV among HIVfected with HIV. Likewise, those who acquire infected men who have sex with men has also HIV by sexual exposures are not usually inbeen reported, including several recent outfected with HCV. This point is easiest to apbreaks (6, 7). In a detailed analysis of one preciate when viewing HCV prevalence by recent outbreak in the United Kingdom, 60 HIV risk factor (Figure 1) (3). HIV-infected men who acquired HCV reported more high-risk sexual practices and 90 were more likely to have shared drugs via a 80 nasal or anal route in the preceding year in comparison with 130 controls who did not ac70 quire HCV infection (7). Interestingly, HCV 60 infections occurred in both HIV-infected and 50 HIV-uninfected men in this outbreak (7a). These data must be interpreted in the con40 text of traditionally low rates of HCV in men 30 who have sex with men, even those with HIV infection (8). Although it is difﬁcult to exclude 20 the possibility of unacknowledged or forgot10 ten prior injection drug use in these HCV outbreaks, cumulatively the reports underscore 0 that HCV infection remains a threat to men IDU Heterosexual MSM who have sex with men, and an important reaFigure 1 son not to engage in very high-risk practices. The prevalence of HCV among HIV-infected patients attending the Persons with hemophilia represent a Johns Hopkins HIV Clinic by HIV risk factor (adapted from Reference 3). IDU, injection drug user; MSM, men who have sex with men. distinct but relatively small fraction of % anti-HCV

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EPIDEMIOLOGY OF HIV/HCV COINFECTION

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HIV/HCV-coinfected persons. Transfusion transmission of HCV was a long-standing problem that peaked in the early 1970s and continued until 1991, when effective HCV screening was implemented (9). Transfusion transmission of HIV in the late 1970s and early 1980s led to HIV/HCV coinfection of some, especially those with severe hemophilia (10). As discussed below, rapid progression of liver disease has been documented in HIV/HCV-coinfected persons with hemophilia, so it is important to recognize dual infection in this population. Each virus also can be transmitted from mother to infant and by heterosexual intercourse. Mothers infected with both HIV and HCV are more likely to transmit each virus to their infants (11, 12). Infants who acquire both infections develop persistent hepatitis C, but little is known about their long-term risk of liver disease (13). It is not clear whether the risk of heterosexual transmission of either virus from HIV/HCV-coinfected persons is greater than from persons with just one infection (10). Although it is not uncommon to detect HCV infection in women who acquired HIV from heterosexual exposures, it is difﬁcult to ascertain how commonly the HCV was also sexually transmitted, since it is difﬁcult to exclude remote illicit drug use.

NATURAL HISTORY OF HEPATITIS C IN PERSONS WITH HIV INFECTION HIV infection adversely affects the natural history of hepatitis C at all stages. Approximately 30% of HIV-uninfected persons spontaneously clear HCV in the ﬁrst year of infection. Although spontaneous HCV clearance has been demonstrated in small HCV outbreaks among HIV-infected men, HIV infection reduces the likelihood of spontaneous clearance several-fold (6, 14). In one study of Baltimore IDUs, HCV persistence was detected ﬁve times more often in HIV-infected than HIV-uninfected IDUs, and the risk of

persistence was even greater in those with low CD4+ lymphocyte counts (14). This ﬁvefoldincreased risk of persistence represents cumulative cycles of reinfection among multiplyexposed IDUs rather than the increased risk attributed to a single HCV infection (15). It is not clear whether the risk of HCV persistence is measurably increased in HIV-infected persons with preserved CD4+ lymphocyte counts (e.g., >500/mm3 ). Among persons who develop persistent hepatitis C, HIV infection increases the viral “set point” as indicated by the plasma level of HCV RNA (16, 17). In some but not all studies, the viral set point continues to increase as HIV-related immunosuppression advances (18). Interestingly, effective antiretroviral therapy is actually associated with a slight increase in HCV RNA, especially in persons with pretreatment CD4+ lymphocyte counts <350/mm3 (19). HIV infection also increases the risk of cirrhosis and liver failure in persons with chronic hepatitis C. The effect of HIV on hepatitis C natural history has been most apparent in cohorts of persons with hemophilia (17). In one large hemophilia cohort, the 16-year cumulative risk of liver failure was 14.0% in HIV/HCV-coinfected persons compared to 2.6% in those with just HCV infection (17). In a meta-analysis that included many of the hemophilia cohorts, the average risk of liver failure was estimated to be sixfold higher in HIV-infected persons than in HCV-infected persons without HIV (20). The impact of HIV on HCV natural history was less apparent in some IDU cohorts, probably because of very high competing mortality (14). However, as the use of antiretroviral therapy has increased among IDUs and overall mortality has diminished, the effect of HIV on liver disease has become more visible.

Highly active antiretroviral therapy (HAART): use of three or more antiretroviral compounds, which generally began after 1996

ANTIRETROVIRAL THERAPY AND HEPATITIS C There are important interactions between highly active antiretroviral therapy (HAART) www.annualreviews.org • Hepatitis C and HIV

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and liver disease. Since the advent of HAART, liver disease has become a leading cause of death among HIV-infected persons (21–23). Estimates vary, but in most large series, liver disease is the second leading cause. In the large D:A:D study, 23,441 HIV-infected persons were followed from December 1999 through February 2004, and 1246 died (23). The primary causes of death were AIDS (31.1%) and liver failure (14.5%) (Figure 2). Many of those who died of liver-related causes had effective treatment of HIV. In this series, 54.6% of those who died of liver-related causes had achieved HIV RNA suppression <400 c/ml and half had CD4+ lymphocyte counts >200/mm3 . This is especially important because these individuals would not be expected to have died of opportunistic infections. The relative contribution of liver-related mortality is likely to increase. In a populationbased study in Denmark, mortality among HIV-infected persons dropped from 124 per 1000 person-years pre-HAART to 38 per 1000 person-years in the early HAART period (1997–1999) and to 25 per 1000 person-years once HAART use was established (2000–2005) (24). Despite this overwhelming overall success, mortality among

HIV/HCV-coinfected persons did not decline as much. From 2000 to 2005, mortality in HIV/HCV-coinfected persons was 57 per 1000 person-years compared to 19 per 1000 person-years in HCV-uninfected persons. Likewise, a recent review of death certiﬁcates in New York City demonstrated a 32.8% increase in non-AIDS-related mortality in HIV-infected persons between 1999 and 2004 (25). Clearly, the primary effect of HAART on liver-related mortality is a reduction in AIDSrelated mortality, which both increases the proportion of mortality attributed to liverrelated causes (denominator effect) and allows HIV/HCV-coinfected persons to live long enough to develop liver-related mortality. The ﬁnding that half of persons dying of liver-related causes in the D:A:Ds study had HIV RNA suppression <400 c/ml and CD4+ lymphocyte counts >200/mm3 underscores this point (23). What remains unclear is whether HAART directly affects HCV-related liver disease progression. On the one hand, HIV/HCVcoinfected persons are more likely to have grade 3–4 liver toxicity when taking HAART (26). It is conceivable that, like alcohol exposure, HAART-induced liver inﬂammation

60

Figure 2 Cause-speciﬁc mortality (per 10,000 person-years) among 23,441 HIV-infected persons followed a median of 3.5 years from December 1999 through February 2004 in the Data Collection on Adverse Events of anti-HIV drugs (D:A:D) study. From Reference 23. 476

Mortality/10,000 person-years

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50 40 30 20 10 0 AIDS

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Liver

CVD

non-AIDS cancer

Other

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could accelerate HCV-related liver disease. Some antiretroviral drugs can also cause hepatic steatosis, which has been linked to accelerated progression to cirrhosis (27). However, signiﬁcant liver enzyme elevations occur in only ∼10% of persons initiating HAART and are often self-limited, and the drugs that cause steatosis (d4T and ddI) are now infrequently used (28). On the other hand, some data suggest that HAART diminishes progression of HCVrelated liver disease, presumably by offsetting the deleterious HIV effect. If this were true, HAART might be indicated in HIV/HCVcoinfected persons at a higher CD4+ lymphocyte count to forestall progression of liver disease rather than the current threshold focused on prevention of AIDS. Reduced liver disease was ﬁrst associated with HAART by Benhamou et al. in a cross-sectional study and has been conﬁrmed by some but not all subsequent work (29–31). One recent prospective study compared HAART exposure and effectiveness in 184 HIV/HCV-coinfected persons who had at least two liver biopsies between January 1998 and July 2006. Fortyone (24%) had signiﬁcant progression (≥2point increase in the Ishak scoring system). Between biopsies, HAART was used by 51% of progressors (median exposure 0.6 years) and 65% of nonprogressors (median exposure 1.5 years), a difference that was not statistically signiﬁcant. Similarly, no signiﬁcant difference was observed in the proportion of visits with HIV RNA suppression between biopsies among progressors (43%) and nonprogressors (60%) and the median CD4+ cell count nadir between biopsies. Methodological differences between studies could explain the apparent discrepancies. Unfortunately, our limited understanding of the pathogenesis of each viral infection and the nature of HAARTinduced immune restoration makes it difﬁcult to speculate. At this point, it is fair to conclude that the timing of HAART should chieﬂy be determined by HIV RNA and CD4+ lymphocyte count, not by HCV or liver status.

PREVENTION AND TREATMENT OF HEPATITIS C IN PERSONS WITH HIV INFECTION The accelerated course of liver disease associated with HIV infection underscores the importance of hepatitis C prevention and treatment. There is no vaccine licensed for prevention of HCV infection and no approved (or effective) form of postexposure prevention of HCV infection. Thus, primary prevention has to be achieved by risk reduction (32). Because most HCV infection results from injection drug use, efforts to reduce drug use or make it safer are central to preventing HCV infection. Recent outbreaks of HCV among HIV-infected men having sex with men demonstrate that, in counseling HIVinfected persons, HCV prevention should be included as one of the reasons to practice safe sex. This advice may be especially important if inhibitions are diminished either because one already has HIV or because one is less concerned about transmitting HIV in the era of HAART. The goals of HCV treatment are suppression of plasma HCV RNA to an undetectable level (e.g., <50 IU/ml) by the end of treatment and maintenance of suppression six months after treatment is stopped, an end point referred to as a sustained virologic response (SVR) (32, 33). Among both HIV-infected persons and those without HIV, 99% of persons who achieve SVR remain free of active infection ﬁve years later (34, 35). Data are sparse, but we generally infer that persons who achieve SVR have a markedly lower risk of developing end-stage liver disease than those with persistent infection. Although acute HCV infection is uncommonly detected in HIV-infected persons, it is noteworthy that interferon alfa treatment appears to be more effective at this early stage, as is the case in persons without HIV (6, 36). The ideal timing of treatment of acute hepatitis C, the best regimen, and the optimal duration are not known even for patients

www.annualreviews.org • Hepatitis C and HIV

Sustained virologic response (SVR): HCV RNA undetectable six months after therapy is stopped; correlates with long-term “cure”

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without HIV. Nonetheless, there is growing consensus that a 24-week course of peginterferon alfa 8–12 weeks after exposure in persons with persistent HCV RNA is reasonable. Beginning treatment even earlier in HIV/HCV-coinfected persons may be justiﬁable because of the greater risk of persistent infection and lower likelihood of treatment response once persistence has been established. The best available treatment of chronic hepatitis C in HIV-infected persons is the combination of peginterferon alfa and ribavirin, as shown in 2004 by four “pivotal” studies in which HIV/HCV-coinfected patients were randomized to receive 48 weeks of peginterferon alfa (Table 1) (37–40). In the largest study (APRICOT), 868 persons were randomized to receive either standard interferon alfa-2a (3 mU, tiw) plus ribavirin (800 mg daily), peginterferon alfa-2a 180 μg per week plus placebo, or peginterferon alfa2a 180 μg weekly plus ribavirin 800 mg daily. The SVR rates were 12%, 20%, and 40%, respectively (38). For persons with genotype 1 infection, the SVR rate was 29% with peginterferon alfa and ribavirin, whereas SVR was observed in 62% of those with genotype 2 or 3 infection. Table 1

The pretreatment level of HCV RNA is an important determinant of response likelihood. In the APRICOT study, SVR rates were >60% in genotype 1–infected persons who were randomized to peginterferon alfa and ribavirin for 48 weeks and had a HCV RNA level ≤800,000 IU/ml compared to 18% for genotype 1–infected persons with higher HCV RNA levels and the same treatment (38). Interestingly, the SVR rate in APRICOT for HIV/HCV-coinfected persons with genotype 1 infection and HCV RNA level ≤800,000 IU/ml (61%) is similar to that reported in a study of HIV-uninfected persons with the same genotype, HCV RNA level threshold, and therapy (55%) (38, 41). As in persons without HIV infection, failure to suppress HCV RNA on treatment has a high negative predictive value. In the four studies summarized in Table 1, <2% of persons whose HCV RNA was still detectable 12 weeks after starting treatment (or whose HCV RNA level was not reduced by at least 2 logs) went on to SVR (37–40). Other important information also came from the APRICOT study. Pre- and posttreatment liver biopsies were evaluated on 401 subjects (42). A histologic response (reduction of at least 2 points from baseline in the

Four pivotal studies of treatment of chronic hepatitis C in HIV-infected persons, published in 2004

Characteristic

APRICOT

ACTG 5071

RIBAVIC

∗

Barcelona

Number enrolled

868

133

412

95

Peginterferon

2a

2a

2b

2b

Ribavirin

800 mg

600 up to 1g

800 mg

0.8 g, 1 g, 1.2 ga

>200/mm3

>100/mm3

>200/mm3

>250/mm3 and HIV RNA <10,000 c/ml

ALT

“elevated” twice

NA

NA

>1.5 ULN

% Genotype 1b

60

77

48

55

% bridging ﬁbrosis or cirrhosisb

12

11 (cirrhosis)

39

29

29%

14%

17%

38%

HIV and

CD4+

status

or 100–200/mm3 and HIV RNA <5000 c/ml

Genotype 1 peg-ribavirin SVR ∗

ratec

and HIV RNA <10,000 c/ml

ALT, alanine aminotransferase; ULN, upper limit of normal; c/ml, copies/ml; NA, not applicable. Based on body weight <65, 65–75, >75 kg. b Taken from peginterferon and ribavirin arm; cirrhosis deﬁned as F4–6 MHAI or F3–4 metavir and Scheurer. c Refers to the sustained virologic response (SVR) rate for HIV-infected persons taking peginterferon and ribavirin. Rates are for patients with genotype 1 hcv infection except for the RIBAVIC and Barcelona studies that grouped genotypes 1 and 4. a

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24-point Ishak modiﬁed HAI score) was observed more often in those who received peginterferon plus ribavirin (57%) than in those who received peginterferon plus placebo (39%) or standard interferon plus ribavirin (41%). Among those without SVR, 28% had a histologic response, 48% were unchanged, and 25% worsened. Medication was discontinued in 25% of those taking peginterferon alfa and ribavirin; in 15% this was due to adverse events. The CD4+ lymphocyte count dropped an average of 157 cells/mm3 , but median CD4+ lymphocyte percent did not decline. Hepatic decompensation occurred in 14 of the 860 patients. In each instance, subjects had Child-Pugh scores of 5 or higher at baseline; also associated with decompensation were other markers of cirrhosis such as low platelet counts and ddI use (43). Among those in whom HIV RNA was detected at baseline, HIV RNA levels actually declined an average of 0.696 log10 copies/ml in the peginterferon and ribavirin arm. It is possible that higher SVR rates can be achieved in HIV-infected persons by longer therapy or by higher doses of peginterferon or ribavirin. In many studies, the ribavirin dose was 800 mg per day to diminish ribavirinassociated anemia, which is a greater problem in HIV-infected persons, especially those taking azidothymidine (AZT) (44). However, ribavirin doses similar to those used in HIV-uninfected persons (1.0 g ≤75 kg and 1.2g >75 kg) are currently recommended for HIV/HCV-coinfected persons (33). HIV/HCV-coinfected persons have additional safety concerns with peginterferon and ribavirin therapy. Ribavirin-associated anemia may be a greater problem in coinfected persons than in the HIV-uninfected, a problem that is compounded by concomitant AZT use (44). Ribavirin also raises ddI levels and causes toxicity including fatal hyperlactatemia (45). Although interferon alfa therapy is associated with a dose-related reduction in white blood cell count and absolute CD4+ count, the percentage of CD4+ cells remains essentially unchanged, and interferon alfa therapy is not as-

sociated with the development of opportunistic infections (37–40). Liver failure has also occurred in HIV/HCV-coinfected persons on peginterferon alfa and ribavirin therapy, especially when treatment is started in persons who already have Child’s B cirrhosis (43). Peginterferon alfa is contraindicated in persons who already have liver failure. Thus, liver transplantation is the only treatment available for HIV/HCV-coinfected persons with decompensated cirrhosis (that is, Child’s B or C cirrhosis) (46). However, transplant remains an investigational procedure, is very expensive, and is unavailable for many HIV/HCV-coinfected persons. Clearly, prevention of liver failure must remain the primary goal. Unfortunately, the overall effectiveness of HCV treatment of HIV/HCV-coinfected persons is even lower than the efﬁcacy mentioned above. In one university-based HIV clinic in Baltimore, a hepatitis C treatment center was established in 1998, but through 2003 only 277 (33%) of 845 HIV/HCVcoinfected persons were referred to this center for HCV care (Figure 3) (47). The overall referral rate increased from <1% in 1998 to 28% in 2003; however, even in 2003, 65% of HIV/HCV-coinfected persons with CD4+ cell count >200 cells/ml were not referred. Interestingly, only 67% of 277 patients referred kept their appointment, and only 68% of them completed their pretreatment evaluation. Of the remaining 125, only 69 (55%) were medically eligible for treatment, and 29 (42%) underwent HCV treatment; SVR was achieved by six (21%), who represented only 0.7% of the full cohort. Compared to this predominantly genotype 1 (90%) and AfricanAmerican (70%) population, higher effectiveness rates are reported in other locations, such in Spain, where patients are Caucasians with a greater proportion of genotype 3 infection. Nonetheless, these data indicate that the challenge of reducing the burden of liver disease in HIV/HCV-coinfected persons is larger than merely developing more potent antiviral compounds. www.annualreviews.org • Hepatitis C and HIV

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HIV/HCV co-infected patients in regular HIV care N=845

Not referred N=568

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Figure 3

Referred N=277

Not complete >1 appointment N=92

Outcome of 845 HIV/HCVcoinfected patients in regular care (at least one visit per year for at least two years) at the Johns Hopkins HIV Clinic after establishment of a hepatitis C treatment center. From Reference 47.

Completed >1 appointment N=185

Incomplete evaluation N=60

Treatment ineligible N=56

Complete evaluation N=125

Treatment deferred N=40

Non-response N=23

PATHOGENESIS OF HEPATITIS C IN PERSONS WITH HIV INFECTION The pathogenesis of HIV/HCV coinfection is poorly understood. Direct viral interactions are not likely. HCV chieﬂy replicates in hepatocytes, which are not known to express CD4+ and have not convincingly been shown to support HIV replication. HCV replication in monocytes and lymphocytes has been inferred, but only at low levels and in a minority of cells (19, 48). Given that each virus infects at most a minority of CD4+ mononuclear cells, direct viral interactions are unlikely to explain much of the pathogenesis. HIV infection and the subsequent immune activation markedly affect adaptive immune responses to many infections and immunizations. HIV preferentially infects activated CD4+ lymphocytes and affects the function and number of uninfected CD4+ lymphocytes, which are necessary for spontaneous clearance of HCV infection (49). The extent of CD4+ lymphocyte + lymphocyte depletion (so-called nadir) has also been 480

Thomas

Treatment initiated N=29

Sustained virologic response N=6

linked with the magnitude of reduction in HCV-speciﬁc CD8+ lymphocyte responses found in HIV-infected persons (who interestingly do not have lower CD8+ lymphocyte responses speciﬁc to Epstein-Barr virus or cytomegalovirus) (50, 51). CD4+ lymphocyte suppression has been correlated with diminished HCV-speciﬁc humoral responses (52). Thus, HIV-related CD4+ lymphocyte depletion probably contributes to the clinical observation (discussed above) that HIV infection reduces the likelihood of spontaneous HCV clearance. There is very limited understanding of how HIV accelerates liver disease progression. Increased STAT1 activation and Fas ligand expression in HIV-infected persons may lead to increased hepatocyte apoptosis (53). In one study, HIV/HCV-coinfected persons had greater expression of the proﬁbrotic cytokine transforming growth factor beta 1 than HCV-uninfected persons (54). These studies provide clues to the interaction, but it has been very difﬁcult to learn more in the absence of experimental models.

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The nature of CD4+ lymphocyte restoration associated with HAART is also poorly understood. Some studies found that HCVcoinfected persons had less robust increases in peripheral CD4+ lymphocyte number following HAART, even after adjustment for HIV RNA suppression (55). This ﬁnding has not been consistently conﬁrmed, and whether restored peripheral CD4+ lymphocytes affect HCV-speciﬁc adaptive immunity is unknown (56). The study by Kim et al. (50, 51) suggests that, at least in the near term and for CD8+ responses, the degree of initial HIV-related immunosuppression (CD4+ lymphocyte nadir) is more important. Interestingly, Chung and coworkers (57) demonstrated that HCV RNA levels in plasma actually increase in the ﬁrst months after HAART and do so more in persons with low pretreatment CD4+ lymphocyte counts. Whether this represents extrahepatic HCV replication in restored CD4+ lymphocytes is unknown.

FUTURE DIRECTIONS In the future, HCV-related liver disease will continue to be a major cause of morbidity and mortality among HIV-infected persons in settings where antiretroviral therapy is provided. In fact, as integrase inhibitors, CCR5 competitors, and other new compounds magnify the antiretroviral impact (and as HIV-infected persons age), the contribution of HCV-related liver disease to morbidity and mortality is likely to increase. Clearly, the greatest challenge is to improve hepatitis C prevention and treatment in HIVinfected persons. A promising anti-HCV drug pipeline makes it likely that potent antiHCV compounds will be available by 2015. Thus, the major challenge will be to deliver novel treatments to the large proportion of HIV/HCV-coinfected persons not engaged in medical care and to forestall liver disease in the interim by optimizing existing treatment options. A comprehensive synopsis of future

Table 2 Future research priorities to diminish the impact of liver disease in HIV-infected persons (National Institutes of Health Action Plan for Liver Disease Research) Intermediate term (4–6 years)

Long term (7–10 years)

High risk

Deﬁne effects of HIV infection on the liver, including on different populations of liver cells

Short term (0–3 years)

Develop noninvasive means of detecting early hepatic mitochondrial dysfunction

Develop in vitro or in vivo models of HIV-HCV and HIV-HBV coinfection Develop means to reliably attribute causality of drug-induced liver disease in HIV-infected persons

Intermediate risk

Deﬁne safety and efﬁcacy of peginterferon therapy for acute hepatitis C in HIV coinfection

Elucidate mechanisms by which HIV infection accelerates ﬁbrosis and disease progression in HBV and HCV infection Deﬁne factors that lead to reactivation of HBV in HIV coinfection and develop means of prevention

Develop noninvasive means of assessing liver disease stage and activity in HIV-infected persons

Low risk

Develop improved regimens of HAV and HBV vaccination Deﬁne short- and long-term safety and efﬁcacy of peginterferon and ribavirin in different subpopulations of patients with HIV-HCV coinfection

Deﬁne whether long-term peginterferon slows progression of disease in chronic hepatitis C with HIV coinfection Deﬁne prevalence, etiology, and severity of different liver disease in different cohorts of HIV-infected patients

Develop optimal therapeutic regimens for chronic hepatitis B in different stages and patterns of disease in HIV-coinfected patients Determine safety and efﬁcacy of new agents for therapy of hepatitis C in HIV coinfection

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research priorities to diminish the impact of liver disease in HIV-infected persons is maintained as Chapter 6 of the National Institutes

of Health Action Plan for Liver Disease Research (http://liverplan.niddk.nih.gov) (Table 2).

SUMMARY POINTS 1. Because of shared transmission routes, approximately one quarter of HIV-infected persons in the Western world are HCV-coinfected. 2. Liver disease caused by HCV infection is a leading cause of death among HIV-infected persons with access to antiretroviral drugs.

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3. HIV accelerates the natural history of hepatitis C and reduces the likelihood of treatment response. 4. Just as in persons without HIV infection, the combined use of peginterferon and ribavirin is the standard of care for treatment of hepatitis C in HIV-infected persons. 5. The pathogenesis of HIV/HCV coinfection is poorly understood. 6. New and more potent treatments for HCV are anticipated and will increase the importance of expanding access to diminish the global impact of liver disease.

DISCLOSURE STATEMENT The author has served on advisory boards for Human Genome Sciences and Sandhill.

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28. Khella SL, Frost S, Hermann GA, et al. 1995. Hepatitis C infection, cryoglobulinemia, and vasculitic neuropathy. Treatment with interferon alfa: case report and literature review. Neurology 45:407–11 29. Benhamou Y, Bochet M, Di Martino V, et al. 1999. Liver ﬁbrosis progression in human immunodeﬁciency virus and hepatitis C virus coinfected patients. The Multivirc Group. Hepatology 30:1054–58 30. Brau N, Salvatore M, Rios-Bedoya CF, et al. 2006. Slower ﬁbrosis progression in HIV/HCV-coinfected patients with successful HIV suppression using antiretroviral therapy. J. Hepatol. 44:47–55 31. Qurishi N, Kreuzberg C, Luchters G, et al. 2004. Effect of antiretroviral therapy on liverrelated mortality in patients with HIV and hepatitis C coinfection. Lancet 362:1708–13 32. Strader DB, Wright T, Thomas DL, et al. 2004. Diagnosis, management, and treatment of hepatitis C. Hepatology 39:1147–71 33. Alberti A, Clumeck N, Collins S, et al. 2005. Short statement of the ﬁrst European consensus conference on the treatment of chronic hepatitis B and C in HIV-coinfected patients. J. Hepatol. 42:615–24 34. Reichard O, Glaumann H, Fryden A, et al. 1999. Long-term follow-up of chronic hepatitis C patients with sustained virological response to alpha-interferon. J. Hepatol. 30:783–87 35. Soriano V, Maida I, Nunez M, et al. 2004. Long-term follow-up of HIV-infected patients with chronic hepatitis C virus infection treated with interferon-based therapies. Antivir. Ther. 9:987–92 36. Dominguez S, Ghosn J, Valantin MA, et al. 2006. Efﬁcacy of early treatment of acute hepatitis C infection with pegylated interferon and ribavirin in HIV-infected patients. AIDS 20:1157–61 37. Carrat F, Bani-Sadr F, Pol S, et al. 2004. Pegylated interferon alfa-2b vs standard interferon alfa-2b, plus ribavirin, for chronic hepatitis C in HIV-infected patients: a randomized controlled trial. JAMA 292:2839–48 38. Torriani FJ, Rodriguez-Torres M, Rockstroh JK, et al. 2004. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection in HIV-infected patients. N. Engl. J. Med. 351:438–50 39. Chung RT, Andersen J, Volberding P, et al. 2004. Peginterferon alfa-2a plus ribavirin vs interferon alfa-2a plus ribavirin for chronic hepatitis C in HIV-coinfected persons. N. Engl. J. Med. 351:451–59 40. Laguno M, Murillas J, Blanco JL, et al. 2004. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for treatment of HIV/HCV coinfected patients. AIDS 18:F27–36 41. Hadziyannis SJ, Sette H Jr., Morgan TR, et al. 2004. Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose. Ann. Intern. Med. 140:346–55 42. Lissen E, Clumeck N, Sola R, et al. 2006. Histological response to pegIFNalpha-2a (40KD) plus ribavirin in HIV-hepatitis C virus coinfection. AIDS 20:2175–81 43. Mauss S, Valenti W, Depamphilis J, et al. 2004. Risk factors for hepatic decompensation in patients with HIV/HCV coinfection and liver cirrhosis during interferon-based therapy. AIDS 18:F21–25 44. Alvarez D, Dieterich DT, Brau N, et al. 2006. Zidovudine use but not weight-based ribavirin dosing impacts anaemia during HCV treatment in HIV-infected persons. J. Viral Hepat. 13:683–89 45. Lafeuillade A, Hittinger G, Chadapaud S. 2001. Increased mitochondrial toxicity with ribavirin in HIV/HCV coinfection. Lancet 357:280–81

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46. Neff GW, Bonham A, Tzakis AG, et al. 2003. Orthotopic liver transplantation in patients with human immunodeﬁciency virus and end-stage liver disease. Liver Transpl. 9:239–47 47. Mehta SH, Lucas GM, Mirel LB, et al. 2006. Limited effectiveness of antiviral treatment for hepatitis C in an urban HIV clinic. AIDS 20:2361–69 48. Lanford RE, Chavez D, Von Chisari F, et al. 1995. Lack of detection of negative-strand hepatitis C virus RNA in peripheral blood mononuclear cells and other extrahepatic tissues by the highly strand-speciﬁc rTth reverse transcriptase PCR. J. Virol. 69:8079–83 49. Grakoui A, Shoukry NH, Woollard DJ, et al. 2003. HCV persistence and immune evasion in the absence of memory T cell help. Science 302:659–62 50. Kim AY, Lauer GM, Ouchi K, et al. 2005. The magnitude and breadth of hepatitis C virusspeciﬁc CD8+ T cells depend on absolute CD4+ T-cell count in individuals coinfected with HIV-1. Blood 105:1170–78 51. Kim AY, Schulze zur WJ, Kuntzen T, et al. 2006. Impaired hepatitis C virus-speciﬁc T cell responses and recurrent hepatitis C virus in HIV coinfection. PLoS Med. 3:e492 52. Netski DM, Mosbruger T, Astemborski J, et al. 2007. CD4+ T cell-dependent reduction in hepatitis C virus-speciﬁc humoral immune responses after HIV infection. J. Infect. Dis. 195:857–63 53. Balasubramanian A, Ganju RK, Groopman JE. 2006. Signal transducer and activator of transcription factor 1 mediates apoptosis induced by hepatitis C virus and HIV envelope proteins in hepatocytes. J. Infect. Dis. 194:670–81 54. Blackard JT, Kang M, Clair JB, et al. 2007. Viral factors associated with cytokine expression during HCV/HIV co-infection. J. Interferon Cytokine Res. 27:263–70 55. Greub G, Ledergerber B, Battegay M, et al. 2000. Clinical progression, survival, and immune recovery during antiretroviral therapy in patients with HIV-1 and hepatitis C virus coinfection: the Swiss HIV Cohort Study. Lancet 356:1800–5 56. Sulkowski MS, Moore RD, Mehta SH, et al. 2002. Hepatitis C and progression of HIV disease. JAMA 288:199–206 57. Contreras AM, Hiasa Y, He W, et al. 2002. Viral RNA mutations are region speciﬁc and increased by ribavirin in a full-length hepatitis C virus replication system. J. Virol. 76:8505–17

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:473-485. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

Contents

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

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Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection ¨ Luc Geeraert,1 Gunter Kraus,1 and Roger J. Pomerantz2 1

Tibotec BVBA, BE-2800 Mechelen, Belgium

2

Tibotec Inc., Yardley, Pennsylvania 19067; email: [email protected]

Annu. Rev. Med. 2008. 59:487–501

Key Words

First published online as a Review in Advance on September 10, 2007

HAART, latency, viral reservoir, eradication

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.59.062806.123001 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0487$20.00

Abstract The success of highly active antiretroviral therapy (HAART) for HIV-1 infection has sparked interest in mechanisms by which the virus can persist despite effectively suppressive therapy. Latent HIV-1 reservoirs established early during infection not only prevent sterilizing immunity but also represent a major obstacle to virus eradication. When HIV-1 gains a foothold in the immunologic memory or in certain inaccessible compartments of the human body, it cannot be easily purged by HAART and is able to replenish systemic infection on treatment interruption. Because latently infected cells are indistinguishable from uninfected cells, deliberate activation of latent infection combined with intensiﬁed HAART seems to be the best strategy to combat latent infection. Initial hypothesis-driven clinical trials did not achieve their ultimate goal, although they provided valuable insight for the design of future eradication protocols. A more detailed understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 reservoirs will be critical in developing new eradication approaches.

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INTRODUCTION

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HAART: highly active antiretroviral therapy

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The combination of antiretrovirals from different classes in highly active antiretroviral therapy (HAART) has greatly extended survival and improved quality of life for HIV-1-infected individuals (1). The ﬁnding that HAART could reduce plasma viremia to clinically undetectable levels (<50 copies per milliliter plasma) within two months in many patients (2–4) raised hopes for the eradication of HIV-1. However, suppression of viral replication by HAART unveiled the existence of long-lived HIV-1 reservoirs (5–7) that occur with extremely low frequency (106 –107 latently infected cells per individual, i.e., 0.1–1 cell per million lymphocytes) (8). Viral latency is a characteristic of many viruses, including retroviruses, adenoviruses, and many members of the herpesviridae. Unlike other viruses, retroviruses do not have a latent state between episodes of active viral replication in untreated patients. Hence, HIV-1 latency is mainly a consequence of the virus’s replication in activated CD4+ T lymphocytes, which are a critical part of the immune system (9). After binding to CD4 and a chemokine receptor (CCR5 or CXCR4), HIV-1 enters the host cell, and the viral single-stranded RNA template is reverse transcribed into a double-stranded DNA molecule by the viral reverse transcriptase enzyme. This viral DNA is then integrated into the host cell genome, an event that is mediated by the viral integrase enzyme and creates the integrated provirus. While the infected CD4+ T cell remains activated, the proviral DNA initiates new rounds of viral replication (10–12). Such infected activated T cells have a short half-life owing to HIV-1mediated cytopathic effects and host cytolytic effector mechanisms. However, some activated CD4+ T cells return to a resting state and become memory cells that no longer permit viral replication. As such, the integrated provirus persists throughout the lifespan of the infected memory T cell—which may be years, given that these cells form the basis of

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immunologic memory (13, 14). When resting infected CD4+ T cells become activated, virus production resumes. In addition to resting CD4+ T cells, a minor HIV-1 population is suspected to persist in other cell types, discussed later in this review. Because of these long-lived viral reservoirs, current antiretroviral regimens cannot purge HIV-1 from the system (15). The therapy does largely prevent establishment of additional infected cells in the reservoir and restrain viral production from the reservoir; however, it is possible that resistant virus arising because of suboptimal therapy is stored in the reservoir (16–18), making it an archive that encompasses the original wild-type virus as well as drug-resistant mutants. From the reservoir, a very low level of new HIV-1 virus is continuously produced by hidden or cryptic replication (19). Discontinuation of therapy triggers a new systemic infection from the latent reservoir (20). Thus, when the therapeutic goal is viral eradication, every latent reservoir must be targeted and cleared.

VIRAL DYNAMICS AND THE LATENT RESERVOIR HIV-1 replicates extraordinarily rapidly in infected individuals, with a virion half-life in plasma of <6 h (4). Well over 99% of this viral replication occurs in activated and productively infected CD4+ T lymphocytes in the peripheral blood and lymphoid tissue. In HAART-naive patients receiving therapy for the ﬁrst time, analysis of plasma viral loads demonstrates a reduction of viremia in at least three distinct phases. In the ﬁrst phase, the viral load drops by one or two orders of magnitude in two weeks (11, 12). Two processes contribute to this rapid decay: the clearance of free virions and the decay of short-lived CD4+ T cells that were productively infected before initiation of therapy (21). In the second phase, plasma virus loads decline at a slower pace. The loss of longlived infected cells (half-life of 1–4 weeks)

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is a major feature of this phase, whereas activation of latently infected lymphocytes is only a minor one (4). It is presumed that infected macrophages and monocytes form the majority of this population because of their longevity and the minimal cytopathic effect in these cells (22, 23). The viral pool associated with follicular dendritic cells is an additional signiﬁcant reservoir of HIV-1 virions, comprising a large fraction of viral burden in an infected patient (24). In response to HAART, this pool shows biphasic decay with a fast two-day onset followed by a slower steady dissociation rate (3, 25). The loss of virus trapped on the follicular dendritic cell network is another major source of second-phase virus decay, although the long-term clearance kinetics remains unknown (25). The dominant species of HIV-1 DNA in infected resting CD4+ T cells is trapped in its preintegration complex and residing in the cytoplasm. This full-length or nearly full-length DNA can be linear or in the form of long terminal repeat (LTR) circles (8, 26, 27). A small fraction of this unintegrated viral DNA is replication-competent, and because it is prone to degradation (28, 29), this labile reservoir is believed to be cleared during the ﬁrst weeks of HAART. After several months of HAART, plasma viremia is reduced to clinically undetectable levels in many patients (2–4). This stage is considered the third phase of viral decay (30). Although undetectable with current clinical assays, a low level of virus is present in plasma and elsewhere (31, 32). This residual viremia is only detectable with very sensitive methods. Decay rates of the residual viremia are slow and tend to decrease with time, suggesting a heterogeneous and dynamic composition of the latent pool (33). Resting CD4+ T lymphocytes with integrated provirus represent an extremely stable reservoir that is believed to be the prime constituent of the latent pool. These T cells have been observed in the peripheral blood of HIV-1-infected individuals after treatment effectively suppressed most productive infec-

tion (8). Their presence is not conﬁned to any particular location as they populate the lymphoid tissues and circulate continuously. The latent CD4+ T cell reservoir is established soon after primary HIV-1 infection, and even initiation of HAART before seroconversion or during perinatal HIV-1 infection is unable to prevent its establishment (16, 34). Only a minute fraction (<0.05%) of the resting CD4+ T cell population carries integrated provirus because infected CD4+ cells rarely survive long enough to revert back to a resting memory state carrying integrated provirus (8, 9). HIV-1 also infects other cells, which may offer additional opportunities for HIV-1 latency. Low levels of integrated virus were found in resting newly generated CD4+ T lymphocytes in patients on HAART. It was suggested that such naive CD4+ T cells are rarely directly infected but that the naive cells harboring provirus are generated via reversion from a memory phenotype (35) or via direct viral infection of thymocytes in both children and adults (36). Furthermore, HIV-1 integrated in the genome of cells with a proliferative capacity, such as stem cells in the monocyte-macrophage lineage, have been hypothesized to be a source of residual viremia (37). In addition, the gut-associated lymphoid tissue (GALT) appears to be an important site for early HIV-1 replication (38); this implicates mucosal lymphoid tissues with high baseline levels of T lymphocyte activation as probable critical reservoirs outside of the peripheral blood. Another potential reservoir for HIV-1 exists in the tubular epithelial cells of the kidney (39). Tissues that maintain blood-tissue barriers may limit penetration of certain antiretroviral agents and act as partial “drug sanctuaries.” These compartments would potentially include the central nervous system, the retina, and the testes (40–42). Release of virus by activation of latently infected cells replenishes the viral load, and owing to statistical variation around mean HIV-1 levels below 50 copies per milliliter, www.annualreviews.org • Viral Persistence in HIV-1 Infection

LTR: long terminal repeat

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intermittent episodes of clinically detectable viremia (blips) can be found. Based on viral reservoir decay characteristics, it was suggested that patients treated with current standard-of-care therapy need to continue therapy for 60 years for potential eradication (43). Unfortunately, even this estimate may not represent the worst-case scenario for patients, as the analysis did not fully take into account low-level viral replication in CD4+ T lymphocytes and tissues aside from lymphoid organs. The continual replenishment of the latent pool from these sources is a major cause of its persistence, apart from its slow intrinsic decay characteristics. More potent and tolerable drug regimens that can completely block HIV-1 replication will be essential to eliminate the latent reservoir (19).

MECHANISMS OF LATENCY INDUCTION HIV-1 latency is best described as a lack of proviral gene expression. The reasons for this lack of transcription are manifold. Because the provirus is integrated in the host genome, its activity is signiﬁcantly affected by the metabolic and activation state of the host cell. The presence of the transcriptionally silent provirus in the genome remains the

only known difference between latently infected resting CD4+ T lymphocytes and their uninfected counterparts. There are no markers that can distinguish latently infected from uninfected T cells. Integration of viral DNA into the host genome is an essential step in the viral replication cycle. Viral DNA seems to have preferred integration sites and is mainly integrated into introns of transcriptionally active genes (44– 46). The molecular mechanism of integration site selection remains unclear, but the nature of the chosen site inﬂuences HIV-1 transcription levels in active CD4+ T lymphocytes (47). The HIV-1 LTR, located at the 5 end of the integrated provirus, contains the promotor and enhancer elements that regulate HIV-1 expression (48) (Figure 1). Transcription factors include cellular activation signals and the HIV-1 encoded transactivating protein Tat (trans-activator of transcription). The absence of these factors in resting CD4+ T lymphocytes contributes to HIV-1 latency. Tat is encoded by multiply spliced viral transcripts and binds to the transactivation responsive element (TAR), an RNA stem-loop structure located at the 5 end of all nascent viral transcripts. When bound, it recruits the positive transcription

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 1 Mechanisms of latency induction and activation. (A) Organization of the HIV-1 5 -LTR promotor. LTR consists of the U3, R, and U5 regions; transcription is initiated at the junction of U3 and R. Locations of binding sites for cellular transcription factors that interact with the U3 region of LTR are indicated. (B ) In an HIV-1-infected resting T cell, two positioned nucleosomes may prevent transcription factors from accessing LTR. Histone deacetylases such as HDAC1 stabilize nucleosomes by deacetylating regional histones and compacting histone structure; e.g., HDAC1 is recruited to the nucleus by NF-κB1 (p50 homodimer) bound to LTR tandem NF-κB binding sites (93). (C ) TNF-α, NF-κB, or Tat activity results in recruitment of histone acetyl transferases (HATs) and displacement of HDACs; e.g., TNF-α liberates NF-κB (p50/p65 heterodimer), which replaces constitutively LTR-bound NF-κB1, thus removing HDAC1 (93). The resulting disruption of the nuc-1 nucleosome relaxes chromatin and may increase accessibility to transcription factors and RNA polymerase II (RNA pol II). These effects could be mimicked with HDAC inhibitors. (D) T cell activation leads to recruitment of cellular transcription factors such as NF-κB and NF-AT to the nucleus. Consequently, HIV-1 transcription is upregulated. Recruitment of the kinase P-TEFb results in phosphorylation of the C-terminal domain site of RNA pol II, causing transcription elongation. Immune activation therapy also leads to T cell activation and subsequent events. (E ) Tat protein binds to TAR (see text for details), thereby increasing transcription rate and stimulating transcriptional elongation through recruitment of P-TEFb. 490

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YY1

TBP

Sp-1 YY1

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antigen

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GR

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modulatory region

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5'-LTR mRNA

U3 R

enhancer core region promotor

HDAC1 p50

HAT HDAC inhibitors

nuc-0

p65 NF-κB

P-TEFb Tat

nuc-0

U5

leader region

nuc-1

C HDAC1

TAR

nuc-1

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P

nuc-0 RNA pol II

E P

P

RNA pol II

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elongation factor b (P-TEFb) kinase, which enables transcriptional elongation by hyperphosphorylating the cellular RNA polymerase type II (RNA pol II) (49, 50). In the absence of cellular activation signals or Tat, the RNA pol II complex, attached to the HIV-1 promoter, constitutively initiates the synthesis of abortive short RNA transcripts (50–52). In the presence of Tat, the RNA pol II complex generates full-length HIV-1 transcripts (53, 54). HIV-1 gene expression is controlled by cellular transcription factors as well. When T cells are activated by their speciﬁc antigen, cell-cell contact, or cytokine signaling, nuclear factor of activated T cell (NF-AT) and NF-κB are recruited to the nucleus, and virus gene expression is upregulated (55–58). Both NF-AT and NF-κB act synergistically with Tat to upregulate viral gene expression (59). Interaction of nuclear NF-κB with its two tandem binding sites in the LTR has an important role not only in upregulating HIV-1 transcription but also in transcriptional elongation by recruiting P-TEFb. Both active NF-AT and NF-κB are absent in resting T cells. In addition, the HIV-1 LTR contains three binding sites for the ubiquitous transcription factor Sp-1 and putative binding sites for many other transcription factors. The presence of consensus sequences for host transcription factors allows HIV-1 to continuously monitor the host immune status. As HIV-1 DNA is integrated into the host genome, the chromatin conformation at the integration site determines whether the provirus is transcriptionally active. Access of transcription factors to the LTR is critical for high-level HIV-1 transcription, and heterochromatin structure may prevent them from binding (60). In the 5 LTR of integrated provirus, two precisely positioned nucleosomes are present. Activation of HIV-1 gene expression is accompanied by the disruption of the positioned nucleosome nuc-1, immediately downstream of the viral transcription initiation site (61). This nucleosome disruption can be induced by histone acetylation via displacement of histone deacetylases

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HDAC: histone deacetylase

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(HDACs), a consequence of the recruitment of histone acetyltransferases by NF-κB or Tat. This event can be mimicked with HDAC inhibitors (62). Also, by an unknown mechanism, phorbol esters and tumor necrosis factor alpha (TNF-α) speciﬁcally disrupt nuc-1. In general, histone acetylation acts as a signal for the recruitment of chromatin remodeling complexes.

IMPROVING HAART Given the extremely long half-life of the latent reservoir, current antiretroviral drug regimens are unlikely to eradicate HIV-1 from an infected individual. The viral reservoirs established early in HIV-1 infection remain mostly unaffected by HAART and are able to replenish systemic infection when drug treatment is interrupted. The emergence of a new wave of very potent antiviral agents could foster the hope of eradicating the latent pool by sustained antiviral therapy. HAART could be expanded to include potent second-generation protease inhibitors (e.g., darunavir, tipranavir), reverse transcriptase inhibitors (e.g., TMC125, TMC278), or agents aiming at new targets such as HIV-1 fusion (enfuvirtide), viral integrase (raltegravir, elvitegravir), or the CCR5 coreceptor (maraviroc). Adding such agents to standard HAART could potentially reduce the latent reservoir’s half-life to a sufﬁcient extent. However, given the persistent nature of provirus in latently infected cells, it is doubtful that intensiﬁcation of HAART alone will be enough (63–65). Presumably, a deliberate activation of latently infected cells will be required to sufﬁciently accelerate purging of the long-lived latent reservoir. Several treatment strategies have been attempted to this end (Table 1). In theory, a reactivation of HIV-1 expression in latently infected cells would make them vulnerable to elimination by viral cytopathic effects and host immune response. Such an activation approach would not lead to infection of healthy cells because virus spread

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Eradication protocols used in clinical trials

Regimen

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Baseline HAART intensification

Potential mechanism of action

Evidence for potential efficacy

Outcome

References

IL-2

IAT; direct T cell stimulation

Reduction of resting CD4+ T cell pool containing replicationcompetent HIV-1. Instances where no virus could be isolated from peripheral blood, lymph nodes, CSF, resting T cell pool

Re-emergence of latent pool and plasma viremia within 2–3 weeks of therapy discontinuation

68, 70–72

IL-2 and IFN-γ

IAT; direct T cell and macrophage stimulation plus release of proinﬂammatory cytokines

Proviral DNA decrease and positive effects on immune reconstitution

Rebounding plasma HIV-1 RNA levels within a few weeks of therapy discontinuation

73

IL-2 and OKT3

IAT; direct T cell stimulation plus release of proinﬂammatory cytokines

Vigorous immune activation, T cell activation and proliferation, stimulation of HIV-1 replication, long-lasting CD4+ T cell depletion

No signiﬁcant decrease of total HIV-1 DNA

74–76

IAT; direct T cell stimulation

Undetectable replicationcompetent virus after treatment, plasma viral RNA either undetectable or <5 copies/ml

Rebound of plasma virus within weeks of therapy discontinuation

80, 81

Elimination of latently infected cells and available uninfected target cells

Lower CD4+ and total lymphocyte counts

Cyclophosphamide addition to HAART did not result in additional reduction of peripheral blood and lymph node virus

84

HDAC inhibitor; relaxation of heterochromatin structure resulting in transcriptional activation of provirus

Accelerated clearance of HIV-1 from resting CD4+ T cells and pronounced shortening of latent reservoir half-life

Addition of long-term valproic acid did not signiﬁcantly increase the time to virologic relapse after therapy discontinuation

89–91

IL-2 and low-dose OKT3

Hydroxyurea/ didanosine

cyclophosphamide

valproic acid

Enfuvirtide

Abbreviations: IAT, immune activation therapy; CSF, cerebrospinal ﬂuid. www.annualreviews.org • Viral Persistence in HIV-1 Infection

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is prevented by effective HAART, especially when HAART is reinforced by one or more of the powerful new antivirals. Induction of HIV-1 gene expression by cytokines in chronically infected cell lines was demonstrated back in 1987 (66). The feasibility of cytokine-based immune activation therapy (IAT) was proven in vitro with a combination of the proinﬂammatory cytokines interleukin-6 (IL-6) and TNF-α along with the immunoregulatory cytokine IL-2 (67). This combination was a potent inducer of viral replication in highly puriﬁed, latently infected, resting CD4+ T lymphocytes derived from antiretroviral therapy–naive as well as HAART-experienced patients. A similar approach was attempted in HIV-1-infected patients using intermittent IL-2 combined with HAART (68). In vivo, administration of IL-2 alone rapidly induces serum IL-6 and TNF-α (69). As a result, the pool of resting CD4+ T cells containing replicationcompetent HIV-1 was signiﬁcantly smaller in patients on IL-2 therapy than in those receiving HAART alone (68). However, when treatment was interrupted in two patients in whom replication-competent virus could not be found in blood or lymph node cells, high levels of viral RNA were observed in plasma within weeks of stopping therapy (70). Therapy was also interrupted in another group of 18 patients who had undetectable levels of virus for more than one year (71). All patients relapsed to relatively high levels of plasma HIV-1 RNA in 2–3 weeks after stopping HAART. Long-term suppressive HAART alone or in combination with IL-2 did not eliminate HIV-1 infection. As well, a prospective open-label trial of HAART combined with IL-2 in 56 asymptomatic HIV-1-infected subjects did not prevent viral rebound after discontinuation of therapy (72). These studies suggest that when HAART and IL-2 treatment stops, viremia can rebound from as-yet-unidentiﬁed viral reservoirs. We hypothesize that tissue-bound macrophages, which may be relatively resistant to certain intensiﬁcation and stimulation therapeutic

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approaches, are a possible site of residual HIV-1. To affect a broader range of latently infected cells, beyond resting CD4+ T cells, more activators have been added to IAT. A combination of HAART with IFN-γ (to activate monocyte/macrophage scavenger cells) and IL-2 in early HIV-1 infection failed to eradicate the virus in any of the treated patients, although proviral DNA decrease and immune reconstitution were observed (73). When HAART was augmented by IL-2 plus OKT3 (a broad-spectrum T cell–stimulating anti-CD3 murine monoclonal antibody), T cell activation and proliferation resulted, with subsequent long-term depletion of the CD4+ T cell population (74–76). However, early development of OKT3-directed antibodies and serious side effects of the high drug doses hampered this strategy. The OKT3/ IL-2 therapy did not eradicate HIV-1; in fact, one study reported an increase in the latent reservoir (75). HAART intensiﬁcation to abrogate viral replication is indispensable when applying such high-level IAT. Hydroxyurea inhibits HIV-1 DNA synthesis and has been used in several HAART regimens, especially in combination with didanosine, with which it has synergistic effects (77, 78). By selectively blocking ribonucleotide reductase, hydroxyurea depletes intracellular deoxyribonucleotide triphosphate (dNTP) pools. Thus, it inhibits HIV-1 replication, notably in nonactivated CD4+ T cells. The combination of mycophenolic acid with abacavir may have a similar effect on residual HIV-1 replication (79). In a small prospective trial in three HIV-1infected men, low-dose OKT3/IL-2 IAT was combined with hydroxyurea/didanosine intensiﬁcation added to baseline suppressive HAART (80). In contrast to previous OKT3/IL-2 IAT trials, the intensiﬁcation and stimulation therapy used in this study was safe, with only modest side effects. When antiretroviral therapy was discontinued, patients developed plasma viral rebound. An analysis of blood and seminal compartments

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suggested that this HIV-1 eradication protocol caused viral compartmentalization of the semen microenvironment with evidence of viral evolution (81). Another strategy to strengthen HAART is cytoreduction by depletion of the HIV-1 proviral pool after stimulation of proviral transcription. Speciﬁc immunotoxins such as the hybrid toxin CD4-PE40 (82), or HIV-1speciﬁc T cell gene therapy (83), might prove useful. Cytoreduction therapy could also be used to eliminate latently infected cells and available uninfected target cells. Candidates to be incorporated in such therapy include cyclophosphamide as a low-level cytotoxic agent, and hydroxyurea at high concentrations where it is cytotoxic. However, in a small study, addition of cyclophosphamide to antiretroviral therapy did not diminish the cellular reservoir in HIV-1-infected persons (84). An anti-CD45RO ricin immunotoxin offers a very speciﬁc means to target resting CD4+ T cells (85), but the resulting T cell depletion might compromise immunity. Now that potent new antivirals can be added to HAART, cytoreduction strategies might be worth revisiting. An alternative to IL-2, TNF-γ, and OKT3 for IAT is the nontumorigenic phorbol ester prostratin. Prostratin inhibits de novo viral infection and spread, but it can also induce viral expression from latent provirus in the absence of cellular proliferation (86, 87). Moreover, it has the potential to activate the expression of latent HIV-1 in vivo with minimal activation effects on the immune system. These properties suggest that the use of prostratin in IAT holds considerable promise for future clinical trials, although it is still in early stages of development. Because histone deacetylation is important for quiescence of viral gene expression in persistently infected cells, HDAC inhibitors offer an alternative approach to induce provirus. A widely used HDAC inhibitor is the anticonvulsant valproic acid. In vitro, valproic acid induced HIV-1 expression in resting CD4+ T cells of patients receiving HAART, with-

out fully activating cells or enhancing de novo infection (88). In a small proof-of-concept trial, valproic acid was added to enfuvirtideintensiﬁed HAART (89). This combination therapy safely accelerated clearance of HIV-1 from resting CD4+ T cells, and a pronounced shortening of the half-life of the latent reservoir was found in three of four patients. Unfortunately, later studies cast doubt on the additive effect of valproic acid in HIV-1 eradication. In a cross-sectional and longitudinal study of the frequency of latently infected cells in HAART-treated patients receiving valproic acid for neurologic reasons, no difference was seen between the group on combination therapy and the group receiving HAART alone (90). Another report described rapid rebound of viremia in a patient who discontinued HAART plus valproic acid after more than two years of treatment (91). It remains to be seen if IAT and HDAC inhibitors will be able to induce proviral replication along the whole spectrum of latently infected cells, as different cell types might harbor integrated proviral DNA. In principle, a few remaining reservoir cells could rekindle systemic infection. Also, further studies on the penetration of different pharmacologic agents across blood-tissue barriers will be important in attacking potential sanctuary sites.

CONCLUSION The eradication of the HIV-1 cellular reservoirs will remain a major therapeutic challenge for the future because the reasons for HIV-1 latency are still not fully unraveled and can be described at best as multifactorial. To ﬂush out the virus from the latent reservoir will be the key to success in curing the infected individual; otherwise the remaining provirus will reignite the HIV-1 infection as soon as antiretroviral treatment is stopped. Because the intrinsic stability of integrated proviral DNA is the main reason for persistence of the latent reservoir, strategies exclusively based on powerful antivirals are unlikely to rid the body of HIV-1-infected www.annualreviews.org • Viral Persistence in HIV-1 Infection

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cells. Rather, HIV-1 eradication needs to be approached with an oncologic paradigm: Effective HAART would be used as induction therapy, followed by removal of residual disease via approaches directed against HIV-1 latency, cryptic replication, and sanctuary sites. The largest pool of latently infected cells consists of resting CD4+ memory T lymphocytes, with latently infected cells being only a minute fraction of the total resting CD4+ T cell population. In theory, wiping out all resting CD4+ T cells would purge this reservoir, but it would also severely compromise the immune system. Moreover, other residual sites would be missed by such a strategy. Because latently infected cells lack discernible markers, deliberate activation from latency seems inevitable to selectively destroy them. Several activation strategies have been tried. Because cytokine signaling induces cellular transcription factors such as NF-κB and NF-AT, which upregulate virus expression, cytokines seem excellent candidates for IAT. In reality, IL-2-based IAT was unable to purge the latent reservoir. Because IL-2 is known to primarily activate CD4+ T lymphocytes and improve CD8+ T cell function, it was hypothesized that a broad-spectrum activation approach was needed. Unfortunately, supplementing IAT with the monocyte/macrophage activator IFN-γ or the broad-spectrum T cell stimulator OKT3 also failed to purge the reservoir. Moreover, it must be taken into account that such broad-spectrum activation might trigger unwanted side effects, such as autoimmune reactions. Another latencyinducing factor that could be manipulated is the chromatin status at the integration site. Such remodeling of chromatin can be achieved with HDAC inhibitors such as valproic acid. But for this strategy as well, initial enthusiasm was tempered by experience. The combination of HDAC inhibitors with IAT has not been tried yet. One could hypothesize that both are needed (a) to make the U3 region of the LTR accessible for transcription

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factors and (b) to signiﬁcantly increase the concentrations of intracellular transcription factors. The failures in the described eradication trials could be due partly to insufﬁcient virus suppression by current HAART, resulting in new infections after activation of latent virus. Particularly in the OKT3-based protocols, the induced viral burst is difﬁcult to contain. Even with hydroxyurea/didanosinereinforced HAART (80), dramatic spikes of LTR circles were seen in one patient, probably indicating new infection. However, the emergence of exciting new antiretrovirals might offer new opportunities to sufﬁciently increase HAART potency and to develop better eradication protocols for the clinic. New activation strategies would be welcome. Within the restricted armamentarium of drugs currently approved by the US Food and Drug Administration, agents that can activate latently infected cells are scarce. Investigational compounds such as prostratin are promising for future trials. Cytoreduction strategies should also be further investigated. Emerging ﬁelds of research, including therapeutic vaccination, stem cell research, gene therapy, small interfering RNAs, microRNAs (92), and toll-like receptor agonists, could provide new insights and tools for better eradication protocols. Development of a speciﬁc (small-molecule) activator of HIV-1 expression is another tempting strategy. All future activation strategies should take into account the complete spectrum of latently infected cells, beyond resting CD4+ T lymphocytes. Based on current experience and available tools, an eradication cocktail should include IL-2 and low-dose OKT3 to induce T cells and IFN-γ to target latently infected monocyte/macrophages, combined with valproic acid to unlatch LTR, in a background of HAART reinforced with one or more of the new and potent antiretrovirals. Although the initial hypothesis-driven clinical trials did not achieve their ultimate

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goal of purging HIV-1 from the human body, they provide an excellent starting point for fu-

ture research into an effective curative therapy for HIV-1 infection.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

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59. Tong-Starksen SE, Luciw PA, Peterlin BM. 1987. Human immunodeﬁciency virus long terminal repeat responds to T-cell activation signals. Proc. Natl. Acad. Sci. USA 84:6845–49 60. Jordan A, Bisgrove D, Verdin E. 2003. HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J. 22:1868–77 61. Verdin E, Paras PJ, Van Lint C, 1993. Chromatin disruption in the promoter of human immunodeﬁciency virus type 1 during transcriptional activation. EMBO J. 12:3249–59 62. Van Lint C, Emiliani S, Ott M, et al. 1996. Transcriptional activation and chromatin remodeling of the HIV-1 promoter in response to histone acetylation. EMBO J. 15:1112– 20 63. Ramratnam B, Ribeiro R, He T, et al. 2004. Intensiﬁcation of antiretroviral therapy accelerates the decay of the HIV-1 latent reservoir and decreases, but does not eliminate, ongoing virus replication. J. Acquir. Immune Deﬁc. Syndr. 35:33–37 64. Havlir DV, Strain MC, Clerici M, et al. 2003. Productive infection maintains a dynamic steady state of residual viremia in human immunodeﬁciency virus type 1-infected persons treated with suppressive antiretroviral therapy for ﬁve years. J. Virol. 77:11212–19 65. Kim H, Perelson AS. 2006. Viral and latent reservoir persistence in HIV-1-infected patients on therapy. PLoS Comput. Biol. 2:1232–47 66. Folks TM, Justement J, Kinter A, et al. 1987. Cytokine-induced expression of HIV-1 in a chronically infected promonocyte cell line. Science 238:800–2 67. Chun T-W, Engel D, Mizell SB, et al. 1998. Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines. J. Exp. Med. 188:83–91 68. Chun T-W, Engel D, Mizell SB, et al. 1999. Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1-infected patients receiving highly active antiretroviral therapy. Nat. Med. 5:651–55 69. Weidmann E, Bergmann L, Stock J, et al. 1992. Rapid cytokine release in cancer patients treated with interleukin-2. J. Immunother. 12:123–31 70. Chun T-W, Davey RT Jr, Engel D, et al. 1999. Re-emergence of HIV after stopping therapy. Nature 401:874–75 71. Davey RT Jr, Bhat N, Yoder C, et al. 1999. HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression. Proc. Natl. Acad. Sci. USA 96:15109–14 72. Stellbrink H-J, van Lunzen J, Westby M, et al. 2002. Effects of interleukin-2 plus highly active antiretroviral therapy on HIV-1 replication and proviral DNA (COSMIC trial). AIDS 16:1479–87 73. Lafeuillade A, Poggi C, Chadapaud S, et al. 2001. Pilot study of a combination of highly active antiretroviral therapy and cytokines to induce HIV-1 remission. J. Acquir. Immune Deﬁc. Syndr. 26:44–55 74. van Praag RM, Prins JM, Roos MT, et al. 2001. OKT3 and IL-2 treatment for purging of the latent HIV-1 reservoir in vivo results in selective long-lasting CD4+ T cell depletion. J. Clin. Immunol. 21:218–26 75. Fraser C, Ferguson NM, Ghani AC, et al. 2000. Reduction of the HIV-1-infected T-cell reservoir by immune activation treatment is dose-dependent and restricted by the potency of antiretroviral drugs. AIDS 14:659–69 76. Prins JM, Jurriaans S, van Praag RME, et al. 1999. Immuno-activation with anti-CD3 and recombinant human IL-2 in HIV-1-infected patients on potent antiretroviral therapy. AIDS 13:2405–10 77. Lori F, Malykh A, Cara A, et al. 1994. Hydroxyurea as an inhibitor of human immunodeﬁciency virus-type 1 replication. Science 266:801–5

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78. De Boer RJ, Boucher CA, Perelson AS. 1998. Target cell availability and the successful suppression of HIV by hydroxyurea and didanosine. AIDS 12:1567–70 79. Chapuis AG, Rizzardi GP, D’Agostino C, et al. 2000. Effects of mycophenolic acid on human immunodeﬁciency virus infection in vitro and in vivo. Nat. Med. 6:762–68 80. Kulkosky J, Nunnari G, Otero M, et al. 2002. Intensiﬁcation and stimulation therapy for human immunodeﬁciency virus type 1 reservoirs in infected persons receiving virally suppressive highly active antiretroviral therapy. J. Infect. Dis. 186:1403–11 81. Nunnari G, Leto D, Sullivan J, et al. 2005. Seminal reservoirs during an HIV type 1 eradication trial. AIDS Res. Hum. Retroviruses 21:768–75 82. Berger EA, Moss B, Pastan I. 1998. Reconsidering targeted toxins to eliminate HIV infection: you gotta have HAART. Proc. Natl. Acad. Sci. USA 95:11511–13 83. Deeks SG, Wagner B, Anton PA, et al. 2002. A phase II randomized study of HIV-speciﬁc T-cell gene therapy in subjects with undetectable plasma viremia on combination antiretroviral therapy. Mol. Ther. 5:788–97 84. Bartlett JA, Miralles GD, Sevin AD, et al. 2002. Addition of cyclophosphamide to antiretroviral therapy does not diminish the cellular reservoir in HIV-infected persons. AIDS Res. Hum. Retroviruses 18:535–43 85. McCoig C, Van Dyke G, Chou C-S, et al. 1999. An anti-CD45RO immunotoxin eliminates T cells latently infected with HIV-1 in vitro. Proc. Natl. Acad. Sci. USA 96:11482–85 86. Kulkosky J, Culnan DM, Roman J, et al. 2001. Prostratin: activation of latent HIV-1 expression suggests a potential inductive adjuvant therapy for HAART. Blood 98:3006–15 87. Korin YD, Brooks DG, Brown S, et al. 2002. Effects of prostratin on T-cell activation and human immunodeﬁciency virus latency. J. Virol. 76:8118–23 88. Ylisastigui L, Archin NM, Lehrman G, et al. 2004. Coaxing HIV-1 from resting CD4 T cells: histone deacetylase inhibition allows latent viral expression. AIDS 18:1101–8 89. Lehrman G, Hogue IB, Palmer S, et al. 2005. Depletion of latent HIV-1 infection in vivo: a proof-of-concept study. Lancet 366:549–55 90. Siliciano JD, Lai J, Callender M, et al. 2007. Stability of the latent reservoir for HIV-1 in patients receiving valproic acid. J. Infect. Dis. 195:833–36 91. Steel A, Clark S, Teo I, et al. 2006. No change to HIV-1 latency with valproate therapy. AIDS 20:1681–82 92. Triboulet R, Mari B, Lin Y-L, et al. 2007. Suppression of microRNA-silencing pathway by HIV-1 during virus replication. Science 315:1579–82 93. Williams SA, Chen L-F, Kwon H, et al. 2006. NF-κB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation. EMBO J. 25:139–49

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:487-501. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

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Advancements in the Treatment of Epilepsy Annu. Rev. Med. 2008.59:503-523. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

B.A. Leeman and A.J. Cole Epilepsy Service, Massachusetts General Hospital, Boston, Massachusetts 02114; email: [email protected]; [email protected]

Annu. Rev. Med. 2008. 59:503–23

Key Words

First published online as a Review in Advance on September 20, 2007

seizures, neuroimaging, anticonvulsants, epilepsy surgery

The Annual Review of Medicine is online at http://med.annualreviews.org This article’s doi: 10.1146/annurev.med.58.071105.110848 c 2008 by Annual Reviews. Copyright All rights reserved 0066-4219/08/0218-0503$20.00

Abstract Diagnostic tools and treatment options for epilepsy have expanded in recent years. Imaging techniques once conﬁned to research laboratories are now routinely used for clinical purposes. Medications that were unavailable a few years ago are now ﬁrst-line agents. Patients with refractory seizures push for earlier surgical intervention, consider treatment with medical devices, and actively seek nonpharmacologic alternatives. We review some of these recent advances in the management of epilepsy.

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EEG/fMRI: concurrent electroencephalography and functional magnetic resonance imaging MEG: magnetoencephalography

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PET: positron emission tomography

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INTRODUCTION Epilepsy, operationally deﬁned as having or being at risk of having recurrent unprovoked seizures, affects an estimated 1.1–2.3 million people in the United States. The diagnosis of epilepsy confers a greater likelihood of injury and sudden unexplained death, as well as a loss of independence and perceived stigmatization. Patients with epilepsy report poorer overall health-related quality of life, and exhibit activity limitations, psychiatric comorbidities, increased unemployment rates, and lower educational attainment and income than those without seizures (1–3). The goal of treatment is to attain seizure freedom without side effects and return patients to normal, healthy lifestyles. The past several years have brought new diagnostic aids, medication options, and devices for the treatment of seizures. It is not just the armamentarium that has changed but also the way it is used. Imaging techniques once conﬁned to research laboratories are now routinely used in clinics. Medications that were new and unfamiliar a few years ago are now ﬁrst-line treatments. Patients and physicians push for earlier surgical intervention to treat refractory seizures and actively seek alternatives in diets and implantable devices. This is an exciting time in epileptology, and below we share some of the recent developments.

DIAGNOSIS Successful treatment requires a precise diagnosis. Recent advances in imaging technology help to identify subtle lesions that may represent epileptogenic foci. Magnetic resonance imaging, for example, now with phased array surface coils (PA MRI) and ﬁeld strengths of 3 Tesla (3T), has signal-to-noise ratios 6 to 8 times that of conventional 1.5T scans. PA MRI better deﬁnes the gray-white junction and creates a more uniform signal intensity of normal cortex, allowing better visualization of abnormalities such as cortical dysplasias. A study of 3T PA MRI detected brain lesions 504

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in 65% of patients with focal epilepsy and reportedly normal standard 1.5T scans. In addition, in 33% of patients with lesions revealed by 1.5T scans, abnormalities were better deﬁned on the 3T images (4). A greater ability to detect or characterize subtle lesions may alter treatment strategies. Although still largely a research tool, concurrent EEG and functional MRI (EEG/fMRI) is now used for clinical purposes at some centers. Functional MRI uses blood oxygenation level dependent (BOLD) pulse sequences, which map cerebral blood ﬂow with millisecond temporal resolution (5). With simultaneous EEG, it is possible to correlate areas of altered blood ﬂow with the occurrence of interictal or ictal discharges (6). This may help to identify foci or networks that are functionally abnormal during spikes and sharp waves, although they may not be recognized as structural lesions on standard MRI scans (7). Whereas EEG detects electrical potentials, magnetoencephalography (MEG) senses magnetic ﬁelds created by activity of apical dendrites, providing a novel technique for the localization of ictal and interictal epileptiform activity (8). An advantage of MEG is the relative lack of signal attenuation by the skull and scalp compared with EEG. MEG, however, only measures those ﬁelds tangential to the scalp. It is most sensitive to discharges in the neocortical convexities and relatively poor at detecting more mesial sources. Although MEG may identify epileptiform discharges missed by routine scalp EEG, its utility continues to be debated. Nuclear imaging modalities have also gained popularity, as a growing number of centers now have access to this technology. Positron emission tomography (PET) scanning, for example, is commonly used to aid in localization of seizure foci. Interictal injection with a radio-labeled tracer, most often ﬂuorodeoxyglucose-18 (FDG), may reveal regions of cellular hypometabolism that signify underlying abnormal, potentially epileptogenic, brain tissue. In contrast, ictal

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single-photon emission computed tomography (SPECT) uses injection of a technetium99-labeled tracer at the start of a seizure. This lipophilic tracer is 90% extracted on ﬁrst pass into lipid-rich tissue, including brain, in proportion to local perfusion. Uptake of the tracer by a brain region with actively ﬁring cells, evident on the scan as a focal area of hyperperfusion, may indicate the region of seizure onset. SISCOM, a technique in which SPECT images obtained during ictal and interictal periods are subtracted and coregistered with MRI, further improves localization. The utility of ictal SPECT may be limited, however, by the logistics of appropriately timing the injection and by the sensitivity of various tracers. Nevertheless, this method is often useful in settings in which the MRI is unrevealing (9). The availability of minimally invasive techniques provides additional tools for seizure localization. Older methods for focus identiﬁcation, such as foramen ovale electrodes and epidural pegs, have seen resurgence. Foramen ovale electrodes are placed percutaneously through the foramen so that they lie along the inferomesial aspect of the temporal lobe. This allows recordings to be obtained from deep medial structures without the need for surgical placement of depth electrodes into brain tissue. Epidural pegs provide a method for obtaining recordings over the convexities through small burr holes, which eliminates the muscle artifact that may contaminate scalp leads. This technique may be used to plan for the optimal placement of grids or strips, or it may potentially obviate the need for further invasive recording. These methods, either separately or in conjunction, have been helpful in many cases in which scalp EEG data have been inconclusive (8).

MEDICATIONS Historically, treatment options for epilepsy have been limited. Major pharmacologic options included carbamazepine, phenobarbital, phenytoin, primidone, ethosuximide, and

valproic acid. Though often effective, relatively affordable, and familiar, these older medications carry the risks of hepatic dysfunction, drug interactions, and other significant side effects. Between 1978, when valproic acid was introduced, and 1993, when felbamate was approved, no new anticonvulsants were approved by the US Food and Drug Administration (FDA). In contrast, in the past decade, eight new anticonvulsants have been approved in the United States. These agents typically offer equal efﬁcacy but may have fewer adverse effects and drug interactions than the older generation of medications (9a, 9b). Newer anticonvulsants (listed in reverse order of FDA approval) include pregabalin, oxcarbazepine, zonisamide, levetiracetam, tiagabine, topiramate, lamotrigine, gabapentin, and felbamate. What may be more striking than the number of new medications, however, is how quickly these drugs have become ﬁrstline therapies. In 2005, Karceski et al. (10) surveyed 43 epileptologists regarding their prescribing practices. Lamotrigine was considered a ﬁrst-line treatment for idiopathic primary generalized tonic-clonic, absence, simple partial, and secondarily generalized tonic-clonic seizures, and it was the treatment of choice for complex partial seizures. Topiramate was a ﬁrst-line medication for treatment of idiopathic primary generalized tonic-clonic seizures. Oxcarbazepine had become the treatment of choice for simple partial and secondarily generalized tonic-clonic seizures, and levetiracetam was also an acceptable initial option for treatment of these partial-onset seizure types. The survey also marked a shift in thinking from as little as ﬁve years earlier, when lamotrigine, topiramate, and levetiracetam were often considered to be second-line agents. With new treatments, however, often comes confusion and a lack of consensus regarding prescribing practices. The American Academy of Neurology (AAN) and American Epilepsy Society (AES) have published recommendations for use of many of the newer www.annualreviews.org • Treatment of Epilepsy

SPECT: single-photon emission computed tomography AAN: American Academy of Neurology AES: American Epilepsy Society

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medications (11–13). The choice in a given patient, however, continues to be guided by seizure type, patient characteristics and side effect proﬁles. Below we summarize the indications, associated adverse events, mechanisms and pharmacokinetic properties of the more recently approved anticonvulsants. These drugs are discussed in the reverse order of their approval in the United States (Table 1).

Pregabalin Annu. Rev. Med. 2008.59:503-523. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Pregabalin is the newest anticonvulsant. It is structurally similar to gabapentin but has a slightly longer half-life. Although structurally related to gamma-amino-butyric acid (GABA), the drug does not have GABA-ergic activity. Pregabalin binds to the alpha2 -delta subunit of voltage-gated calcium channels, reducing calcium inﬂux and thereby decreasing release of several neurotransmitters, including glutamate. Clinical experience with pregabalin is limited because it was only approved by the FDA in June 2005. The drug is currently indicated for adjunctive treatment of partialonset seizures in adults, on the basis of studies demonstrating seizure frequency reduction up to 47.8%–54% in this population (14–16). Our experience suggests that pregabalin may worsen idiopathic generalized epilepsy. Initial studies suggest a few potential advantages of pregabalin. The drug is reasonably well tolerated at starting doses up to 600 mg/day (14), although many patients complain of dizziness at initial doses above 100–150 mg/day. In urgent situations, steady state can be achieved in as little as 48 h. Pregabalin is renally excreted, so it is suitable for patients with hepatic disease. A lack of hepatic enzyme induction or inhibition is also advantageous in that the drug poses little risk for interaction with other medications. In addition, pregabalin is generally safe. Serious adverse events included a maculopapular rash in one patient, which resolved after discontinuation of the drug, as well as 506

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isolated reports of cholestatic jaundice. There is no known association of pregabalin with cardiovascular dysfunction; cases of peripheral edema have been reported, but detailed cardiac testing of those patients has not been described (16). These adverse events are typically dose-related. They tend to occur shortly after the medication is started, often within one week of initiation, and are generally self-limited, even if the drug is continued. Weight gain has been reported in a substantial fraction of patients chronically treated with pregabalin.

Oxcarbazepine Oxcarbazepine is FDA approved for use as monotherapy and adjunctive treatment in children (17, 18) and adults (19–21) with partial seizures. Oxcarbazepine has rapidly become a ﬁrst-line agent for those with partial seizures and is often preferred in patients with psychiatric comorbidities because of its apparent mood-stabilizing effects. Studies comparing oxcarbazepine monotherapy with phenytoin (22, 23), valproic acid (24), and carbamazepine (25) demonstrate equivalent rates of seizure freedom in populations with both partial-onset and primary generalized tonic-clonic seizures. The AAN/AES guidelines suggest that although oxcarbazepine monotherapy is appropriate in populations that include patients with newly diagnosed partial and/or generalized seizure disorders, there is insufﬁcient evidence to determine efﬁcacy for those with primary generalized seizures alone. The drug was developed as a structural variant of carbamazepine, designed to eliminate the carbamazepine epoxide metabolite thought to cause side effects. Carbamazepine and oxcarbazepine are distinct drugs, with separate metabolic pathways and somewhat different modes of action and side effect proﬁles (26). Oxcarbazepine exerts much of its effect through 10-monohydroxy metabolite (MHD), an active metabolite not present

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Table 1

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FDA-approved indications for new anticonvulsants

Medication

Use in partial seizures (simple, complex, secondarily generalized)— adjunctive

pregabalin

adults

oxcarbazepine adults, children ≥ 2 years of age

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Use in Use in partial primary seizures general(simple, ized complex, tonicUse in secondarily clonic absence generalized)— seizures— seizures— monotherapy monotherapy adjunctive

Use in primary generalized tonic-clonic seizures— monotherapy

Use in LennoxGastaut (tonic/atonic and tonic-clonic seizures)— adjunctive

Use in myoclonic seizures of JMEa — adjunctive

adults, children ≥4 years of age

zonisamide

adults

levetiracetam

adults, children ≥ 4 years of age

tiagabine

adults, children ≥ 12 years of age

topiramate

adults, children ≥ 2 years of age

adultsb , children ≥10 years of ageb

lamotrigine

adults, children ≥ 2 years of age

adultsc ,(d)

gabapentin

adults, children ≥ 3 years of age, ≥12 years of age if secondary generalization

(adults and adolescentsb )

felbamatee

adults

adults

adults, children ≥6 years of age

(children)

adults, children ≥12 years of age

adults, children ≥2 years of age

adultsb , children ≥10 years of ageb

adults, children ≥2 years of age

adults, children ≥2 years of age

(adultsd )

adults, children ≥2 years of age

adults, children ≥2 years of age (≥4 years per AAN)

a

Juvenile myoclonic epilepsy. For initial monotherapy. c Only FDA approved for conversion to monotherapy in patients receiving treatment with carbamazepine, phenytoin, phenobarbital, primidone, or valproate. d For initial monotherapy of partial and mixed (partial and generalized) seizure types. e According to AAN guidelines, children with partial or generalized epilepsies and patients with Lennox-Gastaut syndrome under age 4 years who are unresponsive or intolerant to ﬁrst-line agents may also consider use in certain situations when risk/beneﬁt ratio unclear; data limited. ( ) = Appropriate for use based on AAN/AES guidelines but not FDA approved for this indication. b

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with carbamazepine. Oxcarbazepine blocks voltage-sensitive sodium channels, stabilizing neuronal membranes and inhibiting repetitive ﬁring at lower concentrations than that required by carbamazepine. Increased potassium conductance and modulation of voltageactivated calcium channels may also play a role in its anticonvulsant effect. The interaction occurs at the N-, P-, and R-type calcium channels rather than the L-type channels affected by carbamazepine. Metabolites of oxcarbazepine, unlike those of carbamazepine, are excreted in the urine, so dosing must be adjusted in patients with renal disease. The effect on P-450 hepatic enzyme subtypes is minimal, causing only limited drug interactions (see Table 2). Of particular concern, however, is the induction of CYP3A4 and CYP3A5 enzymes, which may reduce concentrations of oral contraceptives. Nevertheless, oxcarbazepine is associated with fewer hepatic enzyme and drug interactions than carbamazepine or other older agents. In general, oxcarbazepine carries lower risk of adverse events than the prior generation of anticonvulsants. Clinical experience, however, suggests that oxcarbazepine does have adverse effects, often causing headache (26a). Side effects are typically dose-related, and slow titration is recommended to minimize risk. Serious side effects include rash and hyponatremia. Rash, when it occurs, generally appears within one month of use. Because cross reactivity of allergic reactions occurs in up to 27% of patients, those with exfoliative dermatitis from use of carbamazepine should not receive oxcarbazepine. The incidence of hyponatremia is greater with oxcarbazepine than with carbamazepine, and is of particular concern in the elderly and those on sodiumwasting agents. The hyponatremia is most often chronic and asymptomatic, developing gradually over the ﬁrst six weeks of treatment. Sodium levels typically normalize with reduction or cessation of oxcarbazepine treatment and may respond to ﬂuid restriction. Sodium levels should be obtained prior to initiating treatment and should be monitored during

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the ﬁrst three months of use in those at risk. No consensus exists as to a threshold for discontinuation of the drug, although levels less than 128 mEq/L and a continuing downward trend are worrisome.

Zonisamide Zonisamide is FDA approved for adjunctive treatment of partial seizures in patients 16 years of age and older (27, 28). Although some studies demonstrate efﬁcacy in children with partial and generalized epilepsy (29), including infantile spasms (30) and myoclonic seizures (31), AAN/AES guidelines caution that there is insufﬁcient evidence to recommend use of zonisamide in children or patients with primary generalized seizures. Further data are also required to conﬁrm utility as monotherapy, although zonisamide is often used as such for treatment of partial seizures in adults. Zonisamide contains a sulfonamide chemical structure, precluding its use in patients with a sulfa allergy. It produces its anticonvulsant effect by blockade of sodium and T-type calcium channels, thereby stabilizing neuronal membranes. Zonisamide also has weak carbonic anhydrase–inhibiting activity, but this does not appear to substantially contribute to its antiseizure properties. The medication offers several advantages. First, although the drug undergoes primarily hepatic elimination, it does not alter hepatic metabolism. Hence, zonisamide has fewer drug interactions than older medications do. Second, estimates of the half-life are as great as 24–60 h. This long duration enables onceper-day administration, improving compliance. Third, the drug may cause weight loss, as opposed to weight gain caused by many alternative medications. The drug is now available as a generic formulation, potentially making it more affordable. Finally, open-label data and anecdotal experience suggest that zonisamide may have antimigraine properties, providing a reasonable option for those with comorbid headaches.

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Table 2

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Properties of new anticonvulsantsa

Medication

Half-life

Pregabalin

6h

Mechanism

Clearance

Hepatic enzyme effects

Notable drug interactions

Cautions

Likely related to effects at voltage-gated calcium channels

Renal

None

None

None

Oxcarbazepine 4–9 h

Sodium channel blockade; possible contribution of effect on potassium and calcium channels

Renal and hepatic

Minimal induction and inhibition of subtypes

Decreases levels of: carbamazepine dihydropyridines oral contraceptives lamotrigine (?) Increases levels of: phenobarbital phenytoin MHD decreased by: verapamil carbamazepine phenobarbital phenytoin valproate

Cross reactivity of hypersensitivity to carbamazepine; risk of hyponatremia

Zonisamide

24–60 h

Sodium and calcium channel blockade

Hepatic more than renal

None

Levels decreased by hepatic enzyme-inducing medications: carbamazepine phenytoin phenobarbital

Do not use if patient has sulfa allergy or history of nephrolithiasis

Levetiracetam

6–8 h

Binding at SV2A synaptic vesicle protein and high-voltage calcium channels, modulation of GABA and glycine receptors

Renal, hydrolysis

None

None

Risk of psychiatric side effects

Tiagabine

4–9 h

Inhibition of GABA reuptake

Hepatic

None

Levels decreased by hepatic enzyme-inducing medications: carbamazepine phenytoin phenobarbital

Risk of “spike-wave” stupor

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Table 2

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(Continued ) Hepatic enzyme effects

Notable drug interactions

Medication

Half-life

Mechanism

Clearance

Topiramate

15–23 h

Blockade of voltagedependent sodium channels, inhibition of carbonic anhydrase, antagonizes AMPA/kainate glutamate receptors, and modulates GABAa-mediated chloride activity

Renal more than hepatic

Induction and inhibition

Decreases levels of: oral contraceptives (no effect with topiramate doses <200 mg/d), lithium, digoxin, valproate Increases levels of: haloperidol, phenytoin Levels decreased by hepatic enzyme-inducing medications: carbamazepine, phenytoin, valproate

Do not use if patient has sulfa allergy or history of nephrolithiasis Potential side effects include cognitive impairment, open-angle glaucoma, metabolic acidosis, weight loss

Lamotrigine

15–35 h

Likely related to inhibition of voltage-sensitive sodium channels

Hepatic

Minimal induction

Decreases levels of: valproate, oral contraceptives Levels decreased by: phenytoin, oral contraceptives Levels increased by: valproate

Risk of rash, particularly with concurrent use of valproate

Gabapentin

4–6 h

Unknown; may be related to voltage-activated calcium channels

Renal

None

Levels decreased by: Maalox-TC

None

Felbamate

20–23 h

Antagonist of glycine recognition site of NMDA receptor

Renal and hepatic

Induction and inhibition

Increases levels of: valproate, phenytoin, carbamazepine, epoxide,b phenobarbital Decreases levels of: carbamazepine, oral contraceptives Levels increased by: valproate Levels decreased by: phenytoin, carbamazepine

Risk of aplastic anemia, hepatic failure, and rash

a b

Abbreviations: MHD, 10-monohydroxy metabolite; GABA, gamma-amino-butyric acid. Elevated levels of the epoxide may cause toxicity.

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Cautions

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Adverse reactions are typically dosedependent. Most commonly, patients report cognitive clouding. Nephrolithiasis may be associated with zonisamide use. Concomitant administration of other carbonic anhydrase inhibitors, such as acetazolamide or topiramate, may increase the risk for renal stone formation. The drug should be avoided in patients with a history of nephrolithiasis, and those taking the medication should be encouraged to drink sufﬁcient amounts of water. Another serious risk is that of hypohydrosis with resultant hyperthermia, a rare side effect that occurs primarily in children. Zonisamide may also cause a rash and, rarely, Stevens-Johnson syndrome.

Levetiracetam Levetiracetam is indicated for adjunctive treatment of partial epilepsy in adults (32, 33) and children aged 4 years and older (34, 35) with refractory seizures. Data also support conversion to monotherapy in patients with refractory partial epilepsy (36). In clinical practice, levetiracetam is often prescribed as monotherapy on the basis of its success as an add-on treatment. Use as monotherapy, however, is not currently FDA approved or AAN/AES recommended owing to insufﬁcient data regarding efﬁcacy. Levetiracetam is indicated for add-on therapy of myoclonic seizures in patients aged 12 years and older with juvenile myoclonic epilepsy (37, 38). Most recently, levetiracetam also received FDA approval for adjunctive treatment of primary generalized tonicclonic seizures in adults and children aged 6 years and older with idiopathic generalized epilepsy. Levetiracetam has rapidly gained popularity owing to its ease of administration. Taken orally twice per day, the drug is at steady state within three doses and can be rapidly titrated to therapeutic levels. A new intravenous formulation is available, indicated for adjunctive treatment of partial seizures in adults and as an alternative when oral administration is

temporarily prohibited. Access to intravenous levetiracetam may increase its off-label use in acute situations, as case reports have indicated beneﬁt in status epilepticus (39). Because it has no hepatic effects, levetiracetam is a ﬁrst-line medication for those with liver dysfunction. The lack of hepatic effects also minimizes the potential for drug interactions, making levetiracetam a preferred agent for patients taking multiple medications, such as those who are elderly, HIV-positive, or on chemotherapy. Levetiracetam’s mechanism of action is unknown. It does not act at the receptors typically affected by antiepileptic medications. The drug binds to a presynaptic protein, SV2A, located on synaptic vesicles. The protein is probably involved in vesicle fusion to the presynaptic membrane and may reduce neurotransmitter release, but the relationships between SV2A binding and anticonvulsant properties are unclear. The anticonvulsant effect of levetiracetam may also be related to other atypical mechanisms, such as reduction of current through neuron-speciﬁc high voltage–activated calcium channels and modulation of the effects of zinc and betacarbolines on inhibitory GABAa and glycine receptors. No serious adverse reactions to levetiracetam have been reported. Common side effects include irritability and behavioral changes, and in some patients these adverse effects may be treatment limiting. Levetiracetam has a particularly wide therapeutic range. Hence, levetiracetam levels are typically not clinically useful, except to determine compliance.

Tiagabine Tiagabine exerts its anticonvulsant effect via a novel mechanism involving inhibition of GABA reuptake into neurons and glia. The medication is indicated as adjunctive therapy in patients aged 12 years and older for the treatment of partial seizures (40–42). However, little evidence supports www.annualreviews.org • Treatment of Epilepsy

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its use as monotherapy, in younger children, or for treatment of generalized-onset seizures. There are four reasons why tiagabine is not commonly prescribed. First, the drug may not be as effective as others (40–42). Although it is not possible to directly compare trials owing to differences in study populations, dosages, and design, tiagabine seems to have somewhat lower responder rates than the other newer medications. Second, tiagabine undergoes hepatic elimination. This makes it a less desirable option for patients with liver disease. Tiagabine itself does not alter hepatic enzyme function, but its metabolism may be affected by enzyme inducers and inhibitors, which raises concern about drug interactions. Third, the medication takes several weeks to titrate to a therapeutic dosage. Fourth and most important, the drug carries the potential for serious side effects. Use of tiagabine may cause a paradoxical increase in seizure activity. New seizure types may develop, along with an increased incidence of nonconvulsive status epilepticus (NCSE) or so-called spike-wave stupor. In a recent retrospective study of patients with refractory partial seizures, 7.8% of those treated with tiagabine experienced episodes of NCSE, conﬁrmed by spike- and polyspikewave discharges on EEG that resolved after discontinuation of the drug (43). In fact, the FDA has issued a safety alert in response to reports of new-onset seizures and status epilepticus in patients without epilepsy who were prescribed tiagabine for other indications. Some patients on tiagabine have developed an encephalopathy that is probably related to seizure activity. These effects do not appear to be dose-related and may develop more than three months after treatment is initiated. Assertions that frontal lobe epilepsy increases these risks are controversial and not well documented in the literature. The mechanism for development of encephalopathy is uncertain, but is probably related to GABAmediated pathways.

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Topiramate Topiramate blocks voltage-dependent sodium channels, inhibits carbonic anhydrase, acts as an antagonist of 2-(aminomethyl) phenylacetic acid (AMPA)/kainate glutamate receptors, and modulates GABAa-mediated chloride activity. The mode of action underlying its anticonvulsant effect, however, remains unknown. Topiramate is approved by the FDA as initial monotherapy for partial-onset or primary generalized tonic-clonic seizures in patients 10 years of age and older. Studies supporting this indication have shown that patients with partial (44) and primary generalized tonicclonic (45) seizures randomized to higher doses of topiramate had signiﬁcantly greater rates of seizure freedom than those on lower doses of the drug. Topiramate is also approved as adjunctive therapy for patients 2 years of age and older with refractory partial seizures, primary generalized tonic-clonic seizures, and Lennox-Gastaut syndrome. Randomized double-blind add-on studies support its use in these populations, demonstrating greater reduction in partial-onset seizures (46), primary generalized tonic-clonic seizures (47), and drop attacks (48) compared to placebo. Efﬁcacy and safety of conversion from another antiepileptic medication to topiramate monotherapy have not been adequately studied. Topiramate is advantageous in its broad range of efﬁcacy. It may also help prevent migraines and is at times favored for patients with comorbid headaches and seizures. The medication also has mood-stabilizing effects. Topiramate may be less preferable as a ﬁrstline agent, however, owing to its long list of potential side effects. Common adverse reactions to topiramate include cognitive dysfunction, often associated with word-ﬁnding difﬁculties. Paresthesias of ﬁngertips and toes may occur shortly after the medication is initiated, probably related to carbonic anhydrase inhibition. Paresthesias are typically self-limited, resolving

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over weeks. If bothersome or prolonged, isolated case reports indicate that potassium supplementation at 20–40 mEq per day may be helpful (49), although no placebo-controlled trials have been published to date. Serious side effects include nephrolithiasis in 1%–5% of patients, and the drug should be avoided in those with a history of renal stones. The acute onset of diminished visual acuity or ocular pain should prompt concern about open-angle glaucoma associated with topiramate use, typically occurring within the ﬁrst month of treatment. Other possible side effects include hypohydrosis, particularly in children, and a hyperchloremic, nonanion gap metabolic acidosis. Transient but signiﬁcant weight loss may also occur. The risk of the above side effects appears to be dose-related and may be minimized by slow titration. Despite the mood-stabilizing properties of topiramate, there are reports of rare topiramateinduced suicidality. In addition, the drug is a sulfa derivative and is contraindicated in patients with sulfa allergies.

Lamotrigine Lamotrigine has rapidly become a ﬁrst-line agent for many seizure types and patient populations. It is currently FDA approved as adjunctive therapy for partial (50–53) and primary generalized tonic-clonic (54) seizures, as well as generalized seizures of Lennox-Gastaut syndrome (55), in patients 2 years of age and older. Lamotrigine is also indicated for conversion to monotherapy in adults with partial seizures receiving treatment with carbamazepine, phenytoin, phenobarbital, primidone, or valproate (56). Conversion to monotherapy from other anticonvulsants or from multiple anticonvulsants is not currently approved. Nor is the drug approved for initial monotherapy, although there is evidence of efﬁcacy in this situation (57–59). In practice, lamotrigine is often used as initial monotherapy for partialonset and primary generalized tonic-clonic seizures. The AAN/AES guidelines also in-

dicate that lamotrigine is effective for newly diagnosed absence seizures in children (60), although it is not FDA approved for this indication. Anecdotal reports suggest that in some patients, lamotrigine may worsen myoclonic jerks (61). Lamotrigine has become a preferred agent for women planning pregnancy. In multiple pregnancy registries, the rate of fetal malformations in children born to mothers on lamotrigine monotherapy has been low, 2.5%– 2.9%. A single report suggests that within that rate, there may be an overrepresentation of a speciﬁc malformation, cleft lip or palate (62). Because this ﬁnding has not been conﬁrmed in other pregnancy registries, many epileptologists consider lamotrigine to be a ﬁrst-line agent in pregnancy. This issue is likely to generate further discussion as additional data become available. Lamotrigine offers several advantages. The drug is considered to be effective and generally well tolerated. Although it is metabolized by the liver, lamotrigine elicits little hepatic enzyme induction and no enzyme inhibition. Hence, there are relatively few drug interactions, making lamotrigine a reasonable option for the elderly, patients with HIV, and patients with other underlying medical problems in whom polypharmacy is an issue. Owing to its mood-stabilizing effects, the drug has also become a popular choice for patients with comorbid depression or bipolar disorder. Lamotrigine is a ﬁrst-line agent for patients with renal or hepatic dysfunction, as well. Although its metabolism may be affected by hepatic disease, the drug itself causes little renal or hepatic toxicity. A few notable drug interactions, however, do exist. Lamotrigine causes a modest reduction in levonorgestrel levels, and ethinyl estradiol may decrease lamotrigine levels. Adequate lamotrigine levels may be difﬁcult to attain in the presence of phenytoin, as this drug induces the metabolism of lamotrigine. Very high dosages of lamotrigine may be required to yield therapeutic blood levels with concomitant phenytoin use. In www.annualreviews.org • Treatment of Epilepsy

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contrast, valproate may signiﬁcantly increase lamotrigine blood levels. When converting to lamotrigine monotherapy from these anticonvulsants, lamotrigine levels should be monitored carefully. The therapeutic range begins at 4–6 mcg/ml. Some patients may beneﬁt from higher levels, although our clinical experience suggests that levels in the teens pose a greater risk for side effects. Although the drug is typically well tolerated, a few potential side effects deserve mention. Use of lamotrigine carries a risk of rash. The risk appears similar to that associated with phenytoin and carbamazepine, but if a lamotrigine-associated rash occurs, it may be very serious and patients should be encouraged to contact a physician immediately. Cases of Stevens-Johnson syndrome and toxic epidermal necrolysis have been reported. The risk is highest for children and those on concurrent valproate. Uncommon side effects also include cough and insomnia. Risks are minimized by slow titration. It takes several weeks to properly attain a therapeutic dosage; the titration schedule may be confusing for some patients, and various blister starter packets are available to simplify the regimen. An even slower titration schedule should be employed for those also taking valproate, and separate starter packets are available for that purpose. The chemical structure of lamotrigine is unrelated to those of the other anticonvulsants. The mechanism underlying lamotrigine’s anticonvulsant effect is uncertain. The drug is postulated to act by inhibiting use- and voltage-sensitive sodium channels, thereby stabilizing neuronal membranes and modulating release of excitatory neurotransmitters such as glutamate.

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Gabapentin Although gabapentin is structurally related to GABA, the drug does not act via GABAergic mechanisms. The mechanism of its anticonvulsant activity is unknown. Animal data suggest that it binds to a subunit of voltage514

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activated calcium channels, but the functional importance of this is unclear. Gabapentin is FDA approved as adjunctive therapy for partial seizures in patients aged 3 years and older and for seizures with secondary generalization in those aged 12 years and older. These indications are supported by double-blind randomized placebo-controlled studies (63–65). According to the AAN/AES evidence-based guidelines, gabapentin is also a reasonable option as initial monotherapy for adolescents and adults with newly diagnosed partial-onset seizures (66), although it is not yet FDA approved for this indication. Insufﬁcient evidence exists to support its use as monotherapy in those with refractory partial seizures. The drug has not been shown to be effective for primary generalized epilepsies and may in fact worsen myoclonic jerks (67), absence, and primary generalized tonic-clonic seizures. Gabapentin is typically quite well tolerated with few drug interactions. Our clinical experience suggests, however, that it is less efﬁcacious than other available anticonvulsants.

Felbamate Felbamate, an N-methyl-D-aspartate (NMDA) receptor antagonist, was approved by the FDA in 1993 for use as adjunctive treatment (68) or monotherapy (69–71) in adults with partial-onset seizures, and as adjunctive treatment for seizures associated with Lennox-Gastaut syndrome in patients 2 years of age and older (72). In addition, limited data suggest efﬁcacy as add-on treatment for typical and atypical absence seizures (73, 74), partial seizures in children (75), and generalized tonic-clonic seizures in adults (76). Although one study demonstrated efﬁcacy as monotherapy or adjunctive treatment for myoclonic, typical absence, and generalized tonic-clonic seizures associated with juvenile myoclonic epilepsy in adolescents and adults (77), the results must be interpreted with caution given the small sample size.

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The drug appeared to be well tolerated and to cause less sedation than other anticonvulsants. In 1994, however, a “Dear Doctor” letter proposed that physicians discontinue use of the drug owing to cases of aplastic anemia, with recent estimates suggesting a prevalence of 27–209 cases per million. The course of aplastic anemia depends on the severity of bone marrow suppression. Reports also identiﬁed instances of hepatic failure and rash, including Stevens-Johnson syndrome and toxic epidermal necrolysis, associated with felbamate use. Although rare, these reactions were serious and prompted many physicians to discontinue felbamate therapy in their patients. In light of these ﬁndings, the AAN issued a guideline for felbamate use (78). The guideline suggests that felbamate may be an appropriate choice for patients over age 4 with Lennox-Gastaut syndrome, and for patients over age 18 with partial seizures refractory to ﬁrst-line anticonvulsants. One may also consider use of felbamate in certain situations for children with partial or generalized epilepsies and patients with Lennox-Gastaut syndrome under age 4 who are unresponsive to or intolerant of ﬁrst-line agents. Data suggested a better risk/beneﬁt ratio for treatment with monotherapy and for continuation in patients who have taken the drug for more than 18 months. The AAN guideline notes that the risk/beneﬁt ratio should be examined carefully in each patient, however, and that patients should be counseled regarding possible side effects and the recommendations for monitoring. Routine laboratory studies had not been shown to be useful, but the manufacturer and FDA do recommend liver function tests and blood counts. Because the risk of aplastic anemia declines after one year of treatment, the value of routine monitoring after this time period is less clear. The drug should not be used in patients with a history of hematologic abnormalities, liver disease or systemic lupus erythematosus, patients who cannot comply with close follow-up, or

patients and guardians unable to provide informed consent. Should physicians choose to prescribe felbamate, they are encouraged to register their patients in the Felbatol Registry (http://www.guideline.gov, 78a).

Vigabatrin Vigabatrin is a derivative of the inhibitory neurotransmitter GABA and irreversibly inhibits GABA transaminase, preventing breakdown of the neurotransmitter. The drug was initially intended to treat partial seizures in adults. Used in Europe since the late 1980s, it was found to be effective, particularly as adjunctive therapy for complex partial seizures and partial seizures with secondary generalization (79, 80). The medication appeared to be relatively well tolerated with minor side effects. After several years on the market in Europe, however, it was discovered that vigabatrin caused visual ﬁeld defects in up to nearly 50% of adults (81), with additional case reports in children (82). Some patients also developed diminished visual acuity, deﬁcits in color vision, and other retinal abnormalities (81). When FDA approval was sought in 2004, it was denied because of these potential visual effects. As a recent AAN practice parameter suggests, however, the drug may be effective for treatment of infantile spasms, including spasms in the setting of tuberous sclerosis, for which there are few other treatment options (83). Many physicians and parents of these patients obtain the drug from outside the United States despite the potentially serious side effects.

Drugs in Development Several new compounds are currently in development, and the following drugs are quickly moving along the pipeline (84). Brivaracetam, an SV2A ligand related to levetiracetam, recently acquired orphan drug status for symptomatic myoclonus and is undergoing evaluation for add-on treatment of partial seizures and Unverricht-Lundborg www.annualreviews.org • Treatment of Epilepsy

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disease. Eslicarbazepine acetate, structurally related to carbamazepine and oxcarbazepine, is in phase III clinical trials for the adjunctive treatment of partial seizures in adults, with results expected this year. Lacosamide, in both intravenous and oral formulations, is under phase III investigation for the treatment of partial seizures. Thought to be effective in partial-onset seizures, ritigabine and carisbamate are also in phase III trials (85). A New Drug Application has been submitted to the FDA for ruﬁnamide, a compound believed to be efﬁcacious for adults and adolescents with refractory partial seizures as well as for adults and children with Lennox-Gastaut syndrome. Each new drug provides additional hope for seizure freedom in patients with epilepsy.

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Drug Monitoring Routine monitoring of anticonvulsant levels is not recommended except during pregnancy, when an increased volume of distribution causes levels to fall and changes in dosage are often required. Levels should be used to address speciﬁc concerns, e.g., to document compliance, assess for toxicity, or aid in management when changing drug regimens or when breakthrough seizures occur.

ALTERNATIVES TO PHARMACOLOGIC MANAGEMENT A 2001 study of patients with newly diagnosed epilepsy found that 47% become seizure-free with the ﬁrst anticonvulsant prescribed (86). The probability of successful treatment diminishes with successive trials of different medications. Typically patients are considered to be refractory to medications when they have failed three or more anticonvulsants. Failure is deﬁned as breakthrough seizures of any frequency, often quantiﬁed in the literature as at least one seizure within the past year. For these patients, ∼30% of those with 516

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epilepsy, alternatives to pharmacologic management should be considered. The utilization of surgical approaches for refractory epilepsy has increased. Resection is an effective treatment with little associated morbidity. In the ﬁrst randomized controlled trial of epilepsy surgery, 64% of those who underwent anteromesial temporal lobectomy for refractory complex partial seizures were free of seizures that impaired consciousness, compared to 8% of those who received medical management alone. Moreover, 42% of the surgical patients were completely seizure-free (87). A review of the literature regarding anteromesial temporal lobectomy and neocortical resections yielded similar results (88). On the basis of these data, the practice parameter set by the AAN and AES in 2003 states that referral to a surgical center should be considered for patients with refractory, disabling, complex partial temporal lobe seizures. A more recent report demonstrated even more impressive statistics, with 73% of those undergoing resection for mesial temporal lobe epilepsy rendered seizure-free (89). Unfortunately, many potential surgical candidates are not referred for evaluation or are referred after long delays. The appropriate timing of surgical intervention remains in question. A trial to assess outcomes of early surgery (the Early Randomized Surgical Epilepsy Trial, or ERSET) was unable to recruit a sufﬁcient number of subjects. Anecdotal reports and small case series suggest a role for urgent resection in refractory status epilepticus, although randomized controlled trials have not been performed. This highlights the need for studies of treatment strategies for medically refractory seizures. Implantable devices are an alternative for patients who are not candidates for, or do not desire, resection. Vagus nerve stimulation (VNS) was the ﬁrst implantable device developed for treatment of seizures. Its mechanism of action remains unknown. Electrodes wrap around the left vagus nerve and connect to a generator placed subcutaneously in the

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chest wall. The stimulator delivers preprogrammed intermittent electrical pulses to the vagus nerve. It may also be activated by the swipe of a magnet to abort a seizure. During stimulation, however, patients may experience cough, hoarseness, or throat pain. Moreover, VNS requires surgical implantation, a costly procedure that poses a risk of injury to the nerve and carotid sheath. Very few adult patients (only isolated case reports) are rendered seizure-free by the device, and the risks must be weighed against the low probability of signiﬁcant beneﬁt. Overall, VNS is not believed to be useful in adults, although some success has been reported in children with refractory seizures. The AAN practice guidelines state that VNS is indicated for those aged 12 years and older with refractory partial seizures who are not candidates for resection (90). VNS has, however, paved the way for new device-driven therapies. A trial is under way to assess the efﬁcacy of responsive or “closed loop” stimulators. These devices, implanted in the epileptogenic focus, detect epileptiform activity. They then deliver electrical stimulation to abort the abnormal discharges, thereby preventing the evolution of a clinical seizure. Some centers are also investigating use of deep brain stimulation for the treatment of epilepsy. Deep brain stimulation delivers “scheduled” or “open loop” stimulation to structures such as the thalamus, cerebellum, and hippocampus (91). The implantation of any device poses a small risk of infection and hemorrhage. It also requires rigorous workup, as would a surgical resection. For example, one must know the precise location of seizure onset in order to plan the placement of a closed-loop stimulator. This may require invasive EEG monitoring with recording grids, strips, or depth electrodes. Dietary treatments provide an alternative, noninvasive approach for patients with refractory seizure disorders. The classic ketogenic diet, developed in the 1920s, recently gained popularity (92). It involves low carbohydrate and high fat intake, with resultant ke-

tosis. The mechanism of action underlying its efﬁcacy, however, remains unclear. Approximately 20% of children on the diet have a >90% reduction in seizure frequency, with 7% seizure-free at one year. Although the diet is most effective in children, adults have also attained good results. It is typically used for treatment of generalized seizures or multiple seizure types but may also be considered for any patient with refractory seizures or intolerance of medications. The diet requires monitoring of weight, lipid proﬁles, electrolytes, urinalyses, urine calcium, and creatinine every 3–6 months. The risk of hyperlipidemia, however, is relatively low. More common side effects include weight loss, gastrointestinal upset, and acidosis. Unfortunately, the diet also tends to be restrictive and unpalatable. A low-glycemicindex diet, allowing more carbohydrates and less fat, provides a less restrictive alternative. The diet is limited to foods that produce relatively little increase in blood glucose levels. In a study of 20 patients, 50% of those treated with this diet had a >90% reduction in seizure frequency (93). A modiﬁed Atkins diet offers another less restrictive option. This diet yields seizure reduction rates comparable to that of the ketogenic diet but without the limitations on calories, ﬂuid, and protein. At six months, 35% of 20 patients placed on the modiﬁed Atkins diet had a >90% improvement in seizure frequency, and particular efﬁcacy was noted for absence seizures (94). Some suggest that this diet may be appropriate for patients with more recent-onset, less refractory seizures than those treated with the ketogenic diet. Overall, dietary therapies are good options for those resistant to or intolerant of anticonvulsants, with the goal of discontinuing or reducing medications. Our clinical experience with adults, however, suggests that diets are difﬁcult to maintain.

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such as attention, language, and memory, despite otherwise normal intelligence. Factors that may contribute to cognitive difﬁculties include seizure type, frequency, severity, and age of onset; side effects of treatment; and, perhaps most important, psychosocial and psychiatric comorbidities (95–97). Patients with seizures have a greater incidence of anxiety, depression, and bipolar disorder, with suicide rates ten times higher than that of the general population (3). These cognitive and emotional symptoms may be the most troublesome aspects of epilepsy for patients. It has become increasingly clear in recent years that those with epilepsy should be screened for concurrent cognitive and psychiatric disorders.

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FUTURE DIRECTIONS Further advancements in the diagnosis and treatment of epilepsy may stem from the study of genetics. It has long been accepted that many of the primary generalized epilepsies have a genetic basis. A recent trend in genetic studies of epilepsy has been the ﬁnding of gene mutations resulting in channelopathies. Mutations in genes encoding sodium channel subunits were found to underlie various epileptic syndromes of infancy, including generalized epilepsy with febrile seizures plus (GEFS+), severe myoclonic epilepsy of infancy, and benign familial neonatal-infantile seizures. The syndrome of benign familial neonatal convulsions has been traced to gene mutations causing aberrant voltage-dependent potassium channels, and mutations that alter voltage-gated chloride channels have been linked to childhood absence epilepsy, juvenile absence epilepsy, juvenile myoclonic epilepsy, and epilepsy with grand-mal seizures on awakening (98).

Recent studies have also revealed a genetic basis to various focal epilepsies. It is an intriguing notion that such a diffuse process results in a focal brain abnormality, as seen with nicotinic acetylcholine receptor gene mutations in autosomal dominant nocturnal frontal lobe epilepsy and LGI-1 mutations in familial lateral temporal lobe epilepsy (99). The importance of these studies may lie in their implications for optimizing future treatment. For instance, genetic markers that predict anticonvulsant response would be invaluable. Genetic testing might also identify patients at risk for seizure recurrence, allowing more rapid initiation of treatment. Many of the genetic tests are now commercially available, although interpretation of results may be complicated by possible polygenetic mechanisms or variable penetrance. Currently such testing does not typically play a role in clinical management. If patients or their families desire such testing, referral to a genetic counselor should be considered.

SUMMARY Recent years have brought new tools for the diagnosis of epilepsy, with advances in MRI techniques, the advent of MEG, increased availability of PET and SPECT, and use of multimodal imaging studies such as EEG/fMRI. Minimally invasive means of intracranial EEG monitoring are more commonly employed, such as epidural pegs and foramen ovale electrodes. A wider range of treatment options also exists, including new anticonvulsants, dietary therapies, implantable devices, and earlier surgical interventions. Great strides have been made in the management of epilepsy, as many of today’s standard diagnostic procedures and ﬁrst-line therapies were not available just a few years ago.

DISCLOSURE STATEMENT A.J. Cole is a consultant to GlaxoSmithKline, Pﬁzer, Ortho McNeil, and Abbott Laboratories. B.A. Leeman has received a grant from the UCB Young Investigators Research Program. 518

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17. Glauser TA, Nigro M, Sachdeo R, et al. 2000. Adjunctive therapy with oxcarbazepine in children with partial seizures. The Oxcarbazepine Pediatric Study Group. Neurology 54:2237–44 18. Kothare SV, Khurana DS, Mostoﬁ N, et al. 2006. Oxcarbazepine monotherapy in children and adolescents: a single-center clinical experience. Pediatr. Neurol. 35:235–39 19. Beydoun A, Sachdeo RC, Rosenfeld WE, et al. 2000. Oxcarbazepine monotherapy for partial-onset seizures: a multicenter, double-blind, clinical trial. Neurology 54:2245–51 20. Sachdeo R, Beydoun A, Schachter S, et al. 2001. Oxcarbazepine (Trileptal) as monotherapy in patients with partial seizures. Neurology 57:864–71 21. Barcs G, Walker EB, Elger CE, et al. 2000. Oxcarbazepine placebo-controlled, doseranging trial in refractory partial epilepsy. Epilepsia 41:1597–607 22. Bill PA, Vigonius U, Pohlmann H, et al. 1997. A double-blind controlled clinical trial of oxcarbazepine versus phenytoin in adults with previously untreated epilepsy. Epilepsy Res. 27:195–204 23. Guerreiro MM, Vigonius U, Pohlmann H, et al. 1997. A double-blind controlled clinical trial of oxcarbazepine versus phenytoin in children and adolescents with epilepsy. Epilepsy Res. 27:205–13 24. Christe W, Kramer G, Vigonius U, et al. 1997. A double-blind controlled clinical trial: oxcarbazepine versus sodium valproate in adults with newly diagnosed epilepsy. Epilepsy Res. 26:451–60 25. Dam M, Ekberg R, Loyning Y, et al. 1989. A double-blind study comparing oxcarbazepine and carbamazepine in patients with newly diagnosed, previously untreated epilepsy. Epilepsy Res. 3:70–76 26. Schmidt D, Elger CE. 2004. What is the evidence that oxcarbazepine and carbamazepine are distinctly different antiepileptic drugs? Epilepsy Behav. 5:627–35 26a. Palmieri A. 2007. Oxcarbazepine-induced headache. Cephalalgia 27:91–93 27. Faught E, Ayala R, Montouris GG, et al. 2001. Randomized controlled trial of zonisamide for the treatment of refractory partial-onset seizures. Neurology 57:1774–79 28. Schmidt D, Jacob R, Loiseau P, et al. 1993. Zonisamide for add-on treatment of refractory partial epilepsy: a European double-blind trial. Epilepsy Res. 15:67–73 29. Kothare SV, Kaleyias J, Mostoﬁ N, et al. 2006. Efﬁcacy and safety of zonisamide monotherapy in a cohort of children with epilepsy. Pediatr. Neurol. 34:351–54 30. Yanai S, Hanai T, Narazaki O. 1999. Treatment of infantile spasms with zonisamide. Brain Dev. 21:157–61 31. Kyllerman M, Ben-Menachem E. 1998. Zonisamide for progressive myoclonus epilepsy: long-term observations in seven patients. Epilepsy Res. 29:109–14 32. Shorvon SD, Lowenthal A, Janz D, et al. 2000. Multicenter double-blind, randomized, placebo-controlled trial of levetiracetam as add-on therapy in patients with refractory partial seizures. European Levetiracetam Study Group. Epilepsia 41:1179–86 33. Cereghino JJ, Biton V, Abou-Khalil B, et al. 2000. Levetiracetam for partial seizures: results of a double-blind, randomized clinical trial. Neurology 55:236–42 34. Glauser TA, Ayala R, Elterman RD, et al. 2006. Double-blind placebo-controlled trial of adjunctive levetiracetam in pediatric partial seizures. Neurology 66:1654–60 35. Glauser TA, Pellock JM, Bebin EM, et al. 2002. Efﬁcacy and safety of levetiracetam in children with partial seizures: an open-label trial. Epilepsia 43:518–24 36. Ben-Menachem E, Falter U. 2000. Efﬁcacy and tolerability of levetiracetam 3000 mg/d in patients with refractory partial seizures: a multicenter, double-blind, responder-selected study evaluating monotherapy. European Levetiracetam Study Group. Epilepsia 41:1276– 83

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37. Specchio LM, Gambardella A, Giallonardo AT, et al. 2006. Open label, long-term, pragmatic study on levetiracetam in the treatment of juvenile myoclonic epilepsy. Epilepsy Res. 71:32–39 38. Labate A, Colosimo E, Gambardella A, et al. 2006. Levetiracetam in patients with generalised epilepsy and myoclonic seizures: an open label study. Seizure 15:214–18 39. Patel NC, Landan IR, Levin J, et al. 2006. The use of levetiracetam in refractory status epilepticus. Seizure 15:137–41 40. Sachdeo RC, Leroy RF, Krauss GL, et al. 1997. Tiagabine therapy for complex partial seizures. A dose-frequency study. The Tiagabine Study Group. Arch. Neurol. 54:595–601 41. Uthman BM, Rowan AJ, Ahmann PA, et al. 1998. Tiagabine for complex partial seizures: a randomized, add-on, dose-response trial. Arch. Neurol. 55:56–62 42. Richens A, Chadwick DW, Duncan JS, et al. 1995. Adjunctive treatment of partial seizures with tiagabine: a placebo-controlled trial. Epilepsy Res. 21:37–42 43. Koepp MJ, Edwards M, Collins J, et al. 2005. Status epilepticus and tiagabine therapy revisited. Epilepsia 46:1625–32 44. Gilliam FG, Veloso F, Bomhof MA, et al. 2003. A dose-comparison trial of topiramate as monotherapy in recently diagnosed partial epilepsy. Neurology 60:196–202 45. Arroyo S, Dodson WE, Privitera MD, et al. 2005. Randomized dose-controlled study of topiramate as ﬁrst-line therapy in epilepsy. Acta Neurol. Scand. 112:214–22 46. Ben-Menachem E, Henriksen O, Dam M, et al. 1996. Double-blind, placebo-controlled trial of topiramate as add-on therapy in patients with refractory partial seizures. Epilepsia 37:539–43 47. Biton V, Montouris GD, Ritter F, et al. 1999. A randomized, placebo-controlled study of topiramate in primary generalized tonic-clonic seizures. Topiramate YTC Study Group. Neurology 52:1330–37 48. Sachdeo RC, Glauser TA, Ritter F, et al. 1999. A double-blind, randomized trial of topiramate in Lennox-Gastaut syndrome. Topiramate YL Study Group. Neurology 52:1882–87 49. Silberstein SD. 2002. Control of topiramate-induced paresthesias with supplemental potassium. Headache 42:85 50. Matsuo F, Bergen D, Faught E, et al. 1993. Placebo-controlled study of the efﬁcacy and safety of lamotrigine in patients with partial seizures. U.S. Lamotrigine Protocol 0.5 Clinical Trial Group. Neurology 43:2284–91 51. Messenheimer J, Ramsay RE, Willmore LJ, et al. 1994. Lamotrigine therapy for partial seizures: a multicenter, placebo-controlled, double-blind, cross-over trial. Epilepsia 35:113–21 52. Schapel GJ, Beran RG, Vajda FJ, et al. 1993. Double-blind, placebo controlled, crossover study of lamotrigine in treatment resistant partial seizures. J. Neurol. Neurosurg. Psychiatry 56:448–53 53. Duchowny M, Pellock JM, Graf WD, et al. 1999. A placebo-controlled trial of lamotrigine add-on therapy for partial seizures in children. Lamictal Pediatric Partial Seizure Study Group. Neurology 53:1724–31 54. Biton V, Sackellares JC, Vuong A, et al. 2005. Double-blind, placebo-controlled study of lamotrigine in primary generalized tonic-clonic seizures. Neurology 65:1737–43 55. Motte J, Trevathan E, Arvidsson JF, et al. 1997. Lamotrigine for generalized seizures associated with the Lennox-Gastaut syndrome. Lamictal Lennox-Gastaut Study Group. N. Engl. J. Med. 337:1807–12 56. Gilliam F, Vazquez B, Sackellares JC, et al. 1998. An active-control trial of lamotrigine monotherapy for partial seizures. Neurology 51:1018–25 www.annualreviews.org • Treatment of Epilepsy

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57. Steinhoff BJ, Ueberall MA, Siemes H, et al. 2005. The LAM-SAFE study: lamotrigine versus carbamazepine or valproic acid in newly diagnosed focal and generalised epilepsies in adolescents and adults. Seizure 14:597–605 58. Steiner TJ, Dellaportas CI, Findley LJ, et al. 1999. Lamotrigine monotherapy in newly diagnosed untreated epilepsy: a double-blind comparison with phenytoin. Epilepsia 40:601– 7 59. Brodie MJ, Richens A, Yuen AW. 1995. Double-blind comparison of lamotrigine and carbamazepine in newly diagnosed epilepsy. UK Lamotrigine/Carbamazepine Monotherapy Trial Group. Lancet 345:476–79 60. Frank LM, Enlow T, Holmes GL, et al. 1999. Lamictal (lamotrigine) monotherapy for typical absence seizures in children. Epilepsia 40:973–79 61. Janszky J, Rasonyi G, Halasz P, et al. 2000. Disabling erratic myoclonus during lamotrigine therapy with high serum level—report of two cases. Clin. Neuropharmacol. 23:86–89 62. Holmes LB, Wyszynski DF, Baldwin EJ, et al. 2006. Increased risk of nonsyndromic cleft palate amongst infants exposed to lamotrigine during pregnancy. Birth Defect. Res. Part A 76:318 63. Appleton R, Fichtner K, LaMoreaux L, et al. 1999. Gabapentin as add-on therapy in children with refractory partial seizures: a 12-week, multicentre, double-blind, placebocontrolled study. Gabapentin Paediatric Study Group. Epilepsia 40:1147–54 64. UK Gabapentin Study Group. 1990. Gabapentin in partial epilepsy. Lancet 335:1114–17 65. Anhut H, Ashman P, Feuerstein TJ, et al. 1994. Gabapentin (neurontin) as add-on therapy in patients with partial seizures: a double-blind, placebo-controlled study. The International Gabapentin Study Group. Epilepsia 35:795–801 66. Chadwick DW, Anhut H, Greiner MJ, et al. 1998. A double-blind trial of gabapentin monotherapy for newly diagnosed partial seizures. International Gabapentin Monotherapy Study Group 945–77. Neurology 51:1282–88 67. Asconape J, Diedrich A, DellaBadia J. 2000. Myoclonus associated with the use of gabapentin. Epilepsia 41:479–81 68. Leppik IE, Dreifuss FE, Pledger GW, et al. 1991. Felbamate for partial seizures: results of a controlled clinical trial. Neurology 41:1785–89 69. Faught E, Sachdeo RC, Remler MP, et al. 1993. Felbamate monotherapy for partial-onset seizures: an active-control trial. Neurology 43:688–92 70. Sachdeo R, Kramer LD, Rosenberg A, et al. 1992. Felbamate monotherapy: controlled trial in patients with partial onset seizures. Ann. Neurol. 32:386–92 71. Devinsky O, Faught RE, Wilder BJ, et al. 1995. Efﬁcacy of felbamate monotherapy in patients undergoing presurgical evaluation of partial seizures. Epilepsy Res. 20:241–46 72. The Felbamate Study Group in Lennox-Gastaut Syndrome. 1993. Efﬁcacy of felbamate in childhood epileptic encephalopathy (Lennox-Gastaut syndrome). N. Engl. J. Med. 328:29–33 73. Devinsky O, Kothari M, Rubin R, et al. 1992. Felbamate for absence seizures. Epilepsia 33:84 74. Kuzniecky R, Thompson G, Faught E, et al. 1991. Felbamate add-on therapy in intractable atypical absence. Epilepsia 32:10 75. Carmant L, Holmes GL, Sawyer S, et al. 1994. Efﬁcacy of felbamate in therapy for partial epilepsy in children. J. Pediatr. 125:481–86 76. Leroy RF, Castain T. 1991. Pilot study of felbamate in adult medically refractory primary generalized seizure patients. Epilepsia 32:13 77. Sachdeo RC, Wagner ML. 1991. Felbamate in generalized tonic-clonic seizures. Epilepsia 32:54

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78. French J, Smith M, Faught E, et al. 1999. Practice advisory: the use of felbamate in the treatment of patients with intractable epilepsy. Report of the Quality Standards Subcommittee of the American Academy of Neurology and the American Epilepsy Society. Neurology 52:1540–45 78a. Pellock JM, Faught E, Leppik IE, et al. 2006. Felbamate: consensus of current clinical experience. Epilepsy Res. 71:89–101 79. Dean C, Mosier M, Penry K. 1999. Dose-response study of vigabatrin as add-on therapy in patients with uncontrolled complex partial seizures. Epilepsia 40:74–82 80. Bruni J, Guberman A, Vachon L, et al. 2000. Vigabatrin as add-on therapy for adult complex partial seizures: a double-blind, placebo-controlled multicentre study. The Canadian Vigabatrin Study Group. Seizure 9:224–32 81. Miller NR, Johnson MA, Paul SR, et al. 1999. Visual dysfunction in patients receiving vigabatrin: clinical and electrophysiologic ﬁndings. Neurology 53:2082–87 82. Iannetti P, Spalice A, Perla FM, et al. 2000. Visual ﬁeld constriction in children with epilepsy on vigabatrin treatment. Pediatrics 106:838–42 83. Mackay MT, Weiss SK, Adams-Webber T, et al. 2004. Practice parameter: medical treatment of infantile spasms. Report of the American Academy of Neurology and the Child Neurology Society. Neurology 62:1668–81 84. Bailer M, Johannessen SI, Kupferberg HJ. 2007. Progress report on new antiepileptic drugs: a summary of the eighth Eilat Conference (EILAT VIII). Epilepsy Res. 73:1–52 85. Porter RJ, Partiot A, Sachdeo R, et al. 2007. Randomized, multicenter, dose-ranging trial of retigabine for partial-onset seizures. Neurology 68:1197–1204 86. Kwan P, Brodie MJ. 2001. Effectiveness of ﬁrst antiepileptic drug. Epilepsia 42:1255–60 87. Wiebe S, Blume WT, Girvin JP, et al. 2001. A randomized, controlled trial of surgery for temporal-lobe epilepsy. N. Engl. J. Med. 345:311–18 88. Engel J Jr., Wiebe S, French J, et al. 2003. Practice parameter: temporal lobe and localized neocortical resections for epilepsy. Epilepsia 44:741–51 89. Yasuda CL, Tedeschi H, Oliveira EL, et al. 2006. Comparison of short-term outcome between surgical and clinical treatment in temporal lobe epilepsy: a prospective study. Seizure 15:35–40 90. Fisher RS, Handforth A. 1999. Reassessment: vagus nerve stimulation for epilepsy. A report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology. Neurology 53:666–69 91. Morrell M. 2006. Brain stimulation for epilepsy: Can scheduled or responsive neurostimulation stop seizures? Curr. Opin. Neurol. 19:164–68 92. Sinha SR, Kossoff EH. 2005. The ketogenic diet. Neurologist 11:161–70 93. Pfeifer HH, Thiele EA. 2005. Low-glycemic-index treatment: a liberalized ketogenic diet for treatment of intractable epilepsy. Neurology 65:1810–12 94. Kossoff EH, McGrogan JR, Bluml RM, et al. 2006. A modiﬁed Atkins diet is effective for the treatment of intractable pediatric epilepsy. Epilepsia 47:421–24 95. Pavone P, Bianchini R, Triﬁletti RR, et al. 2001. Neuropsychological assessment in children with absence epilepsy. Neurology 56:1047–51 96. Motamedi G, Meador K. 2003. Epilepsy and cognition. Epilepsy Behav. 4(Suppl. 2):S25–38 97. Meador KJ. 2002. Cognitive outcomes and predictive factors in epilepsy. Neurology 58:S21–26 98. Gardiner M. 2005. Genetics of idiopathic generalized epilepsies. Epilepsia 46(Suppl. 9):15–20 99. Andermann F, Kobayashi E, Andermann E. 2005. Genetic focal epilepsies: state of the art and paths to the future. Epilepsia 46(Suppl. 10):61–67 www.annualreviews.org • Treatment of Epilepsy

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Annual Review of Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inﬂuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131 Childhood Obesity: Adrift in the “Limbic Triangle” Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p187 v

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soﬁa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkin’s Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237 Annu. Rev. Med. 2008.59:503-523. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/06/08. For personal use only.

Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p251 Cancer Epigenetics: Modiﬁcations, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381 Medical Management of Inﬂuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p397 Bacterial and Fungal Bioﬁlm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443 vi

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Antiretroviral Drug–Based Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Günter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503 Indexes Cumulative Index of Contributing Authors, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p525 Cumulative Index of Chapter Titles, Volumes 55–59 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

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