Trends in Brain Cancer Research (Horizons in Cancer Research)

TRENDS IN BRAIN CANCER RESEARCH HORIZONS IN CANCER RESEARCH VOLUME 28

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HORIZONS IN CANCER RESEARCH Volume 1: Prostate Cancer John N. Lucas (Editor) ISBN 1-59454-100-0 Volume 2: Trends in Ovarian Cancer Research A. P. Bardos (Editor) ISBN 1-59454-023-3 Volume 3: Percutaneous Cryotherapy of Renal Cell Carcinoma under an Open MRI System Junta Harada, Kazuo Miyasaka and Sajio Sumida (Editors) ISBN 1-59454-169-8 Volume 4: Focus on Colorectal Cancer Research Julia D. Martinez (Editor) ISBN 1-59454-101-9 Volume 5: Focus on Leukemia Research Rafael M. Romero (Editor) ISBN 1-59454-093-4 Volume 6: Progress in Bladder Cancer Research A. M. Mallory (Editor) ISBN 1-59454-129-9 Volume 7: Trends in Prostate Cancer Research John N. Lucas (Editor) ISBN 1-59454-265-1 Volume 8: Tumor Budding in Colorectal Cancer Recent Progress in Colorectal Cancer Research Tadahiko Masaki (Editor) ISBN 1-59454-189-2 Volume 9: Trends in Breast Cancer Research Andrew P. Yao (Editor) ISBN 1-59454-134-5 Volume 10: Trends in Leukemia Research Rafael M. Romero (Editor) ISBN 1-59454-311-9 Volume 11: Liver Cancer: New Research Felix Lee (Editor) ISBN 1-59454-182-5 Volume 12: Focus on Lung Cancer Robert L. Carafaro (Editor) ISBN 1-59454-082-9 Volume 13: Treatment of Ovarian Cancer A. P. Bardos (Editor) ISBN 1-59454-022-5 Volume 14: Focus on Kidney Cancer Research Kelvin R. Nunez (Editor) ISBN 1-59454-110-8 Volume 15: Focus on Pacreatic Cancer Research Maxwell A. Loft (Editor)

ISBN 1-59454-270-8 Volume 16: Peritoneal Carinomatosis from Ovarian Cancer Kostantinos N. Chatzigeorgiou and John N. Bontis (Editors) ISBN 1-59454-398-4 Volume 17: Trends in Pacreatic Cancer Research Maxwell A. Loft (Editor) ISBN 1-59454-524-3 Volume 18: Trends in Kidney Cancer Research Kelvin R. Nunez (Editor) ISBN 1-59454-141-8 Volume 19: Ovarian Cancer: New Research A. P. Bardos (Editor) ISBN 1-59454-241-4 Volume 20: Gene Therapy in Cancer Grace W. Redberry (Editor) ISBN 1-59454-288-0 Volume 21: Lung Cancer New Research Robert L. Carafaro (Editor) ISBN 1-59454Volume 22: New Developments in Bone Cancer Research Catherine E. O’Neil (Editor) ISBN 1-59454-337-2 Volume 23: New Developments in Breast Cancer Research Andrew P. Yao (Editor) ISBN 1-59454-809-9 Volume 24: Trends in Bone Cancer Research E. V. Birch (Editor) ISBN 1-59454-346-1 Volume 25: Focus on Brain Cancer Research Andrew V. Yang (Editor) ISBN 1-59454-973-7 Volume 26: Cancer Vaccine: New Research (Editor) ISBN 1-59454-968-0 Volume 27: Brain Cancer Therapy and Surgical Interventions Andrew V. Yang (Editor) ISBN 1-59454-974-5 Volume 28: Trends in Brain Cancer Research Andrew V. Yang (Editor) ISBN 1-59454-972-9

TRENDS IN BRAIN CANCER RESEARCH HORIZONS IN CANCER RESEARCH VOLUME 28

ANDREW V. YANG EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2006 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and as Table 2 ssions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data Trends in brain cancer research / Andrew V. Yang (editor). p. ; cm. Includes bibliographical references and index. ISBN: 978-1-60876-242-2 (E-Book) 1. Brain--Cancer--Research. I. Yang, Andrew V. [DNLM: 1. Brain Neoplasms. 2. Biomedical Research--trends. 3. Brain Neoplasms-secondary. QZ 206 T794 2006] RC280.B7T74 2006 616.99'4810072--dc22 2006001263

Published by Nova Science Publishers, Inc. New York

Contents Preface Chapter I

vii New Therapeutic Strategies in Low- Grade Gliomas (WHO Grade 2 Gliomas) Luc Taillandier, Laurent Capelle and Hugues Duffau

Chapter II

Strategies in Brain Cancer Research Luciano Neder, Andre Luiz Vettore, Oswaldo K. Okamoto, Rodrigo Proto-Siqueira, Luiz Gonzada Tone, Carlos Scridelli, Silvia Toledo, Suzana M. F. Malheiros, Suely K. Nagagashi Marie, Sueli Mieko Oba-Shinjo, Carlos Gilberto Carlotti Jr., Paulo Lotufo, Sergio Rosemberg, Wilson Araujo Silva and Marco A. Zago

Chapter III

Perspectives in Astrocytic Tumor Molecular Research Sergio Comincini

Chapter IV

Targeting the Renin-Angiotens in System and the Endothelin Axis in Human Brain Cancer Lucienne Juillerat-Jeanneret

Chapter V

Central Nervous System Lymphoma Andrew Lister, Lauren E. Abrey and John T. Sandlund,

Chapter VI

Epigenetic Mechanisms in the Development of Malignancies of the Central Nervous System (CNS) Sabrina Schlosser and Michael C. Frühwald

Chapter VII

Chapter VIII

Neurotrophin Receptors and Heparanase: A Functional Axis in Human Medulloblastoma Invasion Dario Marchetti, Adam J. Kaiser, Bryan E. Blust, Robert E. Mrak and Neeta D. Sinnappah-Kang Psychiatric Manifestations of Brain Tumors Subramoniam Madhusoodanan, Abhishek Sinha, Despina Moise and Sidhartha Sinha

1 89

119

145 167

193

253

281

vi Chapter IX

Index

Contents The Waterjet Instrument in Neurosurgery: A Detailed Account of its Clinical Potential after More than 150 Procedures Joachim Oertel, Jürgen Piek, Henry W.S. Schroeder and Michael R. Gaab

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323

Preface There are two types of brain tumors: primary brain tumors that originate in the brain and metastatic (secondary) brain tumors that originate from cancer cells that have migrated from other parts of the body. Primary brain cancer rarely spreads beyond the central nervous system, and death results from uncontrolled tumor growth within the limited space of the skull. Metastatic brain cancer indicates advanced disease and has a poor prognosis. Primary brain tumors can be cancerous or noncancerous. Both types take up space in the brain and may cause serious symptoms (e.g., vision or hearing loss) and complications (e.g., stroke). All cancerous brain tumors are life threatening (malignant) because they have an aggressive and invasive nature. A noncancerous primary brain tumor is life threatening when it compromises vital structures (e.g., an artery). In the United States, the annual incidence of brain cancer generally is 15-20 cases per 100,000 people. Brain cancer is the leading cause of cancer-related death in patients younger than 35. This new book brings together the leading research in this dynamic area of research. Low-grade glioma (LGG) (grade 2 or G2G) is a brain infiltrative neoplasia, often invading cortical and subcortical functional structures, while displaying as a rule a somewhat indolent course initially (no patent deficit). It affects essentially young, fully active patients, who usually present with seizures. However, these lesions progress relentlessly, and their final fate is anaplastic transformation, leading to neurological impairment and death, with an overall median survival of around 10 years since the onset of symptoms. Due to their apparent biological variability, commonly admitted spontaneous prognostic factors are of limited use if not questionable; consequently, the management of LGGs remains difficult to define (individually), and subject to controversies in the literature. However, most studies have evaluated the eventual impact of treatment(s) independently of the individual natural history and of the global therapeutic strategy. Thus, the goal of chapter one is to give new insights regarding the different therapeutic strategies that need to be considered for each patient, and the parameters that can help the decision making. First, it is now possible to benefit from data allowing a better understanding of the natural history of a given LGG: (1) initial tumoral volume (2) tumoral growth rate evaluated on at least two MRIs (3) tumoral metabolic profile, using new radiological methods such as PET and SRM (4) tumoral molecular biology, completing the information provided by classical histopathology.

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Second, it is mandatory to perform a complete neurological examination, extensive neuropsychological assessment and evaluation of the quality of life from the time of diagnosis throughout the follow-up. Moreover, the analysis of the brain functional (re)organization and connectivity is needed via the use of new neurofunctional imaging methods (PET, MEG, fMRI, DTI), in order to understand the individual mechanisms of functional compensation in reaction to the glioma growth – explaining the frequent lack of deficit despite a classical invasion of so called “eloquent” areas. Third, the advantages and limits of each treatment have to be considered for each patient. In this way, the use of intraoperative electrical functional mapping as well as the integration, up to the operating room, of preoperative anatomo-functional data, has allowed the minimization of the risk of postoperative sequelae, while improving the quality of tumor removal, even in eloquent regions. However, the actual long-term impact of surgery on survival still remains to ascertain. Concerning radiotherapy, the adaptation of doses, fractionation and volume of irradiation has enabled to decrease its risks, especially regarding cognitive functions. Nevertheless, despite an impact on the progression free survival, the effect on the overall survival is not proven. Finally, the recent use of new chemotherapeutic drugs has allowed a better tolerance and a frequent improvement of the quality of life via an impact on seizures, with a stabilization or even partial regression of the LGG; however, the follow-up is still too short to conclude. On the basis of these (non exhaustive) parameters, the authors propose in the last part of this chapter to consider not “a standard treatment”, but rather alternative “multiple dynamic therapeutic strategies” adapted to each patient, to be evaluated according to the clinicoradiological evolution of the LGG. Diffuse astrocytomas are the most frequent primary neoplasms in the central nervous system and account for more than 60% of all primary brain tumors. Although the precursor lesions of these neoplasms have not yet been identified, genetic studies have shown that malignant transformation of neuroepithelial cells is a multistep process, involving distinct molecular pathways. Genetic testing may thus identify distinct subsets of gliomas with similar histologic patterns, for instance, primary and secondary glioblastomas. Despite recent advances in the field, prediction of clinical outcome and the overall survival of patients with brain cancer have remained dismal. The advent of genomic technologies to study complex diseases such as brain cancer brings a new paradigm to translational medicine. While traditional methods for the study of underlying mechanisms of cancer focus on restricted factors, genomic approaches facilitate discovering of numerous genetic markers and pathways related to cancer pathogenesis in a short time frame. New high-throughput strategies based on gene expression profiling of tumors and corresponding normal tissue have practical purposes of discovering targets for the development of smart drugs and new biomarkers for diagnosis and prognosis. Chapter two attempts to outline some of the steps involved in such strategies, based on studies conducted by an interdisciplinary consortium comprised by clinicians, surgeons, pathologists, and molecular biologists. Starting from a limited number of samples, genes with aberrant expression in tumors can be identified with a combined use of high throughput analyses such as SAGE and DNA microarrays. Data can be further validated by quantitative real-time PCR and immunohistochemistry in additional tumor specimens, allowing the

Preface

ix

identification of potential markers of astrocytomas with distinct levels of malignancy. Besides refining diagnostic classification of brain cancers, this genomic-based approach may improve prognostic assessment and definition of therapeutic strategies, bringing useful knowledge into clinical decision-making routine. Astrocytomas are fairly common tumors of neuroectodermal origin that typically show a high degree of tumor malignancy. Specific pathological features of astrocytomas comprise a high degree of neoplastic cell proliferation and invasivity within the brain peritumoral tissues and, in addition, a prominent angiogenesis in the neoplastic tissue. The modern clinical practice, based on surgical interventions and on radio-chemicals approaches, need an accurate anatomical tumor localization and a well defined tumor grade classification. The classification of astrocytomas is based on morphological and immunohistochemical methods aimed at defining the predominant neoplastic cellular typology. Thus, glial tumors can be composed of astrocytes (giving rise to astrocytomas), oligodendrocytes (oligodendrogliomas) as well as of other different glial cells such as oligoastrocytes (oligoastrocytomas) and ependimal cells (ependimomas). The histological tumor classification is necessary associated with the histopathological inspection that states the malignancy grades according to suggested guidelines. The tumor classification system that is mainly in use is that proposed by the World Health Organization (WHO). According to this classification, astrocytomas are divided into four grades: pilocytic astrocytoma (WHO grade I), low-grade astrocytoma (grade II), anaplastic astrocytoma (grade III) and glioblastoma multiforme (grade IV). An important issue in the classification of astrocytoma is to assess whether the tumor was originated de novo (primary astrocytoma) or arose from the tumor progression of an existing lower-grade astrocytoma (secondary astrocytoma). Astrocytoma present a great heterogeneity of neoplastic cells involved, which makes their classification with regards to tumor progression rather difficult. This is an important matter since the prognosis of patients does correlate with age, tumor type and, mostly, with the malignancy grade. Although several studies have improved the tumor classification with suitable histopathological criteria, recent data suggest that morphologically indistinguishable astrocytomas have distinct classes of causal oncogene activation, and that these subclasses may be targetable by oncogene/signaling pathway specific therapies. Recent technical advances such as the RNA and protein microarrays and the gene expression profiling can be very informative in terms of defining the global biological profiling of the different cancers, in identifying molecular tumor subsets and to develop predictive and prognostic tumor markers. In conclusion in chapter three, the integration of these molecular data networks can be used to improve the knowledge on the genetic processes that regulate the tumor progression, to address to novel therapies, likely to result in significant improvement in the survival of astrocytomas patients. The renin-angiotensin system (RAS) and the endothelin (ET) axis, in addition to controlling blood pressure, may be involved in cell growth and/or death in the brain. In order to question these issues in glioblastoma, the authors in chapter four have compared the expression of the components of the RAS and ET axis in surgical specimens of human brain tumors and adjacent tissue. Human brain tumor cells or rat brain cells in culture were used to evaluate the functions of the RAS and ET axis. From these experiments, they have

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demonstrated that the RAS is involved in maintaining the functions of the cerebral vasculature (the blood-brain-barrier) by controlling the ratio between angiotensin (Ang) II/Ang III production, and the enzyme renin more directly in the survival of glioblastoma cells, whereas the ET axis is mainly involved in the survival of tumor cells. Central nervous system involvement with malignant lymphoma whether primary or secondary is an uncommon but not rare complication observed in the management of patients with hematological malignancy. Its importance lies in the considerable morbidity and mortality with which it is associated and the inadequacy of therapy. In chapter five, in Section I, Dr. Lauren Abrey addresses the totality of the problem of primary central nervous system lymphoma, with emphasis on strategies increasingly dependent on systemic chemotherapy. In Section II, Dr. John Sandlund reviews the success of sequential clinical trials of overall therapy for acute lymphoblastic leukemia in child-hood, identifying those patients at high risk of central nervous system leukemia and the development of a rational therapeutic strategy for prevention. In Section III, Dr. Andrew Lister discusses the issue of secondary central nervous system involvement with lymphoma and the indications for prophylaxis. Malignant tumors of the central nervous system represent a rather heterogeneous group of neoplasms originating from virtually any anatomical structure within the spine and skull. While in adult patients malignant gliomas predominate, it is the group of embryonal malignancies (i.e. medulloblastoma, supratentorial primitive neuroectodermal tumor [sPNET], atypical teratoid, rhabdoid tumor [AT/RT] and pineoblastoma) that is prevalent in childhood. Despite major improvements in the clinical management including timely diagnosis, advanced supportive care and refined multimodality treatment prognosis remains grim for a large group of patients. In adulthood the group of high-grade glioma bears a dismal prognosis. Some authors advocate that the diagnosis of a high-grade glioma is synonymous with a palliative situation and should be managed as such. Thus a change of focus has been introduced into adult neurooncology which is quality of life as an outcome measure rather than survival. In childhood major advances have been made in the treatment of embryonal tumors such as standard risk medulloblastoma, which is defined by the following factors: age above three years, neurosurgical complete resection with minimal residual tumor and absence of metastatis. Other factors such as desmoplastic histology, high level of TRKC mRNA are under discussion as prognostic factors. Consequently the diagnosis of medulloblastoma in small children, with metastasis at diagnosis, recurrent or large residual tumor constitutes an almost inevitably fatal condition. This is also true for malignancies like AT/RT or sPNET. No consistently curative therapy exists for these conditions. Understanding the genetic and epigenetic basis of the origin and progression of these tumors shows great promise for the development of prognostic markers and eventually improved diagnosis and treatment. Certain genetic events such as mutation of the tumor suppressor genes TP53 and PTEN or amplification of the growth factor receptor EGRF are long-known hallmarks of genetic mutations in gliomas of adults. Mutations in members of the sonic hedgehog - patched pathway (SHH-PTCH) have been described in medulloblastomas. Likewise deletions and basepair mutations of the SMARCB1

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gene have been found in AT/RT of childhood. No single genes have been identified in sPNET. Epigenetic events i.e. changes in gene transcription not due to base pair mutations have recently received major attention. Foremost aberrant DNA-methylation and histone deacetylation appear to contribute to the malignant potential of CNS tumors. Gene-by-gene approaches and genome scanning techniques such as chip-based-analysis have identified a number of genetic loci with relevance in the development/formation of neoplasms of the CNS in adults and children. Examples include aberrant methylation of the tumor suppressor gene candidate RASSF1A in medulloblastoma and sPNET, which together constitute the most common malignant brain tumors of childhood. Aberrant methylation of the DNA-repair gene O6-MGMT appears to be an important predictor of response to therapy in malignant gliomas of adults. Additional examples of epigenetically inactivated genes have been described in chapter six. Lesions of the epigenome hold great potential for the elucidation of the pathomechanisms of central nervous system tumors. As epigenetic lesions may be reversed by chemical manipulation epigenetic therapy holds great promise for the management of malignant CNS tumors in adults and children. Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Modern therapy has produced five-year survival rates as high as 70% for some MB patients, but this has come at the cost of significant long-term treatment-related morbidity. The cellular mechanisms involved in metastatic spread of medulloblastoma are largely unknown. Neurotrophins (NT) comprise a family of structurally and functionally related neurotrophic factors that are critical for central nervous system (CNS) development, and nerve growth factor (NGF) is the prototypic NT. NT act through two groups of structurally unrelated neurotrophin receptors (NTR): a family of receptor tyrosine kinases (Trks, mainly TrkA, TrkB, and TrkC) and a tumor necrosis factor receptor (TNFR)-like molecule called p75NTR. TrkC expression is a good prognostic indicator for MB. TrkC binds only to neurotrophin-3 (NT-3) whereas p75NTR binds to all NT family members. Importantly, little is known about the biological functions of p75NTR in primitive neuroectodermal tumors such as MB. In contrast, NT-regulated heparanase (HPSE) is a unique ECM-degrading enzyme associated with tumor angiogenesis and metastasis in a wide variety of cancers. However, the potential role of HPSE in MB and in MB invasive pathways has not been investigated. In chapter seven, the authors have provided, for the first time, evidence of differential expression of HPSE in medulloblastoma, and they have shown a correlation between this expression and the invasive properties of three newly developed medulloblastoma cell lines. Equally important, they have demonstrated heparanase expression in XX of XX (88%) clinical medulloblastoma specimens analyzed by immunohistochemistry. This heparanase expression was found both in the cytoplasm and nucleius, with particularly intense immunoreaction in the latter. Quantitative polymerase chain reaction revealed a negative correlation between expression of HPSE and expression of the NT-3 receptor TrkC, which is associated with a favorable clinical outcome in medulloblastoma. Activation of TrkC or TrkC/p75NTR by NT-3 was found to regulate HPSE activity and invasive properties of medulloblastoma. Taken together, our data provide initial evidence that HPSE functionality, in a context linked to

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TrkC and p75NTR activation, may play critical roles in medulloblastoma invasion and tumor progression. Psychiatric manifestations, even though uncommon with brain tumors may be the presenting symptomatology in some cases. If diagnosed early and treated satisfactorily, there may be complete resolution of the presenting symptoms. Various authors have attempted to categorize psychiatric symptoms based on the location of the tumor. Neuro imaging should be considered in patients with new onset psychosis, recurrence of previously well controlled mental symptoms or occurrence of new mental symptoms and in patients who remain refractory to psychiatric treatment. In chapter eight, our review of the published cases over the past 54 years indicate that neither tumor location nor type is correlated with any particular psychiatric symptoms. Mood symptoms have been noted in a significant number of cases and could be a harbinger to an evolving tumor of the brain. The waterjet instrument is currently under clinical evaluation in neurosurgical procedures, and precise tissue dissection with vessel preservation has been demonstrated experimentally. Chapter nine focuses on the general application technique of the device and on the distinct clinical situations in which the device possesses peculiar advantages compared with conventional techniques based on the experience of more than 150 procedures. The waterjet instrument has been applied in more than 150 intracranial procedures including gliomas (°1-4), metastases, meningiomas, acoustic neurinomas, epidermoids cysts, and epilepsy surgery. The instrument was used in combination with conventional methods for tissue dissection and tissue aspiration. All cases were prospectively followed up to 2 years. Intraoperatively, the waterjet was easy to handle. While it was applied in a similar fashion as the ultrasonic aspirator in most tumours, the instrument possessed peculiar advantages in the dissection of tumours from the intact adjacent brain parenchyma and in the separation of brain tissue from the arachnoid membranes. In the first, the parenchyma was precisely dissected and preserved vessels could be coagulated at a wide distance to the surrounding brain. With this technique a significant reduction of surgical blood loss was observed, and the tissue dissection was minimally traumatic. In the latter, the arachnoid membranes were easily preserved while the brain tissue was precisely cut. Our results indicate (i) that the waterjet enables tissue dissection and subsequent vessel coagulation without damage to the remaining brain tissue, and (ii) that it might be well suited for special indications such as subpial dissections. In all, it appears to be more suited for tissue dissection than the CUSA under certain conditions particularly if minimally traumatic surgery with minimal blood loss is of major importance.

In: Trends in Brain Cancer Research Editor: Andrew V. Yang, pp.1-86

ISBN 1-59454-972-9 ©2006 Nova Science Publishers, Inc.

Chapter I

New Therapeutic Strategies in LowGrade Gliomas (WHO Grade 2 Gliomas) Luc Taillandier1, Laurent Capelle2 and Hugues Duffau2,∗ 1 2

Department of Neurology, CHU Nancy, Hôpital Central, Nancy Cedex, France Department of Neurosurgery, INSERM U678, Hôpital Salpêtrière, Paris, France

Abstract Low-grade glioma (LGG) (grade 2 or G2G) is a brain infiltrative neoplasia, often invading cortical and subcortical functional structures, while displaying as a rule a somewhat indolent course initially (no patent deficit). It affects essentially young, fully active patients, who usually present with seizures. However, these lesions progress relentlessly, and their final fate is anaplastic transformation, leading to neurological impairment and death, with an overall median survival of around 10 years since the onset of symptoms. Due to their apparent biological variability, commonly admitted spontaneous prognostic factors are of limited use if not questionable; consequently, the management of LGGs remains difficult to define (individually), and subject to controversies in the literature. However, most studies have evaluated the eventual impact of treatment(s) independently of the individual natural history and of the global therapeutic strategy. Thus, the goal of the present review is to give new insights regarding the different therapeutic strategies that need to be considered for each patient, and the parameters that can help the decision making. First, it is now possible to benefit from data allowing a better understanding of the natural history of a given LGG: (1) initial tumoral volume (2) tumoral growth rate evaluated on at least two MRIs (3) tumoral metabolic profile, using new radiological methods such as PET and SRM (4) tumoral molecular biology, completing the information provided by classical histopathology. ∗

Correspondence concerning this article should be addressed to Dr. Hugues Duffau, Department of Neurosurgery, INSERM U678, Hôpital Salpêtrière, 47-83 Bd de l’hôpital, 75013, Paris, France. Phone: 33 1 42 16 34 28; Fax: 33 1 42 16 34 16; Email: [email protected].

2

Luc Taillandier, Laurent Capelle and Hugues Duffau Second, it is mandatory to perform a complete neurological examination, extensive neuropsychological assessment and evaluation of the quality of life from the time of diagnosis throughout the follow-up. Moreover, the analysis of the brain functional (re)organization and connectivity is needed via the use of new neurofunctional imaging methods (PET, MEG, fMRI, DTI), in order to understand the individual mechanisms of functional compensation in reaction to the glioma growth – explaining the frequent lack of deficit despite a classical invasion of so called “eloquent” areas. Third, the advantages and limits of each treatment have to be considered for each patient. In this way, the use of intraoperative electrical functional mapping as well as the integration, up to the operating room, of preoperative anatomo-functional data, has allowed the minimization of the risk of postoperative sequelae, while improving the quality of tumor removal, even in eloquent regions. However, the actual long-term impact of surgery on survival still remains to ascertain. Concerning radiotherapy, the adaptation of doses, fractionation and volume of irradiation has enabled to decrease its risks, especially regarding cognitive functions. Nevertheless, despite an impact on the progression free survival, the effect on the overall survival is not proven. Finally, the recent use of new chemotherapeutic drugs has allowed a better tolerance and a frequent improvement of the quality of life via an impact on seizures, with a stabilization or even partial regression of the LGG; however, the follow-up is still too short to conclude. On the basis of these (non exhaustive) parameters, we propose in the last part of this article to consider not “a standard treatment”, but rather alternative “multiple dynamic therapeutic strategies” adapted to each patient, to be evaluated according to the clinicoradiological evolution of the LGG.

Keywords: Low-grade glioma, Tumor surgery, Radiotherapy, Quality of life, Tumor biology.

Brain

mapping,

Chemotherapy,

Introduction LGG – gliomas WHO grade II [353] – are slow-growing primary brain tumors representing approximately 15 to 35% of gliomas (average incidence around 2/100.000/year), which usually affect young adults between 30 and 40 years of age [732]. They are generally revealed by seizures, in patients as a rule with no or slight neurological deficit in the first stage of the disease. However, recent extensive neuropsychological assessments have shown that most patients already have mild cognitive disorders at this time [671]. LGG can follow three ways of evolution: (1) local growth (2) invasion (3) anaplastic transformation. First, recent works demonstrated that before any anaplastic degeneration, LGG show a continuous, constant growth of their mean tumor diameter over time, with an average slope around 4 mm of mean diameter increase per year [435]. Second, LGG have a tendancy to migrate along the main white matter pathways, both within the lesional hemisphere or even controlaterally essentially via the corpus callosum [174, 225]. Third, LGG systematically changes its biological nature and evolves to a high grade glioma, with a median of anaplastic transformation around 7 to 8 years, invariably fatal (median survival around 10 years) [732, 747]. Such better knowledge of the natural history of LGG and their clinical consequences has lead, in the past decade, to propose a more active therapeutic strategy rather than a “wait and

New Therapeutic Strategies in Low-Grade Gliomas

3

see” attitude. Indeed, the vision of a tumor with a “dynamic” behavior needs to be integrated in the therapeutic strategy, in order to adapt the treatment both to the actual biology of the glioma at the time of diagnosis (“tumor mapping”), and to the functional compensation of the brain already induced by the slow-growing glioma before any symptom (“cerebral mapping”) – thus to their interactions. The goal of this article is first to review the advances in the determination of the natural history for each LGG. Indeed, the definition of spontaneous risk factors remains very difficult for each patient using classical clinical and radiological parameters, as demonstrated by many retrospective studies and prospective trials in the literature [7-35 ou Wessels à Whitton]. Hence, the adjunct of complementary individual data allowing a “tumor mapping” is very useful, using recent developement in the field of metabolic neuroimaging, in addition to parallel progress in molecular biology (see chapter) and biomathematical modelisation [313, 435, 662]. Second, we will analyze the progress made in the precise evaluation of the consequences of tumour progression on brain functioning, thus on the quality of life. Indeed, in addition to the classical neurological examination, numerous recent studies have shown that it was mandatory to perform an extensive neuropsychological assessment in LGG. Furthermore, the study of the brain functional (re)organization and connectivity is needed via the use of new neurofunctional imaging methods (PET, MEG, fMRI, DTI), in order to understand the individual mechanisms of functional compensation in reaction to the glioma growth – explaining the frequent lack of deficit despite a frequent invasion of so called “eloquent” areas. Third, on the basis of a better understanding of the individual dynamic interrelationships between tumor progression and brain compensation, we will discuss about new therapeutic strategies, i.e. combined and sequential treatments, adapted to each patient and to the clinicoradiological evolution, with the double goal to preserve (or even improve) the quality of life as well as to increase the median survival.

Advances in the Study of the Natural History of LGG: Towards an Individual Prognosis Introduction - Epidemiology Gliomas account for more than half of the primitive central nervous system tumors, and are the result of the abnormal proliferation of glial cells. They are classified according to the tumoral cell type, mainly astrocytes and/or oligodendrocytes, and to their relative aggressivity, reflected by two to four grades (1 to 4) in the WHO classification [353]. Their incidence is usually reported around 5 to 7/100,000/yr, with a greater frequency in males (especially for astrocytomas), caucasians, northern countries (Scandinavian, North American), rural zones [186, 196, 266, 322, 323, 657, CBTRUS, 732], and increases with age [579, 691]. There appears to be a true elevation of annual incidence of gliomas, and of the grade 2 forms [315], globally or among the oldest population [196]. Its signification is still matter of discussion, but could not be the sole result of an easier detection.

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Luc Taillandier, Laurent Capelle and Hugues Duffau

The grade 1 astrocytomas are quite peculiar, comprising various oncotypes that share more or less a slow growth rate, a gross (spatial) delimitation, and on the whole a favorable prognosis. On the opposite, gliomas of other grades share an infiltrative (invasion, migration) potential which renders their radical and selective treatment uncertain, and a growing biological aggressivity from grades 2 to 4. It is noteworthy that, following the essentially pediatric prevalence of grades 1, grade 2 gliomas affect mainly young adults, and grade 3 then 4, representing more than 60% of all gliomas, affect middle-age and older patients. While the overall prognosis follows an inverse rule, with median survivals decreasing from around 10 years to less than 12 months from grades 2 to 4. Oligodendrogliomatous tumors are generally graded in two categories, low-grade (well differentiated) and high-grade (corresponding to a grade 3 glioma). Grade 2 gliomas are glial neoplasias made of cells resembling their presumed original counterparts; astrocytomas derive from type 1A protoplasmic cortical astrocytes (GFAP+, A2B5-) or more frequently from perinatal progenitor cells O2-A giving rise to fibrillar astrocytes (GFAP+, A2B5+), like oligodendrogliomas (GFAP-) [84, 415]. They present a slightly (or moderately) elevated cellularity and cellular atypia, and usually lack pleomorphism, (significant) cytoplasmic or nuclear atypia, vascular endothelial proliferation, mitosis (one/40 HPF accepted) or necrosis, that characterize high-grade gliomas [353]. With few exceptions [130, 146, 319, 321], grade 2 gliomas are unique, sporadic, and do not metastazise, and familial forms outside neurocutaneous syndromes [86, 95], as well as causal environmental or external factors [301, 527, 584], are seldom recognized. They can sometimes grow in a manner resembling gliomatosis from an initially bulky tumor. Astrocytomas can affect all age groups, with grossly 10% encountered before the age of 20, 60% between 20 and 45 years, and 30% after 45 years, the peak being at 30-40 years [713, 732]. There is a male predominance of almost 1.2:1 [353]. They are preferentially located in the supratentorial compartment, mostly in frontal and temporal sites, then in the brainstem and spinal cord, rarely in the cerebellum [353]. If oligodendrogliomas can be encountered ubiquitarily among the neuraxis proportionally to the amount of white matter [183], they show a great predilection for frontal sites while intra-ventricular or posterior fossa locations are rare. Their distribution with age is more widespread, more often between 30 and 60 years, and the masculine predilection is more marqued at around 1.3:1 [353]. A meaningful peculiarity common to all these tumors is to favor locations in the immediate vincinity of, or originally within, eloquent cerebral areas («secondary» functional areas such as frontal SMA, insula), more frequently than de novo high-grade gliomas [162]. By their infiltrative characteristic, they blurr anatomic boundaries, with a distorsion more than a destruction of invaded structures [137]. A categorical classification is by essence imperfect in terms of biological processes, and the situation with gliomas even more complicated. Indeed, there is an on-going controversy concerning the determination of the constituting cellular type, and even its neoplastic nature, and there are notable overlappings among the grades. Moreover, the natural tendancy of a glioma (grades 1 except) appears to be the acquisition/selection with time of a genophenotype of higher aggressivity (malignancy). That explains why the qualification of benign for grade 2 gliomas has been rightly abandonned. The progress made in the last decade(s) in various fields gives nowadays the opportunity to qualify differently these tumors than with

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the sole anatomopathologic examination, with the aim to reflect more closely the proper potential of a given glioma, and to evaluate more precisely its evolutive stage. Until now, reflecting these difficulties, the biological diversity of gliomas, especially of grade 2, has long been recognized [28, 513, 555], and the poor knowledge of their natural history, the more so at an individual level, explains that the commonly accepted spontaneous prognostic factors are mostly imprecise or questionable, and that the impact of available treatments, if any, is still matter of debate [419, 497, 516].

Methodological Biases Our poor understanding of grade 2 gliomas, despite a prolific literature on the subject, results first of all from the different methodological approaches used in reported series, hampering the possibility of comparisons or pooling of available data [418]. Until the last (two) decades, the series of grade 2 gliomas comprised in fact «low-grade gliomas», ie grouped grade 1 and 2 gliomas. This term should be abandoned once and for all, to ascertain that the focus is on grade 2 gliomas only. Moreover, among grade 2 gliomas, the gemistocytic oncotype, known to behave quite differently although this has been recently challenged, should be excluded from analysis or at least analyzed separately [383, 682, 748, 763]. Similarly, the studies often encompassed cases of all age groups. Apart from the redundancy with the first point, since grades 1 affect primarily pediatric patients, this brought to study genuine grade 2 gliomas arising in children, but whose behavior, hence prognosis, is now well known to be fundamentally different than those arising in adults, as shown by clinical observation up to the study of genetic alterations [51, 101, 110, 194, 213, 511, 523, 524, 533, 612, 664]. Also partly redundant with the first two points, is the mixture of the various possible locations inside the central nervous system in the populations studied; certain sites are essentially those of grade 1 gliomas and/or affects younger subjects (cerebellum, midline, in particular optic pathways and hypothalamus/diencephalon). Controversy and natural evolution in histological concepts has lead to the use of various classifications in the series reported. Moreover, given the known important inhomogeneity of these tumors [119], and the frequent scarcity of tumoral specimen available, especially after sole biopsy, a misdiagnosis or underestimation of grade is always possible [43, 96, 361, 397, 448, 449, 454, 507, 512, 597, 637, 718]. Another aspect of the problem would be the delay at which histological diagnosis is made. Some advocate, in the absence of necessity or will of oncologic treatment, to not perform systematically a diagnostic biopsy, and hence might exclude from analysis a given patient whose tumor will undergo malignant transformation and be histologically examined only at that time [713]. The relative rarity of grade 2 gliomas and their usually long history, result in series sometimes (too) small, or encompassing several decades, during which clinical, radiological, histological and therapeutic aspects have eventually greatly varied. Series are mostly retrospective in nature. Statistical methods used are also diverse and sometimes inappropriate, the value and sometimes definition of some parameters are frequently lacking or differ from one series to another, a multivariate analysis has frequently been done only in the last two decades. In the same manner, the evaluation of treatments has long suffered of

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imprecision, as for example the quality of resection only expressed as the surgeon’s opinion, which is clearly unreliable for a tumor mostly difficult if not impossible to distinguish from normal parenchyma. Endpoints of the studies, as their definitions, can be a drawback to adequate comparisons (anaplasia, survival, recurrence, progression..) [570]. Beginning a follow-up at diagnosis or treatment is first of all eventually quite different from a patient or a series to another, and secondly does not take into account in all cases the pre-diagnostic or pre–treatment period. Hence, post-diagnosis or post–treatment outcomes can simply reflect the fact that the patient was treated at a different delay or evolutive stage of his tumor, and not an eventual greater benefit of the therapeutic strategy [515]. Last, the delay of effective observation is most of the time too short to draw reliable conclusions regarding a tumor whose natural history spans over several years to even decades [418, 515, 570]. For all those (non exhaustive) critics, «the bad results obtained from clinical research have contributed (..) to the notion that low-grade gliomas might represent a very heterogeneous population of patients, for which the prognostic factors could play a crucial role in the determinism of the evolution of the affection» [516]. The vicious circle is about to close on itself, the prognostic factors appearing crucial but poorly known or not definitely adressed, and difficult to determine on populations that can not be adequatly subdivided (stratified) along reliable prognostic factors. Also because of the long observation period that would be needed, prospective, randomized studies are very difficult to realize, and a great number of cases would need to be included in the absence of homogeneous (a priori) riskgroups. Therefore, «the establishment of standards of care, or guidelines, is maybe not possible or not desirable» [25 refering to 464, 514, 610], and «another decade will probably be necessary to shift from a passive and defensive attitude (no significant gain so primum non nocere) to an active, aggressive, attitude» [80]. Nevertheless, thanks to a renewed interest in this pathology and technical progress, prospective, randomized trials have been, and are currently undertaken (EORTC/MRC, NCCTG/RTOG/ECOG), a common language begins to be adopted, significant progress has been made in the last decade and real advances should be definitely achieved in the coming years.

Clinico-Radiological Aspects Grade 2 Gliomas are Constantly Evolving Tumors Menacing Life Grade 2 gliomas, with the probable exception of «minute-gliomas» associated to – refractory- epilepsy and as a rule reported in series dealing with epilepsy rather than in an oncologic context [485], are constantly evolving lesions. More than half of the cases followed after withholding any oncologic treatment at diagnosis, will be ultimately treated at a median delay of around 2 years [541, 713], and the same proportion are expected to manifest significant clinical changes at a delay of 5 years after initial therapy (endpoint chosen for the EORTC trial 22845 –330-). The tendancy to progression does not show a plateau and continues at least during 10 years after treatment [752]. Another and not the least characteristic of grade 2 gliomas is their innate tendancy with time to undergo malignant transformation. It appears usually progressively [430] and is

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expected in about two-thirds of the progressions observed [469, 470], its risk growing with each recurrence [4, 469, 470]. It is observed at a growing rate with time (no plateau either) [454, 710], affecting half of the cases at 5-6 years and more than 75% at 9 years [541], and on the whole its incidence increases with observation or survival time [253, 718], appearing «almost ineluctable» [5, 470]. This process appears to be spontaneous, intrinsic, independant of any external stimulus [5]. When transformed into their malignant (high-grade) counterparts (#20% of «secondary glioblastomas»), these gliomas carry the same prognosis that de novo forms [156–case control study -, in contrast to 753]. With practical differences lying in a younger age (about 10 years less), that grade 3 forms predominate, and that a longer survival can sometimes be obtained, due to the fact in our experience that anaplasia can frequently be encountered at its beginning [«intermediate forms»-123-], seen as microscopic foci among an otherwise seemingly histologically grade 2 tumor [448]. If anaplastic transformation is by far the principal cause of fatal outcome (in this otherwise generally young then healthy population) [513], the sole volumetric evolution, especially in the case of deep-seated or axial tumors, can be responsible of the death of the patient [312, 448]. Life expectancy of (usually young) patients affected is greatly reduced, since overall survivals are in the order of 10 years from the clinical onset or 6-9 years after diagnosis or treatment for astrocytomas [316, 558, 706, 732], and up to 12 to even 16.7 years for oligodendrogliomas [497]. In fact, reflecting their biological diversity [28, 513, 555], the reported survival rates at 5 and 10 years post-diagnosis or treatment vary greatly, first of all from one epoch to another [23, 316]. Indeed, in older series, that is before the advent of CT or MRI (and the easier access to these more performing radiological examinations), they were respectively of 17 to 53% and 6 to 11% [188, 193, 397, 514, 609, 637], while in more recent series they are around 40 to 80% and 20 to 50% for astrocytomas, and 60-85% and 30-60% for oligodendrogliomas [23, 260, 312, 351, 375, 405, 420, 448, 488, 512, 515, 591, 614, 718, 748]. This difference in post-treatment survival rates is mainly the result of an earlier detection [407, 515], but apparently not only [316, 483]. The malignant transformation rate is reported around 50% (20 to 80%) at 3 to 5 years post-diagnosis [63, 71, 339, 732], but its exact incidence is difficult to ascertain in the absence of repeated histological examinations. Grade 2 Gliomas Show Grossly Two Clinical and Radiological Phases The first phase is long when clinically perceptible, affecting a young subject; the lesion is initially asymptomatic or limited to epilepsy, which represents the most frequent symptom, inaugural in 2/3 to 90% of the cases, more frequent with oligodendrogliomas [33, 106, 750]. This is in accordance with the low epileptic threshold of the regions most frequently affected by these tumors (limbic and temporal lobes, SMA, operculae and insulae). Radiological growth is slow, the lesion showing no particular vascularity. The progressive acquisition of genotypic and phenotypic characteristics ending up to malignancy leads to the second phase, with radiologically a faster growth, new or increasing contrast enhancement then edema, and biologically a different level of neoangiogenesis and proliferation [4, 93, 376, 427, 428, 540, 718]. The occurrence of focal neurological deficits or signs of raised intracranial pressure is usually associated with anaplastic evolution [320], as is the appearance of new types of seizures and/or the increase in intensity or frequence of epilepsy [204]. Rarely, the inaugural

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symptomatology is due to a tumoral hemorrhage, as a rule with an oligodendroglioma [3, 55, 411]. The diagnosis and follow-up of grade 2 gliomas must rely now on MRI [621], with T1weighted (and after contrast medium) and FLAIR images [78, 648]. Grade 2 gliomas show as an area of hypointense signal on T1 and hyperintense on T2-weighted/FLAIR sequences, more or less homogeneous. Radiologically, they can appear well demarcated (bulky) or not delineated at all (infiltrative), and if they generate a mass effect, it is usually local (sulci, cortical surface) or relatively moderate with regard to the extension of the signal abnormality. Calcifications are frequent, especially with oligodendrogliomas, irregular, serpiginous more often than nodular [338, 727]. Cystic tumors are rare [417], as the eventuality of a bony calvarial erosion [401]. Contrast enhancement should seldom be seen in grade 2 gliomas [8 to 15% of the cases – 81, 428, 513, 635, 718-], and diminishes in more recent series [25], is of faint or moderate intensity and thickness [4], patchy or in strands, and more often encountered with oligodendrogliomas which behold naturally a greater vascularization [183, 338, 420, 497, 727]. When contrast enhancement is underscored by neoangiogenesis or disruption of the blood-brain barrier, it represents a strong indicator of a malignant phenotype, but a contrast enhancing tumor is not always malignant (apart cases of observed appearance and/or growth of the enhancement), and more importantly the reverse is also true, which stresses the importance of realising a sampling of a tumor focused on the enhancing or «hot» zones, whatever the radiological modalitiy (anatomic, vascular, metabolic). This two-way discrepancy is reported from 8 to 50% [4, 77, 96, 232, 361, 440, 449, 628, 718]. Histology and Molecular Aspects «To gauge therapy and advise patients with intracranial astrocytomas, an accurate measure of prognosis is needed. Histological grading has not been adequate to determine individual outcomes» [57]. This is also true with the other oncotypes of gliomas for which none of the classification system is yet satisfying enough to reflect the natural history effectively observed, in particular at an individual level. Histological Classification and Grading of Gliomas Various histological classifications have been proposed, based on histogenesis, on cellular dedifferentiation or anaplasia, on the best fit to (histo)prognosis, on cytologic and spatial configuration criteria, or on the reproducibility of the classification. So anatomopathological classification can start with the histological (tissular) characterization and adjoin secondly an evolutive grade, or can be based on the assumption that the tumoral behaviour will reflect its histological aggressivity [540], grade 2 gliomas moving along an histological continuum that will lead them at the end to the glioblastoma multiforme (Kernohan, Sainte-Anne Mayo/revision). Based on the WHO classification, oligodendrogliomas appear of better prognosis than astrocytomas, eventhough all authors did not observe significant differences [315, 374, 421]. Mixed gliomas seem to carry an intermediate prognosis between oligodendrogliomas and astrocytomas, but this has been debated, based on the various definition of their oligoglial or

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astrocytic components and/or their grading [253, 384, 608, 610, 611, 635, 728]. With time and new theories regarding glioma classification, and due to the partly subjective aspect of the WHO classification [134, 135, 136, 191], the reported respective proportions of the different oncotypes has varied, with a tendancy toward an overrepresentation in the more recent series of oligodendrogliomas, while astrocytomas largely predominated until the nineties. In the absence of specific marker of cellular lineage, the subject is still matter of debate. Different studies, quantitative and/or qualitative, have been made of various cytological parameters, whose groupings allow a grading reflecting tumoral aggressivity, but the results are diverse among institutions, hence non reproducible. Some advocate a two-tiered system of grading only [360], with low- and high-grade tumors, for all oncotypes. One of the main problems lies in the relative part of subjectivity underlying an anatomopathological diagnosis, due to the lack of precision of the diagnostic criteria, the intra-tumoral heterogeneity, and the biological diversity of these tumors. As a consequence, intra- and inter-observer reproducibility is not as high as one would like, especially in terms of grading [327, central reviews of trials]. The presence of gemistocytes in high quantity in a grade 2 glioma indicates for most authors a more dismal prognosis, with a correlation with advancing age [96, 282, 381, 609, 738, 748], up to the gemistocytic astrocytoma which behaves frequently as a grade 3 glioma eventhough there are no cytological signs of anaplasia, or the proliferative index is low, but with a still ongoing controversy [282, 382, 383, 404, 548, 596, 682, 736, 738, 748, 763]. Complementary Methods of Grading The proliferative potential of tumors was long thought to be more prognostic than the radiological evolution of the tumor or other commonly recognized prognostic factors (age, mitotic index…), evaluated by means of tritiated thymidine or bromodeoxyuridine incorporation or antibodies binding with cell-cycle antigens/enzymes [224, 281, 389]. This has lead to sophisticated models. If a general relation exists between various proliferative indices and outcome, its actual value in terms of prognosis, eventually with the adjunct of flow cytometry data, varies among reported studies, ranging from no significance to superseding other variables [57, 114, 115, 117, 133, 218, 280, 303, 582, 583, 649, 650, 717]. On the whole, the most widely used proliferation index utilizises an antibody directed against the Ki-67 antigen (nuclear DNA-polymerase α protein associated with actively dividing cells, present during phases G1 to M of the cell cycle). MIB-1 (monoclonal antibody directed against Ki-67 and usable on paraffin sections) index can be correlated with tumoral size but not location [399], with mitotic index (but more reliable and easy to use, especially on small samples) [601], with various cytological aspects [615], with histologic grade and age [255, 285]. It is higher in oligodendrogliomas [255, 328]. MIB-1 index (mean rather than maximal value) can complete, refine, the histological grading, at least in two classes (low vs. highgrade) [324, 349, 477, 685], but for some authors can even differentiate tumors of the same histological class [116, 195, 255, 279, 285, 345, 364, 380, 549, 580, 595, 682, 685], while other failed to recognize any significant interest in the MIB-1 index, at least at an individual level, frequently disappearing behind other prognostic factors [122, 267, 384, 463, 559, 595, 597].

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The vascularity or angiogenetic status of a tumor should reflect its aggressivity (malignancy), since (neo)angiogenesis seems mandatory, if not causal, to the significant growth of a tumor [136, 197, 200, 201, 521]. The density, size or aspect of tumoral microvessels, the intensity of, in particular, VEGF staining or of its epitopes and receptors, as well as radiologic studies studying vascularity or blood flow, have often uncovered correlations with prognosis [1, 89, 98, 183, 366, 367, 406, 505, 637], but this still needs confirmation [314]. Since tumoral growth results from the (im)balance between proliferation and cellular death, apoptotic ratios or the presence of molecules implicated in apoptotic pathways (inductors or protectors) have been studied, but does not seem to hold a significant prognostic value [89, 92, 365, 476, 554, 597, 598]. Histological grade of gliomas «grow» with time, spontaneously and/or after treatment, leading to the inexorable death of the patient. Hence tumor aggressivity and histobiological characteristics, of value but already insufficient individually from a static point of view, are even more highly variable when seen in a dynamic perspective, impossible to determine at different stages of the tumor history in the absence of repeated biopsy or resective surgery. Molecular Biology Gliomas are generally of the sporadic (vs. hereditary, by germinal mutation) type of neoplasia, as a result of an accumulation of DNA rearrangements (loss or gain of all or part of a chromosome, dysploïdy) and/or focal mutations (structural alteration of a specific locus), as hypothesized by the multi-hit concept [49, 450]. The clonal theory [489] suggests a single cell of origin [335], while tumoral progression is the consequence of a genetic variability (instability) that autorizes the sequential selection of more aggressive cell lines [203, 207]. Tumorigenesis is the consequence of a desequilibrium (at the DNA synthesis level) between inhibitory growth factors (tumor suppressor genes) and growth inducers (proto-oncogenes) [46]. The oncogenes work in a dominant manner, while the former need the loss (functional or structural) of their two alleles. Ultimately, genetic deletions (allelic losses), inactivation (mutation) or gains of function (amplification, increased number of copies, activating mutation), ie. the genotype, will lead more or less to a quantitative and/or qualitative increase or decrease of its corresponding protein product, at the end defining the tumoral phenotype. It seems that «six essential alterations in cell physiology (..) collectively dictate malignant growth: self-sufficiency in growth signals, insensitivity to growth-inhibitory (antigrowth) signals, evasion of programmed cell death (apoptosis), limitless replicative potential, sustained angiogenesis, and tissue invasion (..)» [250]. There are numerous interconnections between the various processes and genetic and biologic parameters that account for the neoplastic transformation, progression (proliferation/apoptosis, angiogenesis, immunosuppression) and invasivity (invasion, migration) of gliomas. Neoplastic glial cells are able to secrete substances playing different roles. Certain components of the extra-cellular matrix can be induced by reparation mechanisms linked to neoangiogenesis and/or local production of substrates guiding locomotion. The extra-cellular matrix represents a reservoir of growth factors, proteases and their inhibitors that can also be expressed by the tumoral cells. Certain regulating agents are common to angiogenesis, migration and proliferation of endothelial as well as tumoral cells.

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The degradation of the extra-cellular matrix in turn liberates cytokines and growth factors. Last this whole microenvironment can induce activation or inactivation of genes promoting or suppressing tumors… [680]. On the whole, glioma progression corresponds to the transformation of a slow growing, contact-inhibited, mildly tumorigenic and invasive tumor into a rapidly proliferating, anchorage-independant, highly tumorigenic and invasive tumor. Oncogenesis of astrocytic and oligodendrocytic tumors obeys to different mechanisms, as well as the two types of grade IV astrocytomas (primary or de novo glioblastomas –for which the main genetic abnormalities concern the EGF receptor and its gene, in association with a loss of chromosome 10-, and secondary forms ie. malignant gliomas deriving sequentially from a grade II astrocytoma –eventhough the frequency of specific genetic modifications differ in the two types of glioblastoma, the same genetic pathways are altered, and the clinico-histologic pattern is similar-432-). To note that the already known genetic mechanisms/alterations involved in gliomagenesis that will be rapidly reviewed here do not account for all the gliomas encountered. Astrocytoma Formation and Progression [111, 207, 353, 423, 480, 490, 538, 539, 705, 726] The earliest reported chromosomal modifications consist in losses of genetic material on chromosomes 6, 7q, 13, 17p and 22, probably linked to the transformation of normal glia into a grade II astrocytoma. Allelic losses on 9p and 19q seem to parallel the transformation into an anaplastic astrocytoma (grade III astrocytoma), and chromosome 10 loss in fine into a glioblastoma (grade IV astrocytoma). Astrocytoma formation appears to result from an original desequilibrium between an enhanced proliferation (PDGF-A & PDGFR-α -on 4q11-12- overexpressed in #60% of cases –[262]) and a diminished apoptosis (LOH 17p13.1 [52, 182, 726] or TP53 mutation –[402, 423, 623]- in more than 65% of cases [352, 496], the latter of increasing frequency with age, representing the only genomic alterations present with a similar frequency in all grades of astrocytomas [422]). Despite of a still slow growth capacity, there is a facilitation of the genomic instability that allows the transition to higher grades of malignancy. Due to the various roles of (wild type) p53 [reviews in 209, 358], transcriptional regulator, its absence or mutation (poorly studied however in low-grade gliomas) leads to, among other effects, genomic instability reflected by amplifications and aneuploidy [623, 712], loss of DNA reparation capacity, diminished apoptosis probably more than an increased proliferation (alteration of the cell cycle control at the G1-S interface by the TP53-MDM2p21(-p27-p14ARF) pathway [297]), perturbation of chromosomal segregation [209, 490]… Mutations are essentially of the missense type affecting primarily residues crucial to DNA binding [423], the one at codon 175 being of worse prognosis [508]; moreover, there might be a correlation of TP53 mutations with MGMT gene methylation [508]. Progression appears similar whatever the p53 status [534], but the delay of anaplastic transformation seems shorter in case of p53 mutation [737], which could then be more associated with malignant progression of astrocytomas [296]. TP53 mutation frequency is highest in gemistocytic astrocytomas, that usually progress more rapidly towards glioblastoma (Watanabe ANP1998).

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On the other hand, PDGF-R [534] as well as EGF-R (on 7p12-13) overexpression are associated with a shorter survival of grade II astrocytomas, independantly of known prognostic factors [715, controversial for 556]. Last, loss of 1p and 19q could concern about 15% of astrocytic tumors [74, 372, 544, 725]. The progression to anaplastic astrocytoma appears associated to the inactivation of tumor suppressor genes on chromosomes 11p, 19q, 9p and 13q (about 50%); the two latter contain the genes CDKN2/p16 (9p21) and Rb (13q14) (about 50% and 30% alteration respectively), resulting in the loss of one more critical pathway of cell cycle regulation at G1-S, hence an increased proliferation and mitotic activity (CDKN2/p16(-p15)-CDK4(/6)-cyclin D1-pRb1 and p27Kip1 pathway) [630]. LOH 19q seems unique, restricted to gliomas and common to the three oncotypes, in a region containing genes implicated in DNA reparation [311]. Last, progression to glioblastoma seems associated to the inactivation of a putative tumor suppressor gene on chromosome 10 [331, 423] and the overexpression of EGFR [395], with sometimes PTEN mutations –at 10q23.3-, loss of DCC expression –at 18q21-... [295, 334, 539, 599]. To note that most non pilocytic astrocytomas in the pediatric age do not share the same genetic alterations as the adult forms [395, 416, 532, 724], except brainstem gliomas, that resemble secondary glioblastomas [424]. Oligodendroglioma Formation and Progression These tumors present early and frequent losses (deletions) on 1p and 19q, in about 80% of the cases [respectively 40-100% and 50->80% -37, 74, 82, 372, 546, 725]. LOH 19q concerns mostly the totality of the long arm [546], a region where is suspected the presence of a gene implicated in astrocytoma progression, near DNA reparation genes [125], and LOH 1p can be complete or partial [189], of opposite prognostic signification (complete hemizygous loss strongly associated with 19q and oligoglial phenotype vs. partial deletions, essentially seen in astrocytomas, not associated with LOH 19q-298-). LOH 1p with or without LOH 19q is associated with a typical oligoglial phenotype [74, 536, 546, 590, 698, 725, 739], is more frequent in low-grade tumors and younger patients [472], is associated with a higher chemosensitivity to PCV protocol [82, 707], and/or Temozolomide, and even seemingly with a longer progression free survival after irradiation [73, 268, 636]. The codeletion is associated with a longer overall survival [73, 82, 185, 189, 510], eventhough these prognostic effects seem less marked for low-grade than anaplastic oligodendrogliomas [189]. These deletions are associated preferentially with frontal locations [390, 771, controversial for 189], and a lesser invasivity in vitro than their astrocytoma counterparts [501]. TP53 alterations or LOH 17p are rare in oligodendrogliomas [245], the accumulation of p53 protein being associated with a worsened prognosis [379]. Overexpression of EGFR is on the contrary frequent in (low-grade mostly) oligodendrogliomas [545]. Oligodendroglioma progression seems associated with LOH 9p, 10q [telomeric end–683], gain on 12q, deletion of CDKN2A gene (p16) and mutation or deletion of CDKN2B (p15) on 9p21 [50, 236, 269, 636]. PCR quantitative analysis of some proto-oncogenes confirms the early amplification (especially CDK4, MDM2, GAC1).

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To note that, as with astrocytomas, pediatric oligodendrogliomas do not share the same genetic profiles as the adult forms [533]. Mixed Glioma Formation and Progression Mixed gliomas seem to represent more clonal neoplasias of variable phenotypes rather than collision tumors [372], in which TP53 mutations and LOH 1p and 19q appear exclusive [546] or more precisely inversely correlated, associated with a preferential astrocytic or oligoglial phenotype respectively [434]. Combined immunohistochemical testing can help oncotype classification, as pure oligodendrogliomas show tumoral cells that are Olig2+/GFAP-, whereas two main populations Olig2+/GFAP- and Olig2-/GFAP+ are found in astrocytomas and mixed gliomas [461]. (Neo)Angiogenesis Gliomas, especially the astrocytic forms, as invasive tumors moreover often limited to tumoral isolated cells in the normal parenchyma, incorporate initially the normal cerebral vasculature [135, 520, 679, 745]. Eventhough progressive anaplastic transformation is associated with microvascular proliferation [136], since this neoangiogenesis is necessary for tumoral growth and facilitates invasion, and can even represent a self-limiting step in tumorigenesis [249], it does not appear in itself sufficient to define anaplasia [201], in part because anatomy can not reflect exactly the functional importance, leading to conflicting results in the correlations between vascularity and prognosis [65, 317, 406]. Angiogenesis implies angiogenetic factors directing endothelial cell migration (integrins) and proliferation, and vascular maturation, but first local disruption of the extra-cellular matrix by proteolysis (matrixmetalloproteinases, serin proteinases, cathepsins) [59, 124, 152, 198, 314, 317, 520, 521, 745]. Soluble growth factors and cytokines released by tumoral cells act in essentially a paracrine way on endothelial cells [199], and pericytes appear also very much implicated in angiogenesis [487, 746]. Different growth factors are implicated in angiogenesis, as well in proliferation, tumor progression.. (EGF, PDGF, bFGF, TGF-β). VEGF is one of the main angiogenetic factors in gliomas, whose expression along with its receptors is correlated with histological grade, especially in hypoxic conditions [585, 622, 716]; it could represent the final common pathway of neovascularization and progression towards grade IV astrocytoma [239, 692]. Last, angiogenesis could be initiated by the functional loss of (tumor) angiogenesis suppressor gene(s) [521, 711] and/or the upregulation of proangiogenic factors [64, 336]. Or, on the contrary, VEGF expression, known to be hypoxia-induced, and onset of angiogenesis, could follow an initial regression of existing vessels due to angiopoietin-2 expression by tumor cells [432, 767, 768]. Invasion and Migration Sub-population of glioma cells migrates away from the main tumor mass and invade the contiguous brain parenchyma (isolated cells of D-D or “guerilla-cells” of 517,104,627), along various possible routes [227, 229]. This requires cell adhesion to extracellular matrix

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components (with loss of adhesion to the principal neoplastic mass), cell locomotion and the ability to create space into which to move [680]. This process is facilitated by interaction with the extracellular matrix [233], in part provided by the neoplastic cells themselves (laminin, tenascin, hyarulonic acid providing substrates for invasion, cell adhesion to the latter being mediated by specific integrins and hyaluronan receptor CD44, SPARC, BEHAB), when stimulated by gangliosides, cytokines (TGF-β) and growth factors (EGF, PDGF, SF/HGF, insulin-like GF) [39, 102, 103, 237, 363, 393, 446, 517, 680]. Modulation of these extracellular matrix components is facilitated by various proteases (matrix metalloproteinases, hyaluronidases, serine protease urokinase-type plasminogen activator, lysosomal cysteine peptidases or cathepsins) which degrade the surrounding stromal cells and extracellular proteins [35, 53, 107, 192, 409, 517, 537, 714, 693, 759]. These proteases are regulated by biochemical pathways, especially protein kinase C [699]. Individual cells are capable of deformation to fit in the extracellular brain spaces [640], and cell motility is an active process dependent on dynamic remodeling of the actin cytosqueleton [433]. The process of motility/invasion is in fact a normal (regulated) capacity of astrocytes, in the mature [678] as well as developing brain [61, 537]. Factors controlling invasiveness also stimulate angiogenesis [36], tumoral cells taking advantage of neoplastic vascularisation for extension [684], but some anti-angiogenic drugs seem to increase invasion [392]. Cellular migration and proliferation share common intracellular pathways [721], with major cross-links (PI-3 kinase and PTEN, focal adhesion kinase –FAK- and p53). Motility-related genes are often up-regulated in gliomas of advancing grade, and mobile cells show a decreased proliferation rate and a relative resistance to apoptosis [226, 229, 345]. The balance of proteinases and their inhibitors varies from low- to high-grade gliomas [409, 537], while in vitro motility increases with histological grade [103]. Deregulation of invasion gene expression can be an early event, under the dependance of p53 impairment with consequently activation of proto-oncogene Ets-1 dependent invasion-associated genes [225]. Hence, astrocytomas and/or oligodendroglial tumors grade 2 share a type II or III spatial configuration [138], and invasivity is not restricted to malignant forms of gliomas. This invasivity and migration is dependent on the tumoral location, frequently limited in the grey matter (peri-neuronal satellitosis), frequent at the level of superficial pia or subependymal zone [probably passively –680-], and along peri-vascular spaces and myelinated pathways of the white matter, facilitated by an eventual peri-tumoral edema (or cellular loss due for example to seizures) which increases the extra-cellular space [228]. The glycolytic phenotype common to all malignancies seems to play a role in invasion since it allows an adaptation to the microenvironment [222]. Immunosuppression Escape from immune survey is a particularity of glial tumors [571]. Eventhough the central nervous system has long been considered as an immune sanctuary, the relative immunity of gliomas implies at least in part an interaction with immune cells (essential role of T lymphocytes). The antigenic presentation could be mediated by microglial cells, endothelial cells and pericytes, or even normal and neoplastic astrocytes [153]. Immunosuppression is also linked to the capacity by neoplastic cells to secrete soluble

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immunologic mediators (as TGF-β2 initially called “glioblastoma cell-derived T-cell suppressor factor”, Fas interacting avec Fas ligand on T cells leading them to apoptosis) [744], as well as to cytokines and/or growth factors, and even p53 [209]. First Results of Genomic, Transcriptional and Proteomic Studies By cDNA (low density) arrays and DNA microarrays (high density arrays), several genetic alterations (up- and down-regulations), some already known and others novel [see 588], can be found in low- to high-grade gliomas, and between primary and recurrent tumors. On the whole, there are somewhat few differences between grades II and normal tissue as well as between grades II and III, the majority of differences in genetic expression being found in grade IV tumors [581]. Genetic alterations encountered in low-grade astrocytomas can be grouped in three main categories [288]: cellular growth and differenciation/cell cycle control/apoptosis, cytokines/protein kinases/signal transduction/cell surface receptors and their corresponding proteins, and cellular adhesion/basal membrane and extra-cellular matrix proteins. Some modifications are (quantitatively) associated with progression from low- to highgrade tumors, interesting for example p53 in astrocytomas (constant anomalies throughout, but increasing with, tumoral grades) [581, 709], CDKN2A and p14ARF before CDK4 in primary glioblastomas [709], and vimentine and IGFBP2 for oligoglial tumors [581]. The most striking differences between low-grade astrocytomas (sharing similarities with secondary glioblastomas) and (primary) glioblastomas affect various categories of genes; -

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genes suppressor of migration and implicated in cellular adhesion (and cytosqueleton) are more often expressed in low-grade tumors [235, 562] angiogenetic genes are up-regulated in primary glioblastomas (vs. grades II and secondary glioblastomas) [235, 709], while FGF2 is overexpressed in low-grade astrocytomas as in pediatric tumors [235] various genes implicated in inflammatory or immune response are differently expressed by grades II and secondary glioblastomas vs. primary glioblastomas [235] TGFβ2 and IGFBP3 also discriminate low- and high-grade gliomas [235], as well as genes implicated in proliferation, maintenance of minichromosomes, transcriptional family, inhibition of apoptosis, cellular motility [562, 709].

Low- and high-grade oligodendrogliomas also show striking differences, with even some «intermediate» forms eventually defined; dysregulations of genes implicated in cellular adhesion and signaling, immune response and cellular differentiation, down-regulated, and much less up-regulated in the anaplastic forms [740]. Oligodendrogliomas with LOH 1p show similar expression profiles to the normal brain regarding the genes that they express differentially from their counterparts with intact 1p [468]. Another approach consists in defining small sets of highly discriminative genes [348 –for example subunit 2 of tansducine β2 in low-grade oligodendrogliomas-]. Generally, it appears that extensive genomic analysis allows to point out a relatively small subset of differentially expressed genes that can reliably discriminate different oncotypes and/or grades of gliomas [205, 210, 493, 605, 732], with a further refinement of

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classical histology in terms of classification or prognostic information. The same is applicable to the study of cell lines [291]. Since mRNA studies by microarrays does not accurately reflect the protein components (that result of transcriptional but also post-transcriptional controls, post-translational modifications and displacements), proteomic analysis are also of value. Again, protein clustering shows few differences between low-grade astrocytomas and normal tissue, can discriminate high-grade astrocytomas of different outcome, or point to modifications associated with progression or histological grade (proteins implicated in signal transduction, molecular chaperons, transcription and translation regulators, cell cycle mediators, linked to extra-cellular matrix and adhesion) [94, 305]. Last, pharmacogenomics of anti-epileptic drugs is also of importance, allowing a more comprehensive use if needed with greater safety. Molecular imaging (with PET, MRI) will represent another valuable, in vivo tool [308]. Gliomas and Stem Cells [See 586] The cell targeted for neoplastic transformation can be a differentiated, mature, cell (hence not representing a truly terminal event), but also an immature, yet undifferentiated one. Indeed, genetic factors involved in glioma genesis and progression are similar to the regulators of neural stem cells [553] and to developmental events [for example PDGF and PDGF-R, EGFR, pRb, p27 –742,see 432, 655-, Hedgehog pathway and transcription factors –126,see 586-, PTEN…], and their invasive propensity resembles glia and neuron migration during embryogenesis [254]. The frequent presence in gliomas of a biphasic tissue pattern [astrocytic and oligoglial differentiation or gliomatous and mesenchymatous differentiation –353-] argues in favor of (independant transformation of two differentiated cell types, or more likely) the neoplastic transformation of a (common) precursor cell presenting the ability of double differentiation [432]. The more homogeneous genotype than phenotype in histologically heterogeneous tumors [730], the common LOH 1p/19q in both components of mixed gliomas [372], the genetic similarities of the glial and sarcomatous components of gliosarcoma [47, 467, 731], gives further arguments in favor of a common cellular origin, as well as results of cell culture studies [see 432]. In animals as well as humans, neural stem cells have been isolated from cerebral tumors of various phenotypes, identified by the expression of the cell surface marker CD 133 and nestin, that are without expression of neural differentiation markers, are necessary for proliferation and maintenance of tumors in culture, are capable of differentiation in vitro into cellular phenotypes identical to those of the tumor in situ [215, 631, 632, 633], as well as to non-glial, mesenchymatous, cell types [see 432]. Stem cells and progenitor cells in the central nervous system (as in several other somatic or cerebellar sites) seem particularly more prone to tumoral/malignant transformation (as demonstrated in somatic neoplasms) than differentiated cells because they possess the ability to bypass apoptosis and senescence, and activated cellular mechanisms similar to those of initiating or progressing (maintained) tumors, especially through the abnormal functioning of developmental signaling pathways [586]. It is then postulated that the state of glial cell differentiation can affect the biological effects of given genetic alterations, so primary malignant gliomas could represent the direct

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malignant transformation of primitive glial precursor cells (neural stem cell or glial progenitor), while secondary malignant gliomas may arise from somewhat more differentiated cells arising from the same precursors [432, 586]. Progenitor cells can be manipulated in rodents to differentiate into astrocytes and/or gliomas [19, 271], while mature astrocytes are more prone to gliomatous transformation unless dedifferentiated [586]. Models of Formation and Progression of Human Gliomas Animal glioma models (genetically and/or phenotypically similar to human gliomas) combined with genomic and proteomic studies allow the study of the molecular mechanisms, and eventual treatment effects, of the formation, growth, invasivity, angiogenesis… of gliomas [34, 128, 242, 244, 285, 271, 272, 273, 275, 290, 294, 370, 408, 447, 700, 701, 743]. The same is attempted with tumor models based on cell lines [127, 504, 620], eventually chimeric [473], eventhough there are also differences between cell lines and actual tumors in vivo [722], as well as in vitro tumor development (cell gene expression and thus phenotype as defined in vitro is affected not only by in vivo growth but also by orthotopic growth). In mice, genes more expressed in vitro reflect increased proliferation in a more favorable environment, while in vivo there is an upregulation of genes involved in extracellular matrix, cellular interaction and angiogenesis [120]. Nevertheless, in vivo models in close relations with clinical contingencies remain necessary as in other domains of cancer research [343]. Spontaneous low-grade gliomas in animals and notably rodents resemble more or less human glioma growth patterns [238, 647]. The historic model of sub-cutaneous heterotopic xenografts of human tumors with spontaneous immunosuppressed animals (like nude mice) showed its interest in the treatment research and allowed the selection of more than 50 drugs today used in general oncology for a great variety of cancers [446]. It is indeed possible to use it by keeping, as in humans, all the usual response criteria (clinical signs variations, tumor measures and so the impact of the treatment on growth curves, treatment tolerance) while privileging survival as the main parameter. Applied to malignant heterotopic xenotransplanted glial tumors, the robustness of the model was confirmed with more than 60 % of graft success and a confirmed pathologic and genotypic stability of transplanted tumors [408]. Models are now widely used, that can be globally considered as being able to reflect the efficiency of treatments in humans in spite of false positive results (revealing active drugs in the laboratory but not in clinical studies) due to various factors as the loss of the heterogeneity of the grafted samples, increased cell kinetics in the transplanted tumors, or the loss of the usual tumoral environment. The orthotopic transplant of these gliomas took gradually, as in other tumoral locations, a place of choice, in being more close to the reality than other models [270]. The heterotopic transplant of human low-grade glioma in immunodeficient animals remains ineffective [666]. Moreover, it appears obviously impossible to evaluate such parameters as survival because of the slow growth of this tumor type. On the contrary, recent data reflect the use of more and more complex animal models, with the aim of mimicking the greatest number of possible stages of the tumor progression, so to allow the evaluation of new drugs different from those acting in conventional chemotherapy, by their modulatory action of cellular signalisation at different stages of the developpment.

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Experimental models of gliomagenesis have been performed [560, 639], mimicking, on normal human astrocytomas, the genetic alterations most often encountered in gliomas (alteration of p53 and Rb pathways, activation of telomerase maintenance and independance from growth factors), giving way to cells with a greatly expanded life-span, possessing a capacity of growth on soft agar and a tumorigenicity in mice resembling malignant gliomas [560]. In cell cultures and in mice, overexpression of PDGF-B increased proliferation of astrocytes and neural progenitors, and neural progenitors transfered with PDGF can form oligodendrogliomas [127, 128], while GFAP+ astrocytes can form oligoglial or mixed gliomas, more often malignant when arising from Ink4a-ARF null progenitor cells [127]. KRas and Akt can induce formation of astrocytomas and glioblastomas from neural progenitor cells [128], but not differentiated astrocytes unless there is a Ink4a-ARF deficiency [700]. Many experiments give opportunities of new targeted therapies (eg. suppression of Rac1 activity leading to apoptosis of most glioma cells –603-, neural stem cells targeting gliomatous cells –2,499-…). Most of recent theoretical/mathematical modeling is based on the mutator phenotype, and as we have seen, on the multi-step acquisition and accumulation of gene alterations from normal cells to the most malignant phenotype. As well, a glycolytic phenotype is assumed generally for mammalian neoplasias, relying on the anaerobic metabolism of glucose to lactic acid, whose energetical inefficiency is compensated by an increase in glucose/blood flux [222]. Other models rely on the frequent aneuploidy of cancers, with an increased cytogenetic instability in cases of hyperploidy, leading to the activation of growth promoting genes [604]. There are numerous other proposed models of neoplasia, using population ecology approaches, game theory, the interaction of mutator phenotypes with environmental selection parameters [222], the spatial mobility of cells towards more growth-permissive places [agentbased model +/- game theory/geno-phenotype link –437, 438, 439-], the interaction of tumorhost interfacial morphology and physiology with tumor progression [reaction-diffusion model –220-]. There are also models proposed of blood-brain barrier [441], angiogenesis [770]... Drug- and Radiation-resistance Genes Correlations of genetic profiles and anticancer drug sensitivity can be tested on a panel of human cancer cell lines [129, 561, 568, 644, 733, 761, 769], on rodent models [248], which allows further novel anti-cancer compounds discovery and testing [306, 761], and help understanding mechanisms of drug action [292] and defining individual and new combinations therapies [22, 377]. The same approach is currently underway for studying radiation sensitivity [687]. Gene expression profiles determination will progressively lead to phenotypic outcome prediction [577]. The transportome has been shown to play an important role in drug resistance [289], as well attachment or extracellular matrix genes explaining different drug sensitivity of solid tumors and their derived cell lines [646], or the SPARC protein or gene expression [665]. Drug resistance seems closely linked to apoptotic pathways, while general drug sensitivity seems associated to genes linked to cell cycle control and proliferation [561], but also cell adhesion [642]. The drug efflux pump protein P-glycoprotein, multidrug resistance related proteins MRP1 to 5, lung-resistance protein (LRP), glutathiome S-transferase-pi (detoxification

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enzyme), DNA topoisomerase IIα, interferon receptor, interferon regulatory factors (IRF1&2), dihydrofolate reductase, among others can protect the brain from xenobiotics, as well as cisplatine-resistance proteins [83, 465]. Drug or xenobiotics metabolizing enzymes include various phase I (eg. cytochrome P450 or CYP) and II enzymes, whose gene expression is increased in response to inducers or xenobiotics triggering a global cellular stress [577]. Endothelial cells of normal brain and tumors express differently the multidrug resistance genes and some matrixmetalloproteinases [543]. Native drug resistance [acquired during tumorigenesis, then with different or common gene alterations for oncogenesis and drug response, or intrinsic property of the cell of origin ? –494-] appears more important than acquired resistance, no significant difference being evident from primary to recurrent tumors [primary glioblastomas show a higher ration MGMT/beta2-microglobulin than secondary forms or other tumors, while low-grade gliomas show a lower ratio –667-]. There could be a link between loss of p53 function and expression of multiple drug resistance in non-tumor CNS cells [442], p53 protein possibly contributing to the regulation of microtubule composition and function [its dysfunction generating complex microtubuleassociated mechanisms of resistance to tubulin-binding agents like vinca alkaloids or taxanes –217-], but (experimental) modulation of chemosensitivity by p53 appears unlikely [690]. This can be put in parallel to the role of defect or dysregulation of the apoptotic pathways [720]. There appear to exist broad cross-resistance secondary resistance mechanisms, involving altered expression in pro- and anti-apoptotic proteins, and primary resistance mechansims, specific to given anti-cancer agents. As for example depletion of alkylguanine alkyltransferase –AGT or MGMT- by O(6)-benzylguanine or MGMT hypermethylation increases the cytotoxicity of alkylating agents widely used against gliomas, of temozolomide [429] more than the BCNU efficacy [60], but mutant MGMT resistant to O(6)-BG have been identified [21], or the possible less growth inhibition of mutant p53 in the NCI anticancer drug screen, with the exception of anti-mitotic drugs [495]. Rat O-2A progenitors, astrocytomes and oligodendrocytes show various expressions of drug resistance genes, with higher expression of MGMT and MDR in astrocytes and of GSTµ and MT in oligodendrocytes [494], and wild-type p53 astrocytes show greater MGMT activity but a BCNU resistance [492]. Human low-grade astrocytoma and oligodendrogliomas show the same proportion of methylation of MGMT gene [almost 50% 739-] or MGMT is more expressed in astrocytic tumors [494, 656] than in oligodendrogliomas and even more oligoastrocytomas or normal cerebral tissue [626], and there is a slightly but significantly lower MGMT expression in low-grade than high-grade gliomas [656]. MGMT expression is inversely correlated with age and correlated with aneuploidy but not S-phase fraction [626], is associated with a shorter survival time [656], and BCNU [310] as well as temozolomide responsiveness of malignant astrocytomas [256].

Tumoral Size and its Evolution The careful monitoring, at the clinical and radiological levels, of the evolution of a patient harbouring a grade 2 glioma, is bound to reflect best the proper biological behavior of

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the latter interacting with the host, defined by his background and tolerance. Despite an apparent biological diversity, longitudinal clinico-radiological data enables to distinguish some common rules in this somewhat chaotic population of tumor/host pairs. The statements made here are derived mainly from our experience centered on supratentorial hemispheric gliomas affecting adults. Tumor size has been evaluated in various one- to three-dimensional manners: greatest diameter, greatest surface, spherical or ellipsoid approximation, sommation of the actual surfaces on each sectional image, and now available, actual volume obtained by tumor segmentation on MRI that should be the only standard eventhough some have failed, in anaplastic gliomas, to find a prognostic relevance of post-operative volume when studying the mean geometrical while the planimetry method did [757]. The FLAIR sequences appear the best to determine the margins of the glioma [78], especially in peripheral and periventiruclar regions [113 –absence of partial volume effect by the nulled CSF-]. The three directions of growth should be taken in account since, due to the anatomico-biological characteristics of intra-cranial structures and the tendancy of most gliomas (depending on their site of origin) to grow towards the ventricular system and along white fibers, some tumors will demonstrate a preferential cranio-caudal growth that would be underestimated by one or two-dimensional estimations. Moreover, the determination of three diameters reduces the risk of error due to patient’s positioning in the MRI unit, the angulation of the slice planes.. Every technique carries more a less a subjective dimension, and 2D or 3D evaluations overestimate the tumoral surfaces or volumes, the more so as the lesional volume is larger or more irregular (Mandonnet 2005, submitted). Nevertheless, a certain proportionality remains, in terms of volume or growth rates, which allows to draw some conclusions from our series, comprising mainly estimations using the ellipsoid approximation method (volume= half the product of the three biggest orthogonal diameters); indeed our preliminary results with segmentation volumetric measurement do not contradict the results obtained with the former method. Grade 2 Gliomas are Constantly Evolving Tumors The first notion is that all grade 2 gliomas grow with time. This means that any tumor image compatible with a grade 2 glioma that does not show any volumetric variation in one or two years, is probably a dysplasia or DNT. But since grade 2 gliomas grow at different rates, sometimes quite slow, one can be abused, comparing the last radiological exam with the second to last, without precise measurement, and believe that a given tumor is quiescent. There are hopefully attempts at developing pragmatic tools (quick and automated postprocessing accessible to clinicians) to monitor eventual variations of volume [113]. The growth of human tumors depends on the cell cycle time, the proliferation index (cells in cycle/cells in G0 phase), and the cellular losses. During their period of possible observation, is follows usually a constant, exponential, type (in fact somewhat different due to cellular losses which result in the fact that the doubling-time is greater than the intermitotic or cell cycle time, and to the presence of aneuploid cells). Tumor doubling-times have been measured more often on CT-scans, ranging from 19.5=+/-1.9 to 48.1+/-20.9 or 69.7 days for grade 4 gliomas [57, 694 –regrowth-, 760], 66.5+/-29.4 to 140 days for grade 3 gliomas [57, 694 –regrowth-], and 937+/-66.5 days for regrowing grade 2 gliomas [694]

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while others have reported doubling-times for the latter, mixed with grade 1 gliomas [140 days –57-]. By comparison, doubling-times of other oncotypes have been reported at around 30 days for embryonal tumors and lymphomas, 40 days for mesenchymatous sarcomas, 60 days for malpighian carcinomas, and 80 days for adenocarcinomas [695]. Tumor doublingtimes of gliomas do not differ greatly before and after treatment(s) [57]. The tumor doublingtime has been shown to differ according to the ploidy of the tumor, much smaller in aneuploid(/multiclonal) than diploid than euploid tumors, whatever their histological grade [57]. From a theoretical origin as a single neoplastic cell of 10 µm in diameter, it is commonly assumed that a tumor needs 30 doublings to attain 1 cc (230 or around 109 cells) that is a minimal size to be detectable clinically [7, 112]. At diagnosis, the tumor has progressed of a mean of little more than 5 doubling-times, and between diagnosis and death only slightly more than 3 doubling-times are necessary, whatever the histological grade of the glioma [57], since the average fatal volume of a (bulky) tumor seems to be around 100-150 cc. The logistic growth model of Verhulst (1838), as the other models of Gompertz (1825, 24) or Bertalanffy (1941 –growth velocity is the difference between anabolism and catabolism-), are empirical models using mathematical equations (sigmoid) reflect probably more the fact that in the clinical phase of the tumors, the growth is somewhat slower when the tumoral volume is greater [9, 398], depending on the « carrying capacity of the environment, which is normally determined by the available resources» and/or waste product accumulation, with greater cellular losses, decreased proliferation fraction and longer mean G1 phase times, generating an exponentially decaying growth rate after an exponential growth of tumoral cells. The competition for nutrients in a avascular tumor could be a determining factor in generating papillary tumor morphology [190]. Other modelisations are refined, for example with a focus on the emergence of clonal subpopulations in tumors [325, 326], a representation of stress distribution during an anisotropic growth with its consequences on vasculature [12], or are more molecular in nature [620]. But some have challenged the mathematical assumptions based on analysis of the growth of transplantable animal tumors and on averages of tumor growth in human populations [756]. Tumoral growth could be more irregular, with dormant phases or plateaus separated by growth spurts [551]. Other models are more functional, mechanistic, physiology- or biologybased, and offer other tumoral evolution laws [770] or focus specifically on one component of growth, for example proliferation, diffusion [697], invasion [437] eventually as a chemotaxis model [394, 587], angiogenesis [97], take in account ecology, or attempt to modelise at the molecular level the mutual exclusivity of proliferation and migration [16]. Derived from the observation of colonies issued from tumor cell lines, and indirectly confirmed on tumors developing in vivo, it has been shown that the spatio-temporal pattern of tumoral growth seems to obey to fractality, and that it follows a universal rule of linear growth (in terms of its radius), this dynamic behaviour in turn being compatible with the molecular beam epitaxy (MBE) universality class [69]. In this setting, the main mechanism responsible for growth is cell diffusion at the interface with normal parenchyma, with a relatively great inhibition of cellular growth inside the tumor or colony (as attested by differential proliferative indices); while MBE dynamics implies surface diffusion of cells, ie. their movement along the tumor/colony border, and not their free movement away from it

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[69]. Globally, the growth velocity is characteristic of the process, and not of the cell line, hence biological behaviour of similar tumors will vary depending on its external environment (the host), the major conditioner of tumor growth being space competition between tumor and host. So, the most commonly accepted mechanism conditioning tumor growth mainly to nutrient competition (between tumor cells and tumor and host cells), in accordance with a Gompertzian model, is insufficient to reproduce the main features of tumor growth [69]. The linear regime of tumoral growth implies that there are less actively proliferating cells, and that these are homogeneously constrained to the border of the tumor and not randomely distributed throughout its whole volume. Moreover, newly generated cells move to sites with a higher number of neighbouring cells, while the inner cells are prevented from proliferating by the pressure exerted on them through the lack os space [69]. Nevertheless, by analogy with pressure effects (solid state stress) in tumor spheroids, the pressure exerted by the host over the tumor could explain the deviation of the tumor growth rate from a pure linear regime. Other models have been hypothesized, that explain an arrested tumoral growth at a volume around 100-150 cc, an irregular shape in the first stages of tumor development then a spheroidal shape, and a centripetal growth remission [479]. Since glial tumors are infiltrative, the cellular density of the more peripheral parts of the lesion will be insufficient to generate a signal abnormality on MRI, even with the most sensitive sequences. Hence, even if the total number of tumoral cells grows exponentially, the volumetric doubling time used to quantify the radiological growth of (high-grade) gliomas [57, 760] will not be appropriate. Instead, the growth curve of the tumoral volume is more likely cubic, or so to say the growth curve of the mean tumoral diameter will appear linear. That is in accordance with our observations, and with a biomathematical model [proposed initially by Murray in the 1990s –662- then others, 689, 754] of glioma growth as a conservation equation taking in account proliferation and diffusion of tumor cells, later refined [659, 660, 661, 662] to account for brain parenchyma heterogeneities and reflect asymmetric, nonspherical, tumor development and migration facilitated in white matter [227]. Representing then the growth rate of grade 2 gliomas as the slope of the curve reflecting the variations of the mean geometrical tumoral diameter with time, it could be shown, in a highly selected series, that the mean velocity of growth of grade 2 gliomas is around 4 mm/yr [435, 662]. By contrast, for grade 4 gliomas, velocities of 30 mm/yr [662], or from 18 to 110 mm/yr (personal observation of four cases), have been observed before treatment. In a larger series of unselected grade 2 gliomas in our experience, growth rates vary widely in contrast to a small confidence interval in the selected series. Nevertheless, the median growth rates were similar, of 3.8 mm/yr in the whole series against 4.1 mm/yr in the selected series. The Growth Rate of Grade 2 Gliomas is Correlated with their Biological Behaviour The slope of the mean diameter increase of a tumor can first help distinguish «true grade 2 gliomas» from particularly aggressive ones, which will soon demonstrate signs of malignancy [57, 542]. Indeed, in our experience, some tumors that look like grade 2 gliomas on MRI (without any contrast enhancement suggestive of anaplasia), and even at the

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histological level, can in fact be ruled out in a few weeks or months as more aggressive, when their diameter growth rate exceeds 8 mm/yr, and more so at 10 mm/yr or more. This suggests that a period of radiological observation should be the rule before any therapeutic decision, since the absence of radiological signs of anaplasia does not rule out the possibility of a highgrade glioma. This remains valid even if a biopsy is realized, since the growth rate seems more reliable than histology, which can change dramatically in a few months (as the radiological features). Those particularly aggressive cases account for survival curves of grade 2 gliomas that show a decline in the first months or 1(-2) year(s), which seems paradoxical for a slow-growing, initially histologically benign, tumor. They account for almost 12% of our series, and in fact anaplastic gliomas with a radiological pattern suggestive of low-grade glioma have been reported in up to 50% of the cases (more frequently with advancing age). This notion gives arguments to the practicians who do not advocate a systematic histological diagnosis in front of a suspected grade 2 glioma, even in the absence of oncologic treatment decision. Indeed, a (stereotactic) biopsy carries a functional risk (0-1% mortality, 2-5% morbidity –80b-, 3% morbidity in our series-), and the histological diagnosis can be mis- or underestimated (wrong diagnosis, or more frequently underestimation of the grade); this is also possible despite the thorough examination of a surgical (resection) specimen, as we have observed in some cases an anaplastic transformation a few months after extensive resection of a grade 2 glioma with no histologic «hint» of malignancy (proliferation index included). In the same way, the growth rate (slope) appears constant throughout the evolution of a grade 2 glioma. But, when the tumor acquires a genophenotype of anaplastic (high-grade) glioma, its growth rate accelerates (Rees 2005 submitted). This change in the slope occurs, under the condition of a somewhat close follow-up (every 6 months for example), usually before other radiological signs of anaplasia (mainly contrast enhancement, which in turn can take up to 2 years to become patent and imply the most part of the tumor), and even preceed clinical signs (recurrent or increased epilepsy, overt deficit, signs of intracranial hypertension). Hence, aggressive therapeutic measures can be taken at the beginning of the anaplastic transformation, which offers in our series a better chance of result in terms of survival, and also helps maintain a patient in a better functional condition. Also, quite logically, the growth rate of a grade 2 glioma reflects in part its biological behavior. There is indeed a correlation, on univariate analysis, between growth rates and outcome. The Tumoral Volume is of Prognostic Importance The determination of tumoral volume is important in more than one way. First of all, lesional volume, and its correlate of extension in terms of anatomical landmarks, is essential as it represents a major decisional factor for treatement decision-making, both in terms of feasibility (or ratio benefit/risk), and also in terms of chances of success. But a tumoral volume, in conjunction of the tumoral growth rate, reflects also the evolutive stage of a given tumor at time of its observation. Indeed, when one evaluates one way or another the tumoral size, it represents an independant prognostic factor of outcome [43 –univariate-375, 516, our series]. The cut-off value can vary from one series to another

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[20 ml –375-, 10 & 30 cc –43-, 5 to 6 cm of greatest diameter –516-]. A greater volume, meaning a later stage of evolution of a (untreated) glioma, can thus undermean that the tumor is closer to its anaplastic transformation, the common terminal fate of grade 2 gliomas. Indeed, in our series, anaplastic transformation was never detected in (previously untreated) tumors whose mean diameter was less than 5 cm (62.5 cc –one exception at 4.7 cm-). Hence, to accurately reflect the inherent biological potential of an individual tumor, or more precisely the result of the tumor/host interaction, one must take into account the volume as well as the growth rate of the lesion. Tumoral Volumes and Growth Rates are also Valuable PostTreatment Follow-up Tools Radiological follow-up of grade 2 gliomas after treatment(s) is as important as their pretherapeutic evaluation [9], in terms of radiological aspect (enhancement, edema, mass effect, extension) as well as in terms of volume and growth rate. It allows to evaluate objectively the quality of an eventual surgical resection, or more pratically the amount of residual MRI signal abnormality representing the residual tumoral tissue. This permits first to speak a common language, second to appreciate an eventual impact of resective surgery in the context of grade 2 gliomas. Indeed, when surgical cytoreduction is so quantified, and not described on the sole basis of the surgeon’s impression, the everlasting debate on its prognostic value if any shows a trend towards a definite interest of surgery under the condition that the residual tumoral volume is less than 10 to 15 cc (43, our series), this cut-off appearing more interesting in practice eventhough the relation appears one the whole linear with the volume as a continuous variable. A simple law [8] linked tumor doubling times and the amount of resection (50% resected offering a time survival gain of one doubling time, 75% two..), before a more thorough modelisation was made that proved valuable when applied to clinical data on high-grade gliomas [for review 662]. Moreover, assuming that a given tumor follows a constant growth rate (mean diameter curve) pre-operatively, and that it resumes the same growth rate after surgery, which should represent a cytoreduction without any «side effect» that could alter tumor kinetics, one can calculate, and hence predict, the extra (progression-free) survival eventually provided by surgery. With the method of ellipsoid approximation, the post-operative diameter curves that we observe are of linear type, but with a slightly greater slope; this discrepancy could be due to the fact that this measurement technique overestimates more the volumes of (greater and) more irregular tumors (Mandonnet 2005, submitted). Model predictions as well as spatial classification of gliomas [137] show that diffusion represents a paramount, if not predominant, component of gliomas. Hence, ideally the coefficient diffusion should be determined before any surgical decision, since essentially lesions with a low diffusion coefficient will benefit from a large resection. In this respect, oligoglial tumors with LOH 1p/19q could be more suitable to surgical treatment since they grow more as circumscribed lesions [501]. Quantitative static and dynamic evaluation of tumoral volume allows to monitor the effect of chemotherapy if any, along with mathematical modeling [661, 689]. In our experience, the first effect of a «successfull chemotherapy» is to arrest the tumoral growth (horizontalization of the diameter curve). Then, there is is a more or less slow and rarely

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important decrease of the tumoral extension, in a centripetal manner, which can even continue well after the termination of chemotherapy [446, 645, personal observations]. This evaluation of the efficacy of chemotherapeutic drugs on low-grade gliomas seems at the moment the only effective monitoring method [645], since even spectro-MR is not fully tested in this context [reduction of choline seemingly parallel to the volumetric effect –471-]. When it is admitted that the growth of grade 2 gliomas obey a linear regime, that is based on fractality and MBE laws, the proliferative cells as discussed upper are mainly associated with the surface of tumors. Since they represent the cells sensitive to antiproliferating agents, one can deduce two main consequences. The first is that the killed cellular population will be at the periphery of the radiological image, as we have observed (as in other models proposed –479-]. Second, the effectiveness of chemotherapy will decrease with tumor size (as described with glioblastomas –340-], on the opposite of the current log-kill concept assuming a constant effect at random (on cells in proliferation). Moreover, it is well known that hypoxia plays a role in radio- as well as chemoresistance, which can be explained by cells following MBE dynamics and hence migrating to positions where oxygen is less available (higher celleular concentration, lower pH due to lactate production). Other models aim to reflect (and understand) that heterogeneity in drug delivery due to variability in vascular density can lead to an apparent tumor reduction in certain areas while there remains a persistent growth in other areas, eventually beyond the resolution of imaging [661], or attempt to represent drug resistance [502], or attempt to predict in the preclinical phase of development of oncology drugs, empirical (mathematical equations), functional (mechanistic, physiology-based hypotheses) or mixed [629]. The effect of radiotherapy is also monitored in the same way. Even if the importance of tumoral reduction is of no prognostic importance on outcome [29], one can rule out early, as under chemotherapy, an absence of effect of the therapeutic modality, allowing to change the strategy more precociously. As is the rule in the field of oncology, we have observed that the quicker and the more important is the tumoral volumetric reduction after chemotherapy essentially, but frequently after radiotherapy also, the greater the probability that the tumor was more aggressive, as reflected as a rule by a greater pre-treatment growth rate. In this field too, modelisation is of importance [350]. But there Still Remains a Biological Diversity or Hazardous Aspect in the Natural History of Grade 2 Gliomas Despite the common rules described above as lessons learned from the (trivial) radiological follow-up of grade 2 gliomas, we are still unable to predict the outcome, moreover at the individual level, of patients harbouring a suspected or proven grade 2 glioma. In terms of functional prognosis, even if the tumoral site and extension, hence volume, are of paramount importance, there is a well known anatomo-functional variability (tolerance, plasticity) that hampers the possibilities of a precise individual functional prognosis. More importantly, anaplastic transformation is an event that appears yet unpredictable, at least in the terms described in this chapter. The modification or value of the slope of the tumoral diameter curve is a constant predictor of imminent anaplasia, but we have observed cases of malignant transformation, in the absence of, as well as after treatment, without significant

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changes of the tumoral growth rate. Anaplastic transformation seems indeed to be a spontaneous, intrinsic process, independant of any external stimulus [5]. Tumor growth modeling is also limited by individual factors that appear prognostic in clinical series, such as age, performance status [662]. In all events, progress has to be made in this aspect of tumoral dynamic evaluation, and maybe the actual volumetric measurement now accessible to everyone will help to refine and extend these observations. Nevertheless, the notions of volume and growth rates should already be included in the evaluation and studies of grade 2 gliomas, providing additional information that can help defining risk groups a priori after a short period of observation of the natural history of these tumors. This should allow the realization of valuable clinical trials, soon probably spanning shorter times of follow-up to obtain results of comparable quality. Every effort in refining volumetric analysis of tumors should then be encouraged [660].

Metabolic Neuroimaging In addition, the use of various brain radioactive tracers has shed invaluable light on the pathophysiology of gliomas: nature and degree of heterogeneity of the tumor, patterns of growth and extension, risk and delay of anaplastic transformation. First, Single Photon Emission Computed Tomography (SPECT) studies showed a relationship between tracers uptake and tumor grade, using both Thallium-201 [302, 498] or 99m Tc-MIBI [32, 38, 259, 386, 592, 641, 643, 741, 766]. Thus, determination of regions with the highest metabolic activity within the tumor was used to guide surgical biopsy in a stereotactic frame [259]. Some authors also suggested that 99mTc-MIBI SPECT might help in predicting the response to chemotherapy in patients with gliomas [766], and in establishing the prognosis of survival after radiation therapy [32]. However, despite the development of new tracers such as 99mTc- Tetrofosmin [641] or 123I-Alpha-Methyl Tyrosine (IMT), potentially useful for identifying postoperative tumor residue [741] and recurrence [386], SPECT still lacks reliability, and cannot be used as the sole noninvasive diagnostic or prognostic tool in gliomas [38]. Second, less widely available and more expensive, Positron Emission Tomography seems to represent a more reliable and accurate method of metabolic imaging in brain tumors [592]. Beyond recent studies with positron emitters presenting definite research interest in molecular imaging [265], e.g. [I-124]Iododeoxyuridine [58] or [F-18]Fluorothymidine proposed to measure tumor proliferation rate [616], or FIAU as an indicator of gene expression in glioma useful for gene therapy [307], most clinical works have focused their efforts on metabolic substrates such as 11C-choline [252, 619], and above all 18Fluoro-2-deoxy-2-glucose (FDG) and 11C-methyl-methionine (MET). Indeed, FDG PET can predict tumor grade [347, 457], while low-grade oligodendrogliomas and pilocytic astrocytomas can be quite FDG avid – so FDG uptake in such lesions does not necessarily imply a poorly differentiated histology [333]. Also, the metabolic activity of gliomas as shown by the PET-FDG method seems to have a good prognostic significance [143, 277], independent from histology [142, 144]. Furthermore, because brain tumors are histologically heterogeneous, PET-FDG was used to guide stereotactic biopsies [410, 445]. Indeed, while

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LGG are noted to have low levels of FDG uptake, areas of malignant degeneration show increased metabolic activity [204], associated with an unfavorable prognosis [143]. Finally, FDG-PET has been used to document the extent of tumor resection [234], and differentiate brain tumors from necrosis after radiation and/or chemotherapy [72]. In a pilot study, it has even been reported that FDG-PET could differentiate responders from non responders after one cycle of temozolomide in recurrent high-grade gliomas [76]. However, since FDG-PET has limited value in defining the extent of tumor involvement and recurrence of LGG, METPET may be preferable for this group of lesions [108]. Indeed, MET-PET appears to be related to tumor aggressivity [148], with a high uptake statistically associated with a poor survival time [141, 149, 261, 557] and allows to delineate the invasion of tumors (especially LGG) much better than FDG-PET – thus representing a better choice for PET guidance in neurosurgical procedures [518] an for assessing response to therapy [723]. Furthermore, the value of the combination of FDG-PET and MET-PET has been suggested [283], in particular in LGG with a low methionine uptake [144], for instance in astrocytomas – which have lower levels of MET uptake in comparison to oligodendrogliomas [147]. Finally, recent development of 18F-labeled amino acid tracers such as 18F-alpha-methyl-tyrosine, with promising preliminary results in the evaluation of gliomas [300], opens the field for wider use of PET scanning in the management of brain tumors [455, 458]. Third, Proton MR spectroscopy (MRS) represents a new, noninvasive tool recently used in clinical practice to investigate the spatial distribution of metabolic changes in brain tumors [140]. Indeed, several authors have reported increased levels of choline-containing compounds [Cho, a marker of increased membrane turnover or higher cellular density –458-] and a reduction in the signal intensities of N-Acetyl Aspartate [NAA, a neuronal marker mainly contained within neurons –703-] and Creatine [a marker of energy metabolism –342-] in gliomas [264, 478]. The ranges of Cho increase and NAA decrease seem compatible with the range of tumor infiltration [121, 284]. The calculation of metabolic maps by integrating the peak area of a metabolite of interest or some ratios such as the Cho-NAA index for each voxel is currently a common method to visualize these changes [154, 452, 673]. Metabolite profiles have been used to differentiate various types of tumor from one another [284], in particular LGG [519] and gliomatosis [214]. Metabolite maps have also helped to determine brain tumor grade [263] and to predict the length of survival [387], notably using: the phosphocholine / glycerophosphocholine ratio which increases with the grade of glioma [578]; Cho levels which correlate with proliferative potential as determined by immunohistochemical analysis of tumors biopsies using the KI-67 labelling index for gliomas [617]; Cho/Cr ratio which increases with grade [284] while myo-Inositol [91] and Glycine [100] decrease with grade; and the lipids which correlate with necrosis [385] then are increased in high-grade tumors [18]. Also, MRS can monitor response to therapy, since the typical change that occurs when a tumor responds to treatment is a reduction of Cho with possibly an increase in lactate and/or lipids [241], indicating the transformation of viable tumor cells towards necrosis. Following radiotherapy, glioma progression could be predicted on the basis of MRS abnormalities (in particular an increase of Cho/Cr ratio) that were outside the MRI-defined treatment region, and can occur prior to subsequent increase in contrast enhancement [241]. However, the sensitivity of MRS to detect tumor progression drops when there is a mixture of necrosis and recurrent tumor [566]. The improved spatial

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resolution and more detailed spectral information using higher field MR systems could optimize the discrimination between radiation damage and glioma recurrence [531]. Progression to higher grade could be equally documented using MRS [675]. Finally, a combined used of PET and SRM in the evaluation of tumor metabolism was recently proposed [704]. To be noted that the biological behavior of brain tumors can also be indirectly studied using complementary sequences of MRI [525], i.e. diffusion weighted imaging (DWI) [542] and perfusion weighted imaging [344]. In particular, apparent diffusion coefficient values for LGG are higher than those for high-grade gliomas, because more highly cellular gliomas would have a smaller interstitial space and hence more restricted diffusion [362, 654, 762]. Perfusion weighted imaging provides information about tumor tissue perfusion by measuring cerebral blood volume (CBV), and might be used in the preoperative classification and grading of gliomas [653]. Indeed, CBV has been shown to correlate with microvessel cell density [403, 565], and varies with tumor grade in that maximum CBV values of LGG seem significantly lower than those of high-grade gliomas [357, 426, 529]. Moreover, perfusion MR imaging could be helpful in predicting LGG response to radiotherapy [211]. Nevertheless, this technique still lacks sensitivity [391]. Thus, it was suggested that a combination of the perfusion image results with those of DWI and SRM could improve the reliability of these methods, notably for tumor grading [762].

Conclusions Grade 2 gliomas are to be considered as continuously evolving tumors, as a continuum along a spectrum beginning long before their discovery, as initially a very slow-growing, indolent, neoplasia, but ending as a highly malignant, and lethal tumor, with overall a short period of clinico-radiological observation. Most of our diagnostic armamentarium is static in essence, and it is indeed mandatory to reflect the status of the tumor and the host at a given time. But the dynamic dimension of tumoral progression has to be evaluated as well, since it probably represents a paramount factor to take into account in the definition of the most appropriate therapeutic strategy. Along with the other parameters available (clinical, radiological, histological, biological), the evaluation of tumoral volume and growth rate, which does not demand the obtention of repeated tumoral samples (eventhough a biopsy at least at some stages of tumoral evolution – recurrences requiring a treatment for example- would prove very interesting), allows already in daily practice to rule out the most aggressive tumors that in fact behave as anaplastic gliomas. It also provides additional information essential for therapeutic decision making (urgency of treatment, evaluation of the individual efficacy and overall prognostic influence of a therapeutic modality, indication of retreatment, prediction of anaplastic transformation), as well as the building and conduct of clinical trials. In the meantime, progress in genomics to proteomics, albeit accessible only if a surgical act is performed, should provide additional tools to classify, grade and envision the prognosis of an individual tumor. Non invasively, metabolic examinations will also probably complete the possibilities of evaluation of a given tumor. We will at last be able to differentiate the

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proper aggressiveness of a tumor a priori, and this will provide the basis for studies aiming to define the most appropriate therapeutic strategy to propose to a given patient. Animal models allow to study in vivo tumoral behaviour, spontaneous as well under different environmental and therapeutic conditions, but models of low-grade gliomas are nevertheless difficult to establish [666].

Advances in the Study of the Individual Dynamic Organization of the Brain The Functional Brain: A Plastic Potential Despite the description by some pioneers of several observations of post-lesional recovery, the dogma of a static functional organization of the brain was settled for more than a century. This vision was essentially based on anatomo-functional correlations performed in lesional studies, which led to the view of a brain organized in so-called « eloquent » regions, for which any lesion induced a neurological deficit (such as the central, Broca’s and Wernicke’s areas, early identified), and in « non-functional » structures – with no clinical consequence despite their damage. However, through regular reports of improvement of the functional status following damages of cortical and/or subcortical structures considered as « critical », this conception of a « fixed » central nervous system was called in question in the past decades. Consequently, many investigations were performed, initially in vitro and in animals, then more recently in humans since the development of functional mapping methods, in order to study the mechanisms underlying these compensatory phenomena: the concept of cerebral plasticity was born. Therefore, cerebral plasticity could be defined as the continuous processings allowing short, middle and long-term remodelling of the neurono-synaptic organization, in order to optimize the functioning of the networks of the brain – during phylogenesis, ontogeny, physiological learning and following lesions involving the peripheral as well as the central nervous system [157]. On the basis of the recent literature, several hypothesis about the pathophysiological mechanisms underlying plasticity can be considered. At a microscopic scale, these mechanisms seem to be essentially represented by: synaptic efficacy modulations [79], unmasking of latent connections [309], phenotypic modifications [304] and neurogenesis [243]. At a macroscopic scale, diaschisis [481], functional redundancies [177], cross-modal plasticity with sensory substitution [31] and morphological changes [155] are implicated. Moreover, the behavioral consequences of such cerebral phenomena have been analyzed in human in the last decade, both in physiology – ontogeny [318] and learning [332] – and in pathology [99, 359]. In particular, the ability to recover after a lesion of the nervous system, and the patterns of map reorganization within eloquent area and/or within distributed network, allowing such a compensation (especially regarding sensorimotor and language functions), have been extensively studied – notably in stroke [247, 412, 491, 563, 569, 677]. Such knowledge allows a better study of the dynamic reorganization of the eloquent maps induced by LGG, and to select the optimal therapeutic management adapted to each patient.

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Linkage Between LGG Progression and Functional Brain Reshaping Most patients with LGG present with seizures and have no neurological deficit [145]. This is puzzling considering the frequent invasion of eloquent structures [162]. This means that these slow-growing lesions have likely induced progressive functional brain reshaping [159]. Preoperative neurofunctional imaging supports this claim [187]. Interestingly, the patterns of reorganization may differ between patients, a notion very important to know by the neurosurgeon with the goal to optimize both indication of surgery and surgical planning [87, 219]. Indeed, despite the limitation of the preoperative neurofunctional imaging previously detailed, these methods have shown that three kinds of preoperative functional redistribution are possible, in patients without any deficit. In the first one, due to the infiltrative feature of gliomas, function still persists within the tumor, thus with a very limited chance to perform a good resection without inducing postoperative sequelae [17, 594]. In the second one, eloquent areas are redistributed around the tumor [258, 755], thus with a reasonable chance to perform a near-total resection despite a likely immediate transient deficit – but with secondary recovery within a few weeks to months. In the third one, there is already a preoperative compensation by remote areas within the lesional hemisphere [602, 676, 702] and/or by the controlateral homologuous [20, 85, 187, 276, 573, 668]: consequently, the chances to perform a real total resection of this kind of gliomas are very high, with only a slight and very transient deficit. Therefore, in cases of brain lesions involving eloquent areas (i.e. the structures supporting the sensorimotor, language or other cognitive functions), plasticity mechanisms seem to be based on an hierarchically organized model, i.e.: first with intrinsic reorganization within injured language areas (indice of favorable outcome) [258]; second, when this reshaping is not sufficient, other regions implicated in the functional network are recruited, in the ipsilateral hemisphere (close and even remote to the damaged area) then in the controlateral hemisphere if necessary.

The Limit of Brain Plasticity Nevertheless, despite this potential of compensation, LGG growth and migration, even before anaplastic transformation, may induce functional deficits. While sensorimotor and/or language disorders are very unusual (around 10% of cases) [145], cognitive deficits are more frequent than previously thought. Indeed, recent works using extensive neuropsychological assessment in patients harbouring a low-grade tumor, have shown impairments of cognitive functioning in most cases (around 90%) at the time of diagnosis [388, 671, 696]. More precisely, an impact of the glioma on verbal fluency performance [240], picture and word recognition memory [240b], attention [240c] and executive functions [240d] has been demonstrated. It is worth noting that, in addition to the tumor itself, epilepsy may interfer with higher function and quality of life [354b]. Furthermore, it seems that cognitive dysfunction might constitute a prognostic factor [68b]. As a consequence, such a deficit of higher functions needs to be accurately evaluated for each patient before making a therapeutic decision, since the treatment may have a positive or negative impact on cognitive,

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emotional and even social (dys)function [10, 11, 14, 15, 68, 68c, 257, 354, 355, 388, 526, 528, 547, 593, 658, 663, 669, 670, 671, 672, 686], depending on the individual interrelationships tumor / function [159]. Longitudinal neuropsychological assessment can also be useful in the early detection of brain tumor recurrence following a first treatment [13] – thus should be incorporated in the systematic follow-up, in order to adapt a specific dynamic (multistage) therapeutic strategy to each patient, with an optimization of the oncological impact, but also with a preservation of the quality of life.

Advances in Therapeutic Management The Classical View The Wait and See Attitude When the management of a disease implies a choice between different therapeutic strategies with a seemingly similar effect in terms of survival, and in the absence of an actual curative treatment, the quality of life can help decision making, the iatrogenic risks of treatment representing the basis of the controversia [25, 81, 497, 541, 635]. Under the condition that the diagnosis can be confidently made on clinical and radiological data, and that outcome is independant of the timing of treatment(s) [541]. In the grade 2 glioma setting, evidence-based decisions are difficult to make in the absence of definite proofs of the efficacy of the available treatments and/or of their timing, with the exception of radiotherapy whose role and modalities have been explored by prospective randomizeds trials. Hence some authors advocate therapeutic abstention in cases of a typical radiological aspect, a medically controlled epilepsia, without significant neurological deficit in a younger patient [80b, 81, 430, 541, 609, 635, 713], the treatment being delayed until it is deemed «clearly indicated» [430]. Nevertheless, some prone the systematic practice of a biopsy for histological confirmation of the diagnosis [23, 93, 405, 513], since there is always, albeit rarely a risk of misdiagnosis [5 to 10% -232, 323, 361, 541-], and more frequently a risk of underdiagnosis, a great proportion of non enhancing lesions mimicking a grade 2 glioma can be in fact anaplastic (eventhough the reverse can also be true, as we have seen), the risk rising with advancing age [26]. Biopsy guided by anatomical, vascular or metabolic examinations as practiced nowadays seems nevertheless reliable, the target consisting of an enhancing, hypervascular or hypermetabolic region of the tumor [with PET -204-, spectro-MR -518-, vascular MR maps –357-], but also sampling the entire extension of the lesion and even its periphery because the undelimited, infiltrative nature of these tumors and their heterogeneity [137, 356, 732]. The tumoral aggressiveness can further be studied, even on small biopsy specimen, by different convergent means (histology, proliferation indices, apoptosis, karyometry, immunohistochemistry, molecular biology...). An alternative to a systematic biopsy would be a close clinical and radiological follow-up [81], to rule out tumoral evolutions somewhat incompatible with the diagnosis/prognosis of a grade 2 glioma. The more so since there is no difference observed, in terms of survival or quality of life, whether the diagnostic has been ascertained or not [535, 547].

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When dealing with a recurrent tumor, the tendancy to obtain a new histological diagnosis should be encouraged, notwithstanding the ethical issues, knowing the possibility of modifications with time of the cellular components (astrocytic and/or oligoglial) of a tumor, of its aggressivity (malignancy), eventually mixed with iatrogenous changes. This attitude represents the best way to tailor the therapeutic aggressivity according to the evolutive stage that the tumor has attained, and to monitor reliably the effect of treatments (ie. stratifying according to the tumoral aggressivity). There is indeed no available evidence that a «wait and see» [541, 713] or «primum non nocere» [80] attitude is better or worse than a more active, (oncologically) therapeutic strategy, in the absence of prospective and even retrospective comparative studies, and of the knowledge a priori of relatively precise risk-groups or reliable prognostic classes. The variable therapeutic indications among series, among teams/institutions, practicians, patients, and epochs, hamper the possibility of comparisons. Most if not all authors of course agree to be more proactive in front of an older patient (>40 or 50 years), the presence of significant neurological deficits or refractory epilepsy, an extensive tumor with mass effect or evolutive contrast enhancement, but these subgroups account for a small proportion of the cases diagnosed by modern and easily available radiological examinations (and in most cases correspond to an advanced stage, “pre-anaplastic”). On the other hand, a subgroup of patients whose tumor is (para-)limbic/temporal, presenting clinically at a young age, with a longstanding isolated epilepsy (with a normal neurologic examination), has been identified with a good prognosis [27, 397, 513, 514]; to note that these radio-clinical aspects resemble those of DNETs. Surgery Most authors agree on the (functional more than oncological) indication, initially, of resective surgery, if by its volume and extension the tumor at diagnosis exerts a significant mass effect (corresponding as a rule to a tumoral diameter of 4-5 cm) [427], as well as when a midline site or extension compromises CSF (out)flow, or when the radio-clinical aspect is suggestive of a (pre-)anaplastic state, or when one thinks that the follow-up of the patient will prove difficult and/or depending on the wish of the patient [80b]. Some authors prone undelayed resective surgery when a large (subtotal or complete) resection appears feasible, claiming that it has a prognostic value and that it is safe (at least under certain conditions/technical adjuncts). By contrast for others, when surgery carries a greater risk (eloquent areas) and especially in the eventuality of an oligoglial tumor, its indication should be carefully weighted, since there are alternative therapeutic modalities. On the opposite, some do not advocate surgery initially in the absence of pejorative factors as we have seen previously. The rationale for a surgical cytoreduction is to reduce the number of proliferating cells («greater log-kill»), so to diminish the risk of successive genetic alterations leading to anaplastic transformation, thereby delaying progression and malignancy [448]; moreover, this can facilitate the effect of other treatments. Because of the everincreasing infiltrative behaviour of these tumors, surgery should logically be proposed as soon as possible (smaller volume, lesser migration). But the infiltrative, undelimited, mode of growth of gliomas precludes their cure by surgery (see above), and some have stressed the tenuous concept of an

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even (sub)total resection of the radiologically apparent part of these tumors, which could be attained by radiotherapy for example [427]. Unfortunately, for practical and even ethical reasons, a prospective randomized study of the impact of surgery seems impossible to accomplish, in part also because there would be to many selection bias that could not be overcome [339]. The everlasting debate on the prognostic influence, if any, of resective surgery of grade 2 gliomas is fueled for most part by the absence of objective estimation of the post-operative residual tumor (which is preferable to the report of the proportion of tumor removed, particularly when the pre-operative volume is not stated). Among the numerous reports relating the effect of surgery as based on the surgeon’s impression, one can find from no interest at all of a surgical resection [80b, 614], to a prognostic influence, on univariate and sometimes even multivariate studies, only in terms of tumoral progression, and/or in terms of survival [303, 337, 397, 405, 464, 475, 482, 488, 506, 513, 591, 607, 637, 732], rarely prospectively [184, 330]. More recently, there has been reports evaluating objectively (and statistically) the amount of resection (on CT then MRI), among which the majority seem to show a benefit of extensive surgery [review in 339], only studied or more often apparent in univariate analysis [43, 45, 482, 506] than in multivariate analysis [23, 312, 418, 591]. The principal differences shown relate to resection vs. no resection, or (sub)total resection vs. biopsy and/or clearly partial surgery, and to a benefit especially apparent in the first years (no or important reduction of progressions and recurrences after complete resection in the 4-5 years after surgery –43, 482, 506-], the survival curves converging at about 10 years [312]. To note is the fact that (sub)total resections are in general achieved predominantly with smaller tumors [591], more easily accessible and less invasive tumors [488], preceeding determination of the spatial configuration of the tumor is warranted [591], lesions not crossing the midline [516]. In our experience, complete radiological removal, eventhough more frequent with smaller tumors, has similar influence whatever the size or location of a tumor. A difference is indeed difficult to demonstrate, especially when one recalls that a small surgical specimen (biopsy, partial removal) can be underscored in terms of aggressivity [516], eventhough the value of cytoreductive surgery can appear more important with more aggressive lesions [123]. Last, the quality of resection might influence only the delay of a surgery for recurrence (in the absence of other intervening treatment) but not the incidence of anaplastic transformation [600, in contradiction with an initial report of the same team –43- and our experience], which recalls the seemingly effect of radiotherapy in our experience. Hence, for the moment no conclusive evidence is clearly demonstrated about the prognostic impact of resective surgery in grade 2 gliomas [749], but the improvement in the surgical techniques (and quality) has allowed a reduced functional risk for the patients [316]. The extent of surgery favorably influences the immediate functional outcome [506, even more linked to age), not solely when it allows the alleviation of signs of raised intracranial pressure or focal deficits (rarely) due to a compressive mechanism. Epilepsy benefits from surgery in the majority of cases, without even the use of specialized approaches/techniques of corticectomy (with improvement of the quality of life), even in eloquent areas [150], and more so with (sub)total resections [206]. But there are unchanged epilepsies, and the initial efficacy seems to decrease often over the years of follow-up, especially in temporal locations,

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whatever the pre-operative duration of seizures [750]. It is difficult in this setting to appreciate the role of the slowly-growing tumor, sometimes infra-radiological only, in the mid/long-term effect of resective surgery on epilepsy. The functional risk of surgery is notable/evident, in this context of tumors that show a high propensity to arise or extend in eloquent areas, with persistance of functional cerebral parenchyma within the tumor (especially in primary eloquent areas) and/or at the tumor/brain interface [159, 161, 165, 167, 168, 169, 174, 594, 634. The recent advances in pre- and intraoperative anatomo-functional techniques have allowed a reduction of the surgical risk while permitting a more extensive tumoral removal (see after). Radiotherapy Historically, radiotherapy was necessary as the first standard postoperative additional treatment. Modalities Initially, main recommendations concerning radiotherapy were based on heterogeneous retrospective studies [443] and led to propose systematically an additional radiotherapy for biopsied patients or those having benefited from a partial surgery all the more if they were more than 40 years old [404, 609]. The most often proposed dose was from 50 to 55 Gy with fractions of 1,8 to 2 Gy at the rate of 5 fractions per week. Target volume was first the brain in toto and then the X ray scan hypodensity or the T2 MRI signal raised by a 2 (to 3) cm safety margin [609, 735]. This "standard" prevailed until the recent realization of prospective multicentric studies, as two trials allowed to clarify the optimal dose of radiotherapy. The North Central Cancer Treatment Group / Radiation Therapy Oncology Group / Eastern Cooperative Oncology Group trial compared 50,4 Gy in 28 fractions versus 64,8 Gy in 36 fractions for 203 eligible patients included between 1986 and 1994. No survival difference was noted, and a recent actualisation has even demonstrated a better survival, but not statiscally significant, in the low dose group after a median follow-up of 6.43 years for the 120 surviving patients; toxicity seems besides greater in terms of radionecrosis in the high dose group [613]. European Organisation for Research and Treatment of Cancer (EORTC) 22844 trial compared 45 Gy in 5 weeks versus 59,4 Gy in 6,6 weeks for 379 [343 eligible) patients included between 1986 and 1997 [330]. No significant difference between the two groups was observed. The median follow up is now greater than 6 years [706]. The five year survival rates were around 60 % for the two groups, median survival a little more than 7 years. The quality of life seems besides better in the low dose group [346, 516]. These last two trials plead clearly in favor of a low dose radiotherapy (45-50 Gy) in terms of efficiency and of toxicity, notably neurocognitive. Optimal Timing The timing question was recently analyzed by a European multicentric study. European Organisation for Research and Treatment of Cancer 22845 trial compared a conventional 6 weeks/54 Gy radiotherapy immediate versus delayed in 311 [290 eligible] patients included

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between 1986 and 1997 [329]. It was clearly shown that immediate radiotherapy allowed an increase of the five year progression free survival (five year estimates for time to progression of 44 % for the treated group and 37 % for the control group) while it had no impact on the global survival (five year overall survival estimates of 63 % for the treated arm and 66 % for the control arm). Data concerning the quality of life and toxicity are nevertheless not available. So, the choice of treatment strategy is still not so evident [368] and relies mainly on the therapist convictions. The theoretical risk of long-term radio-induced side effects in terms of radionecrosis or cognition, all the more when large volumes are irradiated, would rather plead in favour of withholding radiotherapy initially. Short, Middle and Long Terms Effects of Radiotherapy We saw that radiotherapy had no significant impact on survival, but increases the progression-free survival. Radiotherapy can also improve epilepsy or pre-existent neurological deficits (in 70-80% of cases), although the data of the literature remain on this point very marginal [29, 40, 427, 428, 522, 606]. These positive effects must be opposed to the negative effects represented by alopecia (sometimes definitive), late endocrinopathies, radionecroses (risk of 2.5 to 5% -607-) and especially neuro-cognitive decline (correlated with the degree of leuco-encephalopathy even sometimes difficult to differentiate from a tumoral spread) being able to alter considerably the quality of life. Eventhough studies on this subject are contradictory [70, 567, 672, 719, 764], these cognitive side effects seem bound to the total dose, the dose by session, target volume, age of the patient, vascular risk factors like arterial hypertension or diabete melitus and existence of concomitant treatments like chemotherapy. Most of these factors can be partially controled or limited with the modern focal techniques of radiotherapy. To be noted that chemotherapy was used for a long time only in case of anaplastic transformation, and when the glioma continued to grow or recurred after radiotherapy.

New Therapeutic Strategies Methodological Developments Surgery In addition to a systematic preoperative planning by non invasive neurofunctional imaging (now also possible during surgery via the recent development of intraoperative anatomical and functional MRI), an essential advance in the surgery of LGG was the use of intraoperative direct cortical stimulation (DCS), under general or local anesthesia – due to the frequent location of these tumors in eloquent areas and their infiltrative feature [44, 56, 131, 132, 166, 167, 175, 179, 180, 208, 278, 378, 396, 436, 456, 484, 507, 509, 550, 552, 589, 624, 625, 652, 674, 729, 734, 751]. DCS allows the mapping of motor function (possibly under general anesthesia, by inducing unvoluntary motor response if stimulation at the level of an eloquent site), somatosensory function (by eliciting dysesthesia described by the patient

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himself intraoperatively), and also the mapping of cognitive functions such as language (spontaneaous speech, oject naming, comprehension, etc…), calculation, memory, reading or writing, performed in these cases on awake patients – by generating transient disturbances if the electrical stimulation is applied at the level of a functional “epicenter”. It is important that a speech therapist be present in the operative room, in order to interpret accurately the kind of disorders induced by DCS, for instance speech arrest, anarthria, speech apraxia, phonological disturbances, semantic paraphasia, perseveration, anomia, dysculia, and so on. Thus, DCS is able to identify in real-time the cortical sites essential for the function (i.e. to be imperatively preserved), following the dura-matter opening but before the beginning of the resection, in order to both select the best surgical approach and to define the cortical limits of the glioma removal [175]. Intra-operative mapping has also allowed a better understanding of brain functioning, notably with regard to the cortical organization of the areas involving language, memory, and calculating, as well as the role of the supplementary motor area, the insula and the premotor cortex [163, 164, 164b, 170, 173, 176, 202, 223, 425, 459, 572, 573, 575]. This stimulation also allows study of anatomo-functional connectivity, through the detection of bundles for sub-cortical, motor, somatosensory, language pathways and those for other cognitive functions [41, 42, 45, 168, 171, 172, 178, 181, 231, 246, 341, 634, 765]. Finally, repetition of electrical stimulation gradually during resection allowed the existence of reorganization phenomena of the functional cortical maps to be documented over the short and long term, making it possible to consider a second surgical intervention with the addition of resecting lesions located in the eloquent zones that could not be removed during the first intervention [158, 159, 160, 161, 165, 169, 177, 371]. Indeed, the mechanisms of such a plasticity induced by surgical resection within eloquent areas were studied, by performing postoperative neuroimaging once the patient has recovered his preoperative functional status [371]. In particular, several patients were examined following the resection of gliomas involving the supplementary motor area (SMA), which has elicited a transient postsurgical SMA syndrome (see below). Functional MRI showed in these cases, in comparison to the preoperative imaging, the occurrence of activations of the SMA and premotor cortex contralateral to the lesion: the contrahemispheric homologuous then likely participated to the post-surgical functional compensation and recovery [371]. Integrating this plastic potential into the interventional strategy has thus opened the door to surgery for removing lesions traditionally considered to be “unresectable”, in particular within the primary motor areas, primary somatosensorial areas, the Broca’s area, the dominant insula, the striatum or the corpus callosum [for a review, see 159]. In summary, the integration of a systematic functional mapping, of the on-line study of the effective connectivity and of the individual plastic potential during each surgical procedure has enabled (1) to extend the indications of the surgery for LGG within the socalled eloquent areas, (2) to maximize the quality of the resection and (3) to minimize the risk to induce postoperative permanent neurological sequelae – i.e. to optimize the benefit to ratio risk of the surgery.

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Radiotherapy In practice, the current attitude, although still controversial, is rather to withhold radiotherapy, at any stage after eventual surgery, for a documented clinical progression and/or “significative” radiological progression. An ongoing tendancy is rather to use, before radiotherapy, chemotherapy, eventhough the question of the role/efficacy of the latter treatment option remains open as witnessed by the soon to be opened EORTC phase III trial aiming to compare, in a randomised way, radiotherapy versus chemotherapy at time of progression, with a stratification on the 1p status [708]. Target volume remains the hypersignal T2 or flair more or less increased by a safety margin of 2 cms. Fractionation remains at the moment classical (<=2 Gy by session, 5 sessions per week). Finally a dose between 45 and 50 Gy must be discussed. Open Questions A lot of questions persist concerning radiotherapy. -

-

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What could be the criteria of beginning such a treatment: age, epilepsy, general status, neurological status, specific neurocognitive status, tumor volume, curve of growth, new functionnal imaging data, histologic or biologic data? When a treatment other than surgery is indicated, must radiotherapy be systematically moved in second line after chemotherapy? When the treatment is ended, what could be the criteria of response: clinical criteria, radiological criteria such as volume or impact on the growth curve? Which is the optimal moment to estimate the response? In case of response, would be it possible to reconsider an initial contraindication of surgery? With new focal techniques, what is the risk to expose a patient to delayed toxicities? What about radiosurgery for small postoperative residual tumors? What about new fractionation modalities? What about radiotherapy and brain plasticity?

Chemotherapy Modalities The place of chemotherapy has yet to be defined. Many theoretical arguments must be considered against its potential efficiency like subnormal blood-brain or blood-tumor barrier or low proliferation indices. On the contrary, it is classic to consider chemotherapy as generally more effective when delivered before radiotherapy. In this situation, it could allow, if effective, to delay radiotherapy and its potential side effects (necrosis, cognitive decline). It could also, in case of an objective radiological response, to reduce the fields of radiotherapy.

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Finally we know, eventhough it is possible to obtain radiological responses, that the quantification of the latter seems particularly difficult and that the classical Mac Donald criteria used for malignant gliomas are not applicable. The first and only randomised phase III trial concerning chemotherapy for low-grade gliomas was conducted by the Southwestern Oncology Group. Were compared radiotherapy alone versus radiotherapy plus lomustine chemotherapy after subtotal/partial surgery or biopsy. No benefit was showed and the trial was prematurely terminated [184, 749]. Macdonald has, the first, evoked the possibility of an objective response in low-grade tumors within a serie of aggressive oligodendrogliomas [431]. Six years later, Mason was able to note 9/9 responders (8 patients at presentation and 1 patient for a recurrence after radiotherapy) under Procarbazine + CCNU + Vincristine (PCV) [444] while Soffiety [638] reported 13/13 patients stabilised or responders, also under PCV. More recently, Buckner reported a series of 29 patients treated by PCV and noted 28 % of objective response [73], but in this series chemotherapy was followed immediately by radiotherapy. Brada treated 10 patients with temozolomide for newly diagnosed oligodendrogliomas and observed 2 partial responses and 3 minor responses [62]. The multicentric serie reported by Quinn based on temozolomide chemotherapy comprised 46 patients with tumoral progression, with a majority of astrocytomas (14 radiotherapy and chemotherapy pretreated patients). 61 % of objective responses were noted. The PFS for the all group was 22 months. 76 % were in PFS at 12 months from the beginning of treatment [530]. Hoang-Xuan and al reported their experience concerning the treatment of 60 patients by conventional temozolomide schedule (200 mg/m2/day for 5 days every 28 days). The median number of chemotherapy cycles delivered was 11. More than half of the patients were improved clinically. A radiological objective response was noted in 1/3 of the cases. Only 8 % of the patients were considered in progression during the treatment period. The only factor correlated with the response was the loss of the chromosome 1p [268]. Finally, Stege et al reported their experience with PCV chemotherapy for low grade gliomas. They treated 16 newly diagnosed patients and 5 patients at recurrence, and observed respectively 13 and 3 responses with a median time to disease progression up to 24 months. The 1p19q status did not predict, in this small series, the response [645]. Optimal timing Nevertheless, since the princeps publications [431, 444], the interest carried in such a treatment is increasing [88]. The majority of teams suggest to propose chemotherapy as first and early treatment [62, 73], or at the time of progression (after surgery and before radiotherapy) eventhough the clinical or radiological criteria of progression remain to be defined with more precision than at present [268, 500, 530, 645]. Short, Middle and Long Term Effects of Chemotherapy We know that "historic" association of Procarbazine + CCNU + Vincristine (PCV) or more recent monotherapy with temozolomide can allow clear symptomatic improvements

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concerning epilepsy or pre-existent neurological deficits or headache in more than 50 % of patients. In a general way, chemotherapy seems to bring an improvement of the quality of life in spite of insufficient studies concerning this subject [62, 73, 268, 400, 645]. These treatments, contrary to radiotherapy, do not possess informed cognitive toxicity. Nevertheless, the tolerance is not perfect. The "PCV" association possesses a cumulative hematologic toxicity rendering difficult the administration of more than 4 or 6 courses without serious adverse events. Not to be neglected are the peripheral neurological risk of the vincristine or the risk of lung fibrosis with CCNU. The patients complain frequently of an intense asthenia and/or a loss of weight. Gonadic toxicity seems also frequent. The prescription of temozolomide would eventually be more adapted to this type of tumor with an absence of cumulative hematologic toxicity in spite of very prolonged treatments. It will be necessary to consider the risk of lymphocytopeny especially with continuous treatment plans and to consider a preventive treatment of opportunistic infections and notably pneumocystosis [212]. The risk of cutaneous eruption and even of liver toxicity is not to be underscored, and no data is available concerning potential gonadic toxicity. The future EORTC trial will help answering these questions. Moreover, various modalities of prescription could be discussed [66]. In Practice In practice, the tendancy is, at present, to favor this type of treatment first line for non operable tumors, in case of actual clinical and/or radiological evolution, all the more if there is a 1p deletion, since then expecting a clinical and an even radiological efficiency, while delaying radiotherapy because of its potential neuro-cognitive side effects. It is nevertheless imperative to confirm these hypotheses, to set up clinical trials and to associate to it number of ancillary works connected to biologic, pathologic, radiological, functional and neurocognitives and also economic questions. Open Questions Finally, like for radiotherapy, numerous and essential questions surround the problem of chemotherapy. -

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Must the treatment be proposed very prematurely? Otherwise, what can be objective criteria of beginning such a therapy: age, epilepsy, general condition status (WHO Performance Status or Karnofsky scale), neurological status, specific neurocognitif status, tumor volume, curve of growth, functionnal imaging data, histologic or biologic data? When the treatment is begun, what can be the criteria of response: clinical criteria (efficacy and tolerance), radiological criteria such as volume or impact on the growth curve, functionnal imaging criteria? What is the optimal duration of chemotherapy before this evaluation? In case of clinical and radiological stabilization or response, how long must the treatment be pursued? Would be it possible in this case to reconsider an initial contraindication of surgery?

Luc Taillandier, Laurent Capelle and Hugues Duffau

40 -

-

-

In case of response to chemotherapy, is it licit to propose systematically a radiotherapy at the end of the chemotherapy or is it preferable to postpone until a new progression? What would be the criteria to take that kind of decision? If one decides to treat "for a long time", what is the risk to expose the patient to delay onco-hematologic or gonadic toxicities? Can this treatment have an impact on survival by modifying radically the natural history of these tumors? Will the results be the same as radiotherapy with an impact on the progression free survival without modification of the overall survival? What about chemotherapy and plasticity?

Sequential Combined Strategies: Adapted to the Natural History of the Tumor and the Brain Reaction (Fig 1) What do we know and what do we want to achieve? Since the last two decades we are collectively learning to apprehend better the natural history of WHO grade 2 gliomas, which, since it is a continuously evolving tumor interacting with the brain, an adaptative organ but to a certain extent only to its environment, leads to a initially overt (epilepsy) or insidious (functional impairment) deterioration of the quality of life. We know that, inexorably, a grade 2 glioma will, covertly during a long period, acquire genotypic alterations that will ultimately transform its phenotype into that of an overt highgrade glioma, for which we have still no effective enough treatment. In the same way, we are refining our knowledge of the limits and advantages of each of the three treatment modalities available, and are learning how to evaluate them in terms of quality, and duration of, life, more than simply in terms of, for example, a radiological response judged according to habits developped for higher grade tumors. We are learning how to dismember this rather chaotic and biologically diverse group of lesions, and envision that we should soon be able, on clinico-radiological and/or biological grounds, to define homogeneous and finite risk-groups. From then on only will we be able to really appreciate an (almost) individual spontaneous prognosis a priori at the clinical onset or discovery of a lesion compatible with a grade 2 glioma. And tailor according to cumulated data during a short period of observation, a therapeutic strategy aiming, with a minimized risk, to significantly prolong at least (if not definitely cure) a (young) patient’s life span; while preserving or even ameliorating his quality of life, and interfering as little as possible with his productive socio-professional as well as personal life. Significant progress has yet to be made, which demands to pool data because of the relative rarity of the disease and its long duration of evolution, and because of its biological variety, expressed from the clinical to the genomic or proteomic levels. When prognostic factors will be reliable at (almost) the individual level and our methods of evaluation of efficacy adequate, prospective studies and trials will allow to test the efficacy of elementary treatments as well as different therapeutic strategies. What do we know we can achieve, by what means?

Diagnosis

For each step, a new multidisciplinar opened discussion is mandatory, depending on dynamic parameters: - Clinical and functional status - Imaging (anatomical, functional, metabolical) - (new) Histology and mitotic index - Molecular biology - Tolerance and response to treatment(s)

Surgery

Yes

Complete Resection

Subtotal Resection

Clinico-remnological Follow-up

Recurrence

No

Partial Resection

First Line Chemotherapy

Regression

Continuous Growth

Stabilization

Clinico-remnological Follow-up

Progression

Radiotherapy

Stabilization

Progression

Clinico-remnological Follow-up If (Sub)Total Resection is Possible

If (Sub)Total Resection is Not Possible

If (Sub)Total Resection is became Possible

Re-Growth

Second / Third Lines Chemotherapy Re-Growth

Anaplasia

Fig 1: Therapeutic Strategy in Low-grade Glioma Before Anaplasia

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Luc Taillandier, Laurent Capelle and Hugues Duffau

At the moment, the medical community can probably agree on several facts. That a WHO grade 2 glioma is definitely not a benign tumor, since it interferes with a young subject’s quality of life from the start (of its clinical overt phase), and will be responsible of a fatal outcome in gene”ral in the 5 to 15 years after its discovery. That, if administered, radiotherapy should be done with the best technique available (conformal, fractions of no more than 2 Gy, total dose of no more than 45 to 50 Gy) to avoid significant, (essentially) cognitive risks, and that its timing does not significantly alter its overall influence on outcome. That resective surgery could probably effect the functional and probably vital outcome of a patient under certain conditions; it should also be performed with the best anatomo-functional techniques available, allowing the preservation, and even often the amelioration of, function, and aiming to leave only a small, or no residual signal abnormality on the systematic post-operative control MRI (at two-three months in particular). Last, that chemotherapy seems to represent a valuable tool, already occupying a previous therapeutic «no-man’s land» between eventual surgery and radiotherapy, when the latter was withheld until «clearly indicated». But since all these facts need to be further explored, ascertained and/or refined, we need to collaborate for additional and rapid progress. World-wide collaboration underscores that we talk the same language (or that everyone is understandable, readable, «translatable» by everybody else) when reporting on a patient’s clinical condition, on the (radiological) behavior of his tumor, on the monitoring of his evolution, in the pre- as well as posttreatment periods. With that level of comparability, we can exchange, oppose different attitudes, and lessen the need for formal trials, as long as the prognostic (stratification) criteria are reproducible from one series to another, giving an actual value to smaller coherent cohorts. At the end, we will be thru only when the timing and various combinations of treatments will be explored. What can we propose to a patient harbouring a WHO grade 2 glioma? A «primum non nocere» attitude is of course laudable and represents one of our main concerns as physicians. Nevertheless, that is not ineluctably synonymous with a «wait and see» attitude, the latter denying any effect to treatment(s). There are two intermingled levels of medical reflexion in front of a patient, focusing on his functional and life-expectancy status. There again, no inherent contradiction precludes the simultaneous quest of the two goals, since an active as well as an expectative attitude both carry the same kind of risks, albeit dissimilar factually, menacing an individual harbouring a grade 2 glioma. With what we know nowadays, we can argue in favour of a proactive strategy, since even if some therapeutic measures do not at the end really prolong a patient’s life, they can first of all protect, and even improve his functional status, and second prolong his «disease-free» lifetime (recurrence-free period). Meanwhile, we will be able to collect enough homogeneous, convergent data from multiple sources, to prove as we have the intuition, that active strategies also prolong somewhat, if they are still unable to cure, the life expectancy of a patient, significantly shortened at the announcement of this sort of diagnosis. In the absence of a definitive method to avoid and predict anaplastic transformation, the therapeutic strategy(ies) can only aim to try to «confine the tumor to a grade 2 stage», by limiting the number and/or activity of tumor cells, leading to the progression of the tumor and

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moreover its progressive (multi-step, clonal ?) acquisition of a malignant geno-phenotype. In this setting, radiotherapy has a definite effect on grade 2 progression (with its functional favorable consequences, and a good ratio benefit/risk with up-to-date irradiation techniques), surgical sub- and total radiological resection has the same efficacy, and probably is also capable of delaying anaplasia; if pushed to its functional limits, that is the wall of the surgical cavity abuting essential (non compensable) eloquent areas and pathways, we know that we create or worsen focal deficits, which will take several days to months, under adapted physiotherapy at best, for the patient to recover. Chemotherapy, for which less is known, could have a similar capacity of delaying progression, of grade 2 as well as anaplasia as it seems, with an «acceptable» level of risk (ie. low toxicity rate, while considering also its side-effects). When combined in a one-stage or multi-stage temporal associative strategy, we have learned from our personal experience that radiotherapy, but not chemotherapy, carries the risk, if done after «maximal surgery», to hamper functional recovery, and even to reduce the chance of complete recovery, giving the impression that it reduces somewhat brain plasticity capacity. This has not been our experience with chemotherapy, at least with a single regimen. Chemotherapy (and probably soon other treatment modalities –immunologic, genetic..-) can be delivered locally during surgery, with the aim of a higher local (concentration, distribution, therefore) efficacy while diminishing or avoiding the risk of somatic deleterious effects. Last, radio- and chemotherapy can, as is done with high-grade gliomas, be delivered simultaneously, in an attempt to obtain synergestic, potentialisating or facilitatory effects. Then, when should we propose what to which patient? The discussion with a patient presupposes that we have exposed what we know or envision of his common history with his tumor, and foreseen the different offerable possibilities, with their pros and cons, and the risks attached to the instauration as well as the withholding of a treatment. For our part, the first step is to be as sure as possible, with the greatest inocuity but also reliability, that we are dealing with a grade 2 glioma, and to evaluate its evolutive stage (in the continuum from a slow, indolent tumor, to its malignant counterpart). At the moment, before genotypic or proteomic reliable data, the close MRI follow-up of the lesion during three months or more if necessary, allows to improve the diagnostic accuracy by eliminating the probability of other possible diagnoses, and to appreciate the individual tumoral growth rate. Imminent or already anaplastic lesions mimicking a low-grade glioma can thereby, with very few exceptions, be ruled out, that mandate aggressive, multimodality treatment from the beginning. In the meantime, a thorough anatomo-functional evaluation can be performed (neuropsychological/language examinations, fMRI, perfusion MR, spectroscopic-MR/PET scan, DWI, DTI, MEG). A second step to be discussed early, because the ratio surgical usefulness/statistical unsignificance is tenuous considering even the slow growth/extension of the vast majority of these tumors, is to evaluate the possibilities of surgical resection, by cumulating a priori static anatomo-functional and dynamic data, combined to a patient’s background, and his opinion. If a (sub)total resection seems feasible, we would propose it as first choice treatment. If not, but if the a priori impossibility lies only in an extension «slightly to far» in eloquent areas (confer to the probability map), we might propose a neoadjuvant chemotherapy that could, in

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case of even a small centripetal effectiveness in the reduction of the FLAIR area, render an extensive resection possible. If a initially surgical strategy seems impossible, there is in general no urgency in initiating other types of treatment, namely radio- or chemotherapy, unless the glioma is discovered at a late satge, either clinically or radiologically (refractory epilepsy, cognitive or focal neurological deficit, large volume, mass effect, bilateral extension). One can usually take some time more to refine the dynamic (with time) estimation of the result of the everchanging tumor/host interaction before decision making. Grossly, since radiotherapy can be delivered only once, and since it works so well on the grade 2 progression that the recurrence is immediately or very quickly of the anaplastic type, allowing most of the time no intervening treatment before this event, we prefer to delay it as much as possible. Then chemotherapy would be the prefered «second choice», whatever the genetic status with our current knowledge since LOH 1p seems only indicative of a greater chance of efficacy and does not indicate a tautologic mode of sensitivity, and since the role of MGMT and other drug resistance factors status has not been clarified in low-grade gliomas. In all cases, a close clinico-radiologic monitoring of the effective efficacy of a chemotherapy regimen is mandatory, in order to switch treatment modality early (other chemotherapy protocol or other therapeutic modality), but taking into account the generally slow efficiency in this context, and in order to decide the duration of chemotherapy according to the apparent chemosensitivity of the tumor. In our opinion, an important aspect of the treatment of grade 2 gliomas is the notion of repetitive treatments, in a rather «preventive» manner albeit without precipitation, ie. not waiting for the presence of neurologic impairment, in order to try to delay the evolutive stage of the tumor that generates significant brain function alterations, which usually puts it closer to malignant transformation. The same order by which the various treatment modalities were initially discussed is applied to progression, namely considering surgery, then chemotherapy or radiotherapy. Even more than at first treatment, the results of anatomopathological and (molecular) biological thorough examination of an eventual tumoral specimen, crossed with the clinico-radiological tumor kinetics and tolerance of the patient, are the main ingredients of decision making. At progression more than initially given the amount of our ignorance at the moment, could be discussed simultaneous treatment associations, like for example the focalised irradiation of a small post-operative tumoral residue (in particular by radiosurgery, brachytherapy), or the local delivery of a chemotherapeutic (carmustine implant, other by convection…) or radiosensitizing drug (or in the near future an immuno- or genotherapeutic agent). All along follow-up, functional and quality of life monitoring is mandatory as well as the collection of «classical» clinical and radiological parameters. It allows to refine the evaluation leading to decision making, to explore brain plasticity as a resultant of tumoral growth and treatment imputation(s), and ultimately judge the approprietness of the therapeutic strategy applied. Last but not least, as much as possible the treatment should be tailored to the individual at risk, or even more to the couple host/tumor and its state at a given time (but considered in perspective, ie. with the past evolution and what can be foreseen). But in parallel every effort

New Therapeutic Strategies in Low-Grade Gliomas

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should be made to pool data, and include the patients taken in charge into homogenous cohorts or eventual ongoing trials.

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In: Trends in Brain Cancer Research Editor: Andrew V. Yang, pp. 87-117

ISBN 1-59454-972-9 ©2006 Nova Science Publishers, Inc.

Chapter II

Strategies in Brain Cancer Research Luciano Neder1,∗, Andre Luiz Vettore2, Oswaldo K. Okamoto3, Rodrigo Proto-Siqueira4, Luiz Gonzada Tone5, Carlos Scridelli5, Silvia Toledo6, Suzana M. F. Malheiros7, Suely K. Nagagashi Marie8, Sueli Mieko Oba-Shinjo8, Carlos Gilberto Carlotti Jr.9, Paulo Lotufo10, Sergio Rosemberg11, Wilson Araujo Silva12 and Marco A. Zago4 1

Departments of Pathology, 4Clinical Medicine, 5Pediatrics, 9Surgery, and 12Genetics, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil 2 Laboratory of Cancer Genetics, Ludwig Institute of Cancer Research, São Paulo, Brazil. 3 Instituto Israelita de Ensino e Pesquisa Albert Einstein, Center for Experimental Research, Brazil 6 Institute of Pediatric Oncology, and 7Department of Neurology, Federal University of São Paulo, Brazil 8 Departments of Neurology, 10Internal Medicine, and 11Pathology, School of Medicine, University of São Paulo, Brazil

Abstract Diffuse astrocytomas are the most frequent primary neoplasms in the central nervous system and account for more than 60% of all primary brain tumors. Although the precursor lesions of these neoplasms have not yet been identified, genetic studies have shown that malignant transformation of neuroepithelial cells is a multistep process, involving distinct molecular pathways. Genetic testing may thus identify distinct subsets of gliomas with similar histologic patterns, for instance, primary and secondary glioblastomas. Despite recent advances in the field, prediction of clinical outcome and the overall survival of patients with brain cancer have remained dismal. The advent of genomic technologies to study complex diseases such as brain cancer brings a new ∗

Correspondence concerning this article should be addressed to

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Luciano Neder, Andre Luiz Vettore, Oswaldo K. Okamato et al. paradigm to translational medicine. While traditional methods for the study of underlying mechanisms of cancer focus on restricted factors, genomic approaches facilitate discovering of numerous genetic markers and pathways related to cancer pathogenesis in a short time frame. New high-throughput strategies based on gene expression profiling of tumors and corresponding normal tissue have practical purposes of discovering targets for the development of smart drugs and new biomarkers for diagnosis and prognosis. This chapter attempts to outline some of the steps involved in such strategies, based on studies conducted by an interdisciplinary consortium comprised by clinicians, surgeons, pathologists, and molecular biologists. Starting from a limited number of samples, genes with aberrant expression in tumors can be identified with a combined use of high throughput analyses such as SAGE and DNA microarrays. Data can be further validated by quantitative real-time PCR and immunohistochemistry in additional tumor specimens, allowing the identification of potential markers of astrocytomas with distinct levels of malignancy. Besides refining diagnostic classification of brain cancers, this genomic-based approach may improve prognostic assessment and definition of therapeutic strategies, bringing useful knowledge into clinical decision-making routine.

Introduction Cancer harbors both genetic and environmental components to its etiology. It is wellknown that tumor formation may result from interaction between external (environmental carcinogen exposure) and internal (genetic) factors. Although the precursor lesions of the primary brain tumors have not yet been identified, genetic studies have shown that malignant transformation of neuroepithelial cells is a multistep process, involving distinct molecular pathways (Collins, 2004). The diagnosis and treatment of brain tumors is undergoing a paradigm shift. New molecular approaches are enabling the identification target genes, proteins and signaling pathways that drive tumorigenesis and progression. Moreover, the most recent classification of brain tumors incorporates the characterization of genetic abnormalities beside morphological features to classify the distinct types of gliomas (Kleihues & Cavenee, 2000). Despite recent advances in the diagnosis and treatment, prediction of clinical outcome and the overall survival of patients with brain cancer have remained dismal. For instance, brain tumors continue to be the leading cause of cancerrelated death in patients under 35 years of age. Since the development of specific new anticancer drugs, as the KIT inhibitor imatinib for gastrointestinal stromal tumor (GIST) and trastuzumab for breast cancers that harbors amplification of the HER2 gene, huge efforts in brain tumor research are directed towards identifying critical molecular events, to define putative molecular targets for therapy. Considering the state-of-art, an interdisciplinary consortium comprised by research groups, including clinicians, surgeons, pathologists, and molecular biologists, was created in Brazil. The main goal of this consortium is the molecular analysis of brain tumors and identification of new targets for the development of smart drugs and new biomarkers for tumor diagnosis and treatment. Herein, we describe below the different strategies adopt by the consortium in a country with limited resources, focusing the use of high throughput genetic analyses coupled with immunohistochemical approaches, to characterize the gene expression profile of brain tumors (Figure 1).

Strategies in Brain Cancer Research I. Selection of cases/controls

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II. Microdissection of brain tumors and non-neoplastic samples

Peripheral blood

III. RNA extraction

Data mining SAGE

III. DNA extraction

IV. Microarray Unstained Slides

V. Gene selection • Cell migration • Cell invasion • Apoptosis • Angiogenesis • Other pathways

VI. Real Time PCR

New biomarkers Smart drugs

Methilation Studies

VII. Polimorphisms

VIII. Cytogenetics assays IX. Immunohistochemistry

Figure 1. Diagram of the different strategies focusing the use of high throughput genetic analyses coupled with immunohistochemical approaches.

Besides refining diagnostic classification of brain cancers, this genomic-based approach may improve prognostic assessment and definition of therapeutic strategies, bringing useful knowledge into clinical decision-making routine.

Data Bank Variations in gene expressions are not limited to cancer as compared to normal tissue cells, but may also differ in different stages of cancer development, as well as in variants of the same tumor. The aim of gene expression analysis is the identification of relevant genes

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for diagnosis, prognosis, and also predictors of treatment response or possible targets for developing new therapeutic approaches. To achieve this goal, a close collaboration between researchers, clinicians and surgeons is necessary to collect relevant clinical and neuroimaging data, as well as biological samples. The initial phase of the project was carried out by clinical and surgical groups in four different Brazilian’s medical centers. The research was focused on astrocytomas and over the last three years more than 200 hundred tumor samples of newly diagnosed patients were collected. In general, three approaches were used to study these tumors: gene expression analysis, etiological studies and prognostic studies. a) Gene expression analysis (cross-sectional studies) The gene expression profiles from neoplastic and non-neoplastic tissue samples (obtained from individuals that underwent surgical treatment for refractory epilepsy) were compared using microarray and SAGE (Serial Analysis of Gene Expression) assays. b) Etiological studies (case-control studies) DNA blood samples from cases (patients with astrocytoma) were compared to age and gender-matched controls (patients with diseases other than cancer). In this way, the risk of the disease according to the prevalence of a specific genetic polymorphism was evaluated. c) Prognostic studies (follow-up studies) Treatment response and follow-up data were collected according to a structured questionnaire, as well as clinical, pathological and neuroimaging features. All this data will allow the correlation between clinical information and the gene expression or genetic polymorphisms patterns.

Populational Study Selection of Cases and Controls Cases are patients with newly diagnosed astrocytomas in different grades of malignancy. Each case was interviewed soon after histological diagnosis or in the presence of a strong clinical suspicion. A case could be interviewed before surgery, pending subsequent confirmation. For each case, we recruited two controls among in- or out-patients from the same hospital. Excluding criteria are shown in Table 1. Controls were matched to cases according to age and gender. They were broadly stratified by sex and age (quinquennia) according to the distribution of the cases. Controls were enrolled at the same time as cases, and adjustments to ensure the minimal required number in each stratum were introduced after the end of enrolment of cases. Each affiliated group maintained a routine for active search of controls in selected clinics of their hospitals.

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Table 1. Excluding criteria for controls Any cancer Tobacco and alcohol-related diseases Chronic lung disease Coronary heart disease Venous thrombosis Hepatitis Cirrhosis Occupational-related diseases Asthma Pneumoconiosis Acquired aplastic anemia Chronic non-neoplastic diseases of the oral cavity, the pharynx and the larynx Chronic gingivitis and periodontitis Glossitis, stomatitits and abscess of the mouth Leukoplakia Chronic sinusitis, tonsilitis and laryngitis Polyp of the vocal cord and the larynx Abscess of the pharynx Chronic non-neoplastic diseases of the digestive tract Chronic reflux esophagitis (gastroesophageal reflux disease) Barret’s esophagus Atrophic gastritis Crohn’s disease Ulcerative colitis Chronic mesenteric ischemia Adematous polyps Chronic disabling conditions Diabetes Immunodeficiencies Auto-immune diseases Mental disorders Senile or presenile psychotic conditions Other psychoses, mental retardation Diseases of the central nervous system Infections Hereditary and degenerative diseases Cerebrovascular disease Brain tumors Severely ill or unable to respond Similar proportion of controls in each broad diagnostic category was required. The inclusion of controls of any diagnostic category should not account for more than 15 percent of the overall control group. As for cases, controls should be living in the same study metropolitan areas at least six months before the interview.

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At least two interviewers were selected in each study hospital. All interviews were conducted at the hospital by means of two pre-coded questionnaires: one general and other specific. The general questionnaire was applied to cases and controls and included information on socio-cultural indicators, a residential history, life-time history of smoking, alcohol drinking, a detailed cancer family history, and a few selected dietary pattern indicators. The specific questionnaire was applied only to cases and included detailed clinical and neuroimaging information, as well as treatment response and follow-up data.

Coding of Data and Databank Organization and Management The Bio-informatics group developed a flexible database using an Oracle platform (http://ctc.fmrp.usp.br/clinicalGenomics/appSP/organization.asp). The questionnaires were typed in using a standard data entry package and the consistency of the database was checked systematically. Each individual in the study received a unique identification code (ID) that included the affiliated group code and a subject code. This ID followed the participant throughout the study and was used to identify the questionnaires and biological containers. The case/control status of an individual was not directly apparent from their study number. Instead this information was stored in a separate file. The database was filled with all the information from the questionnaire by the interviewer to reduce the amount of missing data. The information was submitted to common data quality control procedure. Each affiliated group received periodically (or could obtain in the database by themselves through an logon/password code) a copy of the data set with its own data. The study was approved by the Medical Ethical Committees from each one of the Institutions included in the consortium. Written informed consent was obtained from all participating cases and controls.

Clinical Presentation, Diagnosis and Surgical Approaches Patients usually presented with complaint of general symptoms and/or focal neurological manifestations. General symptoms included headache, nausea, vomiting, generalized seizures, and changes in level of consciousness, caused by intracranial hypertension. Neurological examination often provided the first clue to the diagnosis. Patients with signs and symptoms suggestive of an intracranial mass were submitted to neuroimaging studies. Magnetic resonance imaging (MRI) was the neuroimaging technique most frequently used in the evaluation and in patient’s follow-up. Grade II astrocytomas typically presented as nonenhanced tumors (Figure 2A). On the other hand, high-grade tumors presented as contrastenhancing lesions causing mass effect. Glioblastomas (GBMs) typically extended along the white matter tracts of the corpus callosum invading the contralateral hemisphere ("butterfly" form). These tumors usually exhibited heterogeneous signal intensity on MRI (T1 and T2 sequences) caused by cysts, necrosis, and hemorrhage (Figure 2B).

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Figure 2. MRI T1 sequence following gadolinium administration. A- Grade II astrocytoma: nonenhanced right frontal tumor with discrete border identifiable.. B- Glioblastoma: irregular enhanced lesion within the left frontal lobe presenting mass effect.

Surgical treatment was made after medical therapy with corticosteroids. Surgical approach provided histological diagnosis, cytoreduction of tumor mass always seeking preservation of neurological functions. Tumors located in the vicinity of eloquent areas were approached using intraoperative cortical stimulation allowing preservation of function during tumor removal, thus minimizing morbidity (Figure 3A). An extra concern during surgery was to provide to the tumor bank a representative sample, avoiding necrosis or normal tissue (Figure 3B). Intraoperative diagnostic evaluation of the mass is usually performed by cytology (smear or squash type) to histopathologic confirmation of achievement of tumor bulk and diagnosis (Figure 4).

Figure 3. Gross section of the glioma’s specimen. A Intraoperative photography showing cortical stimulation of motor area surrounding tumor mass. B. Photography of resected tumor showing viable tissue (yellow) and necrotic areas (white).

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Figure 4. Smears of pylocitic astrocytoma (grade I). Note the presence of the bipolar “hair cells” for which this lesion is named (circles). Rosenthal fiber (arrow) and nuclear hyperchromasia are readily seen.

Tumor resections were made under operating microscope providing excellent illumination and magnification. During surgery, tumors were identified by color, consistency, and vascularity. Ultrasonic aspiration was used for resection of large tumors. All patients were followed according to a protocol including periodic outpatient consultations. Highgrade astrocytomas received external radiation therapy (total dose of 50 to 60 Gy in 30 fractions) and chemotherapy. Patients with GBMs were treated with carmustine (BCNU) and patients with anaplastic astrocytoma with procarbazine, lomustine, and vincristine standard protocols (PCV).

Tissue Microdissection A prerequisite for any molecular tumor research is the capability of obtaining representative samples. Brain tumor specimens may not be representative, whereas include inadequate amounts of tumor cells or display varying degrees of anaplasia in topographically distinct regions (Kleihues & Cavenee, 2000). Microdissection is a technique that is very useful both in the research setting and for molecular testing in surgical pathology, which can be performed in frozen sections or in paraffin-embedded tissue samples (http://cgapmf.nih.gov/Protocols/#Microdissection). The available techniques range from the simple and inexpensive manual microdissection (MM) to laser-capture (LCM), and laser microbeam microdissections with or without pressure catapulting (LMPC) that require expensive and complex equipment. Independently of the purpose or design of the research, it is necessary to obtained samples immediately after surgery and either rapidly frozen at –80°C or alternatively submerged in RNA stabilization solutions (as RNAlater). In our opinion, it is fundamental to confirm the tumor diagnosis and validate the tissue collected by histological evaluation, i.e., determine if the sample represents the tumor bulk, border zone or adjacent parenchyma with gliosis. Whatever of the chosen technique, some steps are universally required to process frozen tissues: avoid RNA contamination, RNase-free conditions, the tissue must be

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harvested under sterile conditions and kept in frozen conditions during all tissue processing (Hunt & Finkelstein, 2004). In our experience, it is useful to put the cryomolds with OCT tissue embedding into liquid nitrogen just before cut the tissue in the cryostat (Figure 5).

Figure 5. Putting the cryomold with OCT tissue embedding into liquid nitrogen as preparative step before tissue cut in the cryostat.

The simplest method is the manual microdissection of paraffin-embedded or frozen sections, which involves use of a standard or inverted microscope. A standard microscope has the disadvantage the operator’s movements are inverted in the visual field and the distance between the stage and the objective may be very small, yielding some difficulties to manipulate the microdissecting implement. Using inverted microscope (stereomicroscope), the movements are not inverted and the distance between the flat stage and objective are larger than obtained by standard microscope. In case of paraffin embedded tissue sections, it is recommended to avoid the use of fixatives with low pH or those contain heavy metals, and fixation should be as soon as possible and fixation times should be less than 12 hours. Staining of the slides is a question of choice. However, stains, as Delafield and Mayer alum or Weigert iron hematoxylin’s, that can interfere with DNA and RNA extraction after microdissection must be avoided, (Serth et al., 2000). Stains such as methyl green and nuclear fast red are reported to aid in visualization with preservation of nucleic acids (Burton et al., 1998). In case of using unstained slides, microdissection tissue sections should be coupled with a hematoxylin-eosin stained scout section to attain the target. Microdissection from paraffin-embedded tissue will be easiest to perform when the tissue sections are deparaffinized before beginning by immersion in

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xylenes and decreased alcohol solutions (Zhuang et al., 1995). By the same token, if microdissection is performed after deparaffinization, the tissue fragments must be deparaffinized in appropriate tube. It is important that tissue fragments must adhere to the microdissecting implement to be deposited into collection tube. This intent can be achieved either through electrostatic forces (dry tissues) or through hydrostatic forces (if the tissue is damp or wet by a solution of 3% glycerol or deionized water). In our laboratories, we have been used the microdissection of the target area direct on the frozen tissue block using blade incisors and do not in the tissue slides (Figure 6). This approach can be easily performed since some precautions were adopted: the thickness of frozen tissue block should be less than 5 mm; the tissue sections must be representative of the entire frozen sample for matching; and the target area must be relatively well circumscribed from the non-representative areas (extensive hemorrhage, necrosis, border zone and gliotic brain parenchyma). It is redundant to say that the blade cutting area must be changed and the work area must be cleaned after every different sample. The advantages of this way are the simplicity of the method, low cost, easy to perform and not time consuming. The major disadvantage is that the method is gross in comparison with laser-capture microdissection and a pure cell population cannot be achieved. However, in research and routine practices, most molecular applications do not require a real pure cell population, especially if validation of genetic data is followed by immunohistochemistry reactions. In fact, we usually collected and placed frozen tissue sections directly on agarose coated slides to further immunohistochemistry reactions when we are trimming the frozen tissue block into cryostat. Consequently, we can obtain unfixed tissue sections that do not need antigen retrieval for immnuhistochemistry. These cryostat sections must be stored on dry ice while cutting or in paper box in at -80°C freezer. It is beyond the scope of this chapter to present specific details of several commercially LCM equipments. Basically, system components for LCM include an inverted microscope, an infrared laser, control units for the laser and for the microscope stage, a digital camera, and a monitor for target visualization (Fend et al., 2000). At present, there are several types of LCM instrumentation commercially available with several advantages and disadvantages, according the equipment (Burgemeister, 2005). Several microdissection standard protocols have been published in the literature – for details, see the excellent review of Hunt & Finkelstein (2004) and available in the web, as NIH’ protocols (http://cgapmf.nih.gov/ Protocols/#Microdissection). Ideally, in major laboratories or in core facilities, it is practical and reasonable to have both an LCM and a manual microdissection setup available and ready for use at all times (Hunt & Finkelstein, 2004). In practice, the manual methods can provide a sufficiently population of cancer cells for most molecular applications, as used in our consortium (Neder et al., 2004).

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Figure 6. A- Cryomold with OCT tissue in the cryostat. Note the position of the blade (arrow). BObtaining tissue sections. C- Stained slides with H&E. D- Microscopic view of the tumor section. Observe the undesirable area of necrosis. E and F- Removing the area of necrosis using the stained scout slide. G- Microscopic view of tumor bulk post-microdissection. H- Checking the RNA integrity by gel eletrophoresis.

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Nucleic Acid Extraction A 4 µm thick section of each sample is obtained at -25˚C in the cryostat for histological assessment under light microscope after hematoxylin-eosin staining. As described previously, necrotic and non-neoplastic areas are removed from the frozen block of tissue by microdissection prior to the RNA extraction procedure. Total RNA is extracted from tissues using Trizol Reagent (Invitrogen) according to the manufacturer’s recommendations, using a Polytron for tissue homogenization. The RNA quantification was carried out by measuring absorbance at 260 and 280 nm. A260/A280 ratios in the range of 1.8-2.0 were considered satisfactory for purity standards. Denaturing agarose gel electrophoresis was used to assess the RNA quality. DNA from the paraffin-embedded tissue samples is routinely extracted with proteinase K digestion and traditional phenol/chloroform protocol. Alternatively, we isolate DNA from the interphase and phenol phase of RNA extraction procedure. Proteins are isolated from the phenol-ethanol supernatant obtained after precipitation of DNA with ethanol using Trizol Reagent.

Gene Expression Microarrays DNA microarrays are a powerful tool to investigate biological processes through the large-scale analysis of genes from a particular cell, tissue, or organism. This high throughput method of gene expression analysis has boosted the biotechnological and pharmaceutical industries, with the promise of improving diagnostics and treatment options. The medical implications of the microarray technology are illustrated in many recent publications in the literature (Bustin, 2002; Calogero et al., 2000; Gilliland et al., 1990). In this system, thousands of probes (usually cDNA or oligonucleotides), each representing a unique gene, are orderly spotted/synthesized onto glass slides or similar solid supports. This plataform is also known as DNA chips. The principle of the technique relies on the specific hybridization between the probes on the chips and the sample targets (fluorescent-labeled DNA, cDNA or cRNA molecules). Generally, DNA microarrays are used to monitor changes in gene expression levels. In this type of application, a single microarray assay can simultaneously analyze the expression of thousands of genes through the quantification of their respective transcripts (mRNA levels). Gene expression is estimated by comparing the relative amount of mRNA in two distinct cell populations. The target mRNA from control and test samples are labeled with fluorescent dyes and after hybridization, the amount of mRNA bound to the chip is estimated by the intensity of the corresponding light emission. The mRNA levels in control and test samples are then compared and the differential expression data given as a ratio or fold change. Biologically relevant information can be extracted from the microarray data by further computational analysis. Genes similarly expressed may be clustered, generating expression profiles that allow a global view of the cellular transcriptional program in a given condition.

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Furthermore, genes consistently up- or down regulated provide a clue to the pathways involved in a cell’s response to its microenvironment.

Understanding the Molecular Basis of Malignancy in Astrocytomas The advent of genomic technologies to study complex diseases brings a new paradigm to translational medicine. While traditional methods for the study of underlying mechanisms of diseases focus on restricted factors, genomic approaches facilitate discovering of numerous molecular markers in a short time frame. The identification of key genes and regulatory pathways, for instance, may help uncover the molecular and biochemical processes related to certain pathological conditions. In central nervous tumors, one issue that still requires clarification is the mechanisms of malignant transformation of astrocytic gliomas. From a limited number of samples, the use of DNA microarrays may help understand this issue through the identification of genes differentially expressed in astrocytomas of polar levels of malignancy. In the study exemplified, gene expression profiling was conducted in six primary astrocytomas, three pilocytic astrocytomas (grade I) and three glioblastoma multiformes (grade IV). Analyses were carried out with oligonucleotide microarrays representing 10,000 human genes. Independent hybridizations for each tumor sample were carried out in duplicates. The reproducibility of the microarray data was ideal, with average coefficients of variation ranging from 6.5 to 9% and Pearson correlation coefficients higher than 0.98 (Figure 7). The mean fluorescence values of each duplicated sample were used to determine genes differentially expressed in glioblastoma, with pilocytic astrocytoma as reference sample. This strategy eliminated from the analysis many redundant genes differentially expressed in both types of tumors when compared to normal tissue. To minimize random interference due to biological variation, the three samples of glioblastoma were individually compared with each of the three samples of pilocytic astrocytomas, generating nine gene expression profiles in silico. After hierarchical clustering, genes displaying higher than 2-fold differences in expression (p<0.01) in all nine cross hybridizations simultaneously were identified. A total of 110 differentially expressed genes were found in glioblastoma, 65 up-regulated and 45 downregulated relative to pilocytic astrocytoma. Functional classification according to the geneontology categorization revealed that a great deal of the up-regulated genes in glioblastomas is related to cell proliferation and processes associated to tumor progression such as angiogenesis, stress defense, and cell migration. Down-regulated genes in glioblastomas fall within a variety of functional categories with many coding for proteins of unknown function (Figure 8).

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A.

Pilocytic astrocytomas

B.

Glioblastoma ltif

sample# 436 Pearson CC: 0.98 % within 2 fold: 99.7 Average CV: 6.5%

sample# 463 Pearson CC: 0.99 % within 2 fold: 99.7 Average CV: 7.9%

sample# 501 Pearson CC: 0.99 % within 2 fold: 99.8 Average CV: 9.0%

sample# 74 Pearson CC: 0.99 % within 2 fold: 99.9 Average CV: 7.7%

sample# 384 Pearson CC: 0.99 % within 2 fold: 99.9

sample# 397 Pearson CC: 0.99 % within 2 fold: 99.8 Average CV: 6.9%

Figure 7. Reproducibility of microarray data. Normalized fluorescence values of each replica were plotted for three samples of grade I (A) and three samples of grade IV (B) astrocytomas. Red: human genes; Blue: negative and positive internal controls; Light grey: poor spots.

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Overexpressed genes unclassified 25%

cell proliferation 27%

response to pathogen 3%

cell-cell signaling 3%

protein metabolism 11%

response to DNA damage stimulus 4%

phosphorus metabolism 7%

cell organization and biogenesis 4% response to biotic stimulus 5%

Repressed genes

signal transduction 6% organogenesis 5%

organogenesis 10%

transport 8%

unclassif ied 33%

biosynthesis 6%

cell death 6% response to biotic stimulus 5%

protein metabolism 6%

programmed cell death 5% nucleobase, nucleoside, nucleotide and nucleic acid metabolism 5%

signal transduction 6%

electron transport 5%

cell adhesion 5%

Figure 8. Functional classification of genes differentially expressed in glioblastomas relative to pilocytic astrocytomas, according to geneontology terms.

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In addition, microarray-generated expression data can be used as a correlate of a particular cell phenotype. Such defined and specific profiles of gene expression are referred to as molecular signatures, which could be useful to identify certain clinical conditions, class, or phase of diseases. It could facilitate premature and precise identification of tumors, providing the basis for a more efficient therapy. Molecular signatures may provide valuable prognostic information since tumors that are morphologically similar may have different molecular characteristics associated with the clinical outcome of the disease. As the technology becomes more mature, the natural tendency is the incorporation of the generated knowledge into clinical decision-making protocols.

Gene Selection Selected genes are question of choice. Genes similarly expressed may be clustered, generating expression profiles. Genes consistently up- or down regulated provide a clue to the pathways involved in a cell’s response to its environment. After hierarchical clustering, we usually adopted the arbitrary cut off value of 2-fold (microarray) or 4-fold (SAGE) to consider a gene as differentially expressed. Afterward, functional classification is performed according to geneontology classification, revealing a great deal of the up- and downregulated genes related to different cell process (cell migration and invasion, apoptosis, angiogenesis, calcium buffering, drug resistance, cell cycle control and signaling). Finally, considering the goals of the research (find new cancer biomarkers and/or achieve new candidates to drug therapy), the target genes are submitted to further selection according some characteristics of predict proteins (for instance, membranic or transmenbranic protein with small length and related to a crucial process of the cancer cell, as signal-transducing proteins).

Real-Time PCR to Evaluate Gene Expression in Human Gliomas The next step constitutes the validation of the selected genes by polymerase chain reactions (PCR), as shown in Figure 1. PCR-based techniques allow us to obtain genetic information through the specific amplification of nucleic acid sequences starting with a very low number of target copies. These reactions are characterized by a logarithmic amplification of the target sequences i.e. increase of PCR copies followed by a plateau phase. In contrast, the classic molecular biology methods like Northern or Southern blot analysis use nonamplified DNA or RNA, but need large amounts of nucleic acids, in many instances from tissues or cells that are heterogeneous. Reverse transcription (RT)-PCR-based assays are the most common method for characterizing or confirming gene expression patterns and comparing mRNA levels in different sample populations (Orlando et al., 1998). Despite of the wide variability of results characteristic of conventional RT-PCR assays (Reinhold et al. 2001) and its unreliability as a

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clinical diagnostic tool (Colagero et al., 2000), considerable attention is still focused on conventional, competitive protocols with steady improvements to internal standards, references for data normalization as well as the introduction of new mathematical models for data analysis (Bustin, 2002; Gilliland et al., 1990; Halford et al., 1999; Vu et al., 2000). Conceptual simplicity and the promise of high throughput have made the homogeneous real-time fluorescence detection assay (Gibson et al., 1996; Heid et al., 1996) the most widely used mRNA quantification method for research (Cohen et al., 2002; Gibson et al., 1996; Heid et al., 1996; Wall & Edwards, 2002). The real-time fluorescence assay is usually applied in monitoring transcriptional in vitro (Liu et al., 2002) and direct detection of the effects of receptor signaling (Yuen et al., 2002). Its potential as a clinical diagnostic assay (Bustin & Dorudi, 1998) is also being realized, and its use has been reported for the identification of micrometastases or minimal residual disease in colorectal cancer (Bustin et al., 1999), neuroblastoma (Cheung & Cheung, 2001), prostate cancer (Gelmini et al., 2001), leukemia and lymphoma (Matsuchita et al., 2001; Medeiros et al., 2002). There are different techniques available that detect amplified product with about the same sensitivity (Wittwer et al., 1997). They use fluorescent dyes and combine the processes of amplification and detection of an RNA target to permit the monitoring of PCR reactions in real-time during the PCR. Their high sensitivity eliminates the need for a second-round amplification, and decreases opportunities for generating false-positive results (Morris et al., 1996). The simplest method uses fluorescent dyes that bind specifically to double-strandedDNA (as Sybr Green). The other three rely on the hybridization of fluorescence-labeled probes to the correct amplicon. The methods differ in their specificity, although at later amplification cycles all can show artefacts that do not correlate to specific product accumulation. As amplicon detection in the molecular beacon, hydrolysis and hybridization probe assays are obviate specific for the target gene but depends on successful hybridization of the probe (Bustin, 2000). For interpretation of quantitative gene expression measurements in clinical tumor samples, normalization is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies the identification of a valid reference for data normalization remains the most stubborn of problems and none of the solutions proposed are ideal. It is especially difficult when dealing with in vivo samples and comparing gene expression patterns between different individuals (Bustin, 2002). Recently, expression patterns of 13 frequently used housekeeping genes were determined in 80 normal and tumor samples from colorectal, breast, prostate, skin and bladder tissues. These approaches suggest that HPRT gene should be used as a single reference for normalize gene expression quantification by real time (de Kok et al., 2005). When dealing with in vivo samples it is not possible to predict which housekeeping gene might be useful, indeed it is likely that mRNA levels of all housekeeping genes vary to such degree that normalization becomes inaccurate and/or misleading. So, it is recommendable to use the geometric median of two or three house keeping genes, with previously validation (de Kok et al., 2005). When we are using primer and probe based real time PCR to evaluate or corroborate gene expression profile in tumor samples it is necessary to follow a few pre-determined steps: 1) Primers must be designed in different exons with the probe binding to both exons to avoid false-positive results due to possible DNA contamination; 2) All PCR reactions should be

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performed in duplicates using the same amounts of cDNA templates (~20ng/reaction); 3) The transcription should be well standardized to reduce internal variations, high capacity enzymes should be used; 4) Standard curves using serial sample dilutions to ensure amplification efficiency equal or close to 100% should be included; 5) Two or more house keeping should be used, and the geometric mean from house keeping genes is recommended (Vu et al., 2000). An internal control, such as cell lines or a pool of normal samples must be used as a calibrator. Real time PCR can be used to corroborate high throughput analysis of gene expression in astrocytomas, such as SAGE and microarray. In the other hand it is an important tool to elucidate potential targets and genetic prognostic markers, which can be more useful for stratifying glioma patients than classic pathology alone. Recently, an analysis comparing the transcriptional profile of approximately 6,800 genes in primary WHO grade II gliomas and corresponding recurrent high-grade (WHO grade III or IV) gliomas from eight patients using oligonucleotide-based microarray analysis was capable to identify 66 genes whose mRNA levels differed significantly (P < 0.01, > or =2-fold change) between the primary and recurrent tumors. The microarray data were corroborated by real-time reverse transcription-polymerase chain reaction analysis of 12 selected genes, including 7 genes with increased expression and 5 genes with reduced expression on progression. In addition, the expression of these 12 genes was determined in an independent series of 43 astrocytic tumors (9 diffuse astrocytomas, 10 anaplastic astrocytomas, 17 primary, and 7 secondary glioblastomas). These analyses confirmed that the transcript levels of nine of the selected genes (COL4A2, FOXM1, MGP, TOP2A, CENPF, IGFBP4, VEGFA, ADD3, and CAMK2G) differed significantly in WHO grade II astrocytomas as compared with anaplastic astrocytomas and/or glioblastomas (van den Boom et al., 2003). Another recently published study set out to search for genes that are differentially expressed in anaplastic astrocytoma and normal central nervous system tissue by applying a PCR-based subtractive hybridization approach, namely, representational difference analysis. The results of DNA sequencing of a sample (96 cDNA clones) from the subtracted library allowed the identification of 18 different genes, some of which were represented by several cDNA clones, coding for the Np95, LMO1, FCGBP, DSCAM, and taxilin proteins. Quantitative real-time PCR analysis for five of these genes was performed using samples of astrocytic tumors of different grades, confirming their higher expression when compared to non-tumoral central nervous system tissue (Oba-Shinjo et al., 2005). Putative markers of grade IV astrocytomas identified by in silico SAGE screening were validated using real-time PCR in tumors of different grades of malignancy (astrocytomas grade I, II and IV) and non-neoplastic central nervous system samples. The results of this analysis indicated that a number of these markers were common to grade I and IV astrocytomas, despite the discrepancies in histopathology and clinical course of these tumors of polar grades. Further comparison between grade I and IV astrocytomas by DNA microarrays revealed over 100 differentially expressed genes possibly related to malignancy, most involved in cell proliferation (about 40% of over expressed genes in grade IV). Novel candidate markers of astrocytic gliomas were discovered, which included well-characterized genes, as well as members of the Hox family of transcription factors and type II cadherin

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superfamily, as well as genes of unknown biological function such as OLFM1 (olfactomedin 1), BCAS1 (breast carcinoma amplified sequence 1) and AIPC (activated in prostate cancer). The different strategies (SAGE, Microarray) used to elucidate evolution markers, therapeutic targets and differences between normal and neoplastic brain tissues, should be validated by an independent technology, and real time PCR was elicited in all of them. So, the molecular analysis of a limited number of samples by complementary techniques (such as real time and microarray) is capable of generating hypotheses and identifying new targets for disease management.

Polymorphisms In order to develop preventive strategies for cancer progression, it is important to identify inherited and acquired host factors that modify the individual risks. Inherited differences in the capacity to metabolize environmental carcinogens as a result of genetic polymorphism in xenobiotics metabolizing enzymes have been suggested to modify an individual susceptibility to cancer and response to treatment. A variable number of these polymorphisms are single nucleotide polymorphisms (SNPs) and understanding how they are involved in conferring susceptibility or resistance to disease, or in rendering a drug efficacious or toxicity in the patient, is a major goal of the relatively new fields of pharmacogenomics.

Figure 9. PCR-RFLP in 2% agarosis gel to detect CYP2D6-BstNI polimorphism. Lines 1, 3, 4, 6-10: homozygosity for wild allele; Line 2: heterozygosity; Line 5: homozygosity for polimorphic allele. M = Ladder Mark (100 bp).

The genetic polymorphisms studies require methods for the efficiently and accurately detection of gene sequence variations in DNA samples. Among the numerous techniques

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already described for genotyping we can highlight the PCR-restriction fragment length polymorphism (PCR-RFLP), real time PCR, mass spectrometry and Denaturating High Pressure Liquid Chromatography (DHPLC), being the PCR-RFLP and the DHPLC the most useful screening methods. The PCR – RFLP is performed by the amplification of a DNA fragment followed by digestion with specific restriction enzymes to detect RFLP. The Polymorphism may generate or remove some restriction sites resulting in changes in the length or number of the DNA fragments after the digestion. The DHPLC analysis allows the detection of DNA sequence alterations in heteroduplex samples. The principle of the DHPLC is heteroduplex formation through hybridization after heating and cooling the PCR products and the separation of heteroduplexes from homoduplexes is accomplished under partially denaturing conditions. This technique was developed because it has a high sensibility and its easy automated, resulting a high– throughput method and allowing the analysis of a great number of samples. The use of these methods for polymorphisms investigation could be applied to metabolizing enzymes with regard to carcinogen activation (Figure 9). The result of this kind of analyses looks promising for the detection of links between environmental or life style factors and cancer risk.

Cytogenetics Assays Cancer cytogenetics has undergone remarkable advancement as molecular biology techniques have been applied to conventional chromosome analyses. The limitation of conventional banding analysis in the accurate interpretation of certain chromosomes abnormalities have largely been overcome by molecular cytogenetics techniques that include fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH) and multicolor FISH (MFISH or SKY). FISH is the pioneer among molecular cytogentics techniques (Pinkel et al., 1986). This technique, is based on capable of DNA denature and renature forming hybrids. Another advantage related with FISH is that it can also be used in interphase (no dividing) cells, than we can investigate frozen tissue and paraffin imbedded tissue. Alteration in DNA copy number is one of the many ways in which gene expression and function may be modified in cancer, including brain tumors. For this reason, FISH is an important technique to discovery genes involved in tumorigenesis process. Much data about genetic imbalances in tumors have been accumulated by Comparative Genome Hybridization (CGH). CGH is a different type of FISH in which the tumor DNA is a labeled probe. It utilizes two-color FISH to compare, for example, brain tumor DNA with standard probe of normal DNA. One probe is labeled with one molecular tag, such as biotin and another one with digoxigenin. CGH produce a map of relative DNA copy number as a function of chromosomal location by comparing the hybridization of test and reference DNA to metaphase chromosomes (Kalloniemi et al, 1992; Kalloniemi et al, 1994). We can call CGH as competitive FISH, and allows determining complete or partial gain or loss of chromosomes.

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To evaluate the genetic aberrations in an invasive tumor, the normal or necrotic tissue adjacent to the tumor must be carefully removed. Tissue microdissection based on microscopic observation is one alternative to accomplish this goal. The DNA from microdissected tissue can then be used for CGH. While CGH serves as an effective screen for chromosomal imbalance across the genome, fluorescence in situ hybridization (FISH) answers more specific questions, depending on the probes used. Probes of alpha satellite repeat sequences at the centromere have been used to assess chromosome number, unique sequence probes have been used to target specific loci, whereas DNA sequences that are specific to unique DNA sequences along the entire length of the target chromosome have been used to investigate chromosome rearrangements. FISH can be used to confirm data from CGH and can be used to localize specific DNA sequences either in nuclei or on chromosomes. Thus far, FISH has mainly been used in the research rather than the diagnostic laboratory. Although, chromosomal CGH has already made a significant impact on cancer cytogenetics, it can provide only limited resolution at 5-10Mb level. The cytogenetic analysis, classical and molecular, as we know for the past years could be replaced by the quantitative, microarray-based comparative genomic hybridization technology (Pinkel et al, 1998). CGH-array consists in DNA sequences spotted in a slide, provide the possibility to high resolution and automated screening for chromosomal imbalances. Rather than replacing classical cytogenetics, these techniques have extended the range of cytogenetic analyses, aiming to improve the resolution of CGH, enabling researchers to refine and define regions in the genome that may be causal to cancer, and facilitating gene discovery at a rapid rate. The need for high-resolution analysis, at least in cytogenetic analyses, has importance of studying early-stage disease looking for genetic alterations that may be causal to cancer progression and etiology. Epidermal growth factor receptor (EGFR) gene amplification occurs in glioblastomas as double-minutes, it operates to maintain high EGFR copy number over multiple cell divisions. Roerig et al., 2005 established a genomic microarray that consist bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones representing tumor suppressor genes, proto-oncogenes and chromosomal regions frequently gain and lost in gliomas and analyze 70 gliomas. This array could detect amplifications as well as low-level copy number gains and losses, molecular classification was able to distinguish with few exceptions between diffuse astrocytomas and oligodendrogliomas, anaplastic astrocytoma and anaplastic oligodendrogliomas, anaplastic oligodendrogliomas and glioblastoma, as well primary and secondary glioblastomas. Cancer cytogenetics can now combining banding techniques with multicolor FISH, discovery in chromosome, CGH can be refined using FISH and/or CGH-array. The state-ofthe-art moves cytogenetic to another dimension that can improve both diagnosis and research.

Immunohistochemical Evaluation In this strategy for brain cancer research immunohistochemistry (IHC) assays have pivotal importance to determine the cellular location of selected gene products. Localization

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of specific antigens in tissues or cells is based on antigen-antibody recognition, i.e. exploits the specificity of this interaction, which is usually revealed by a chromogen (usually peroxidase) visualized at light microscopy level. Since tissue microdissection were done direct on the frozen tissue block (Figure 6), a “pure” cell population cannot be achieved as performed by single cell laser-capture microdissection. On the other hand, this approach allows us to obtain unfixed tissue sections directly on the frozen tissue block. In some cases we begin the IHC detection of a novel antibody in these unfixed sections, which precludes antigen retrieval techniques (Figure 10). Alternatively, we may quickly fixed these frozen sections in 100% acetone or ethanol just before storing it in -80°C freezer and/or at time to initiate the immunoperoxidase reactions.

Figure 10. Photomicrography of squamous cell carcinoma of larynx. Note the diffuse cytoplasmic staining of stromal cells with F19 antibody. (10 µm - Frozen sections, 1:400).

In our practice, we usually perform the titration of the primary antibody on 10% buffered formalin-fixed, paraffin-embedded tissue sections and we may use the immunolabeling of the unfixed tissue as reference. The titration of the primary antibody usually starts at 1:5 dilution and the optimal dilution is obtained when the greatest contrast is achieve between the expected positive staining and background staining. When it is feasible, we prefer to use primary commercially available antibodies with predilection for using the monoclonal ones. In these cases we usually follow the manufacturer’s recommendations. One of the critical issues in the IHC techniques is related to standardization of a novel reaction in fixed tissue sections – for instance, when using an unknown antibody. In case of using antigen retrieval, we prefer vegetable steamers or microwaves at different pHs retrieval

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solutions, instead of pressure cookers or wet autoclaves. The proteolytic digestion of the tissue sections must be carefully considered in these cases. Usually, the detection system is the avidin-biotin conjugate procedure (ABC), particularly the indirect technique. The others systems (alkaline phosphatase anti-alkaline phosphatase method and enzyme-labeled antigen methods) are performed only in selected cases. Therefore, with IHC it is possible to try answering some basic and fundamental questions such as: 1) What kind of cell is responsible for overexpressing a particular gene/protein?; 2) Is there any labeling of non-neoplastic cells? If so, in which cells?; 3) Which pattern of immunolabeling was revealed? The signal labeling may be cytoplamic, membrane, nuclear, paranuclear, “dot” or punctate and some antigens may be present in more than one location – for instance, galectin-3 may be cytoplasmic or nuclear and is often in both. The subcellular localization of proteins/epitopes cannot be realistically achieved by peroxidase methods. For this proposal, imunogold methods are recommended (Figure 11); 4) The immunolabeling is real or due to tissue background?

Figure 11. Immunoelectron microscopy of a sparsely granulated lactotroph pituitary adenoma. Note labeling of endosecretory granules by silver particles of 10 nm corresponding to PRL reactivity (Courtesy of Dr. Jorge E. Moreira).

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For instance, the comparison of gene expression profile of glioblastomas (GBMs) with anaplastic oligodendrogliomas and non-neoplastic brain tissue by SAGE revealed the overexpression of galectin-3 (Gal-3) for GBM. These data were later confirmed by rt-PCR. In order to answers the above questions, we performed IHC approach in different series of astrocytic and oligodendroglial neoplasms of different grades of malignancy in comparison with non-neoplastic brain tissues (Neder et al., 2004). Distinct Gal-3 expression was observed in pilocytic astrocytomas (PILOs) compared to diffuse astrocytomas, as shown in Figure 12. PILOs showed diffuse Gal-3 expression in tumor cells, both in nuclei and cytoplasm, whereas in diffuse astrocytomas the tumor cells did not show immunolabeling. The non-neoplastic areas presented Gal-3 expression only in the endothelium and eventual entrapped reactive astrocytes.

Figure 12. Gal-3 immunostaining of non-neoplastic brain samples showing expression in endothelial cells and in reactive astrocytes (A, B). C - Pilocytic astrocytoma showed diffuse expression of Gal-3. D - Diffuse astrocytoma exhibited lack of Gal-3 immnoreactivity.

GBMs thus showed a heterogeneous, but intense immunostaining for Gal-3, particularly in the pseudopalisading of tumor cells around serpiginous areas of necrosis (Figure 13). In contrast, anaplastic oligodendroglioma tumor cells were negative, and only macrophages have stained positively. Interestingly, there was no immunostaining of endothelial and muscle cells/pericytes for Gal-3 in the microvascular proliferation of high-grade gliomas, particularly in GBMs. In general, we found more Gal-3 positive endothelial cells in low-grade than in high-grade gliomas. However, even considering the limitations of visual quantification of immunohistology (Leong, 2004), we performed a semiquantitative evaluation of Gal-3

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Figure 13. A- Pseudopalisading in glioblastoma. Gal-3 expression in GBM (B, C) in comparison with anaplastic oligodendroglioma (D). Note the conspicuous Gal-3 expression along the pseudopalisading and the absence of immunolabeling in microvascular proliferation (*). In anaplastic oligodendrogliomas only histiocytes (arrow) exhibited immunoreactivity for Gal-3.

Galectin-3: Mean Labeling Score 4 3,5 3 2,5 2 1,5 1 0,5 0

Pilo

AstII

AstIII

GBM

OligoIII

OligoII OAstII*

Figure 14. Mean Labeling Score of Gal-3 immunoreactivity in a series of gliomas. The case of grade II oligoastrocytoma* showed diffuse immunolabeling in the astrocytic component. (Pilo, pilocytic astrocytoma; AstII, diffuse astrocytoma; AstIII, anaplastic astrocytoma; GBM, glioblastoma; OligoIII, anaplastic oligodendroglioma; OligoII: oligodendroglioma grade II; OAstII: oligoastroctytoma grade II.

expression by mean labeling scores (MLS) obtained in the tumor bulk. The MLS were significantly different in PILOs (3.2 ± 0.2 SD) and GBMs (3.5 ± 0.2 SD) in comparison with

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all other gliomas analyzed that were uniformly negative, except for a case of oligoastrocytoma that labeled with a 2 score in the astrocytoma component (Figure 14). In resume, this case illustrates the use of different genomic approaches coupled with immunohistochemical methods in the identification of a new biomarker that is helpful in distinguish GBMs and anaplastic oligodendrogliomas, and pilocytic astrocytomas and diffuse astrocytomas. The meaning of high expression of Gal-3 in pilocytic astrocytomas (WHO grade I) remains an open question, although the immunohistochemical staining is different from that observed in glioblastomas (WHO grade IV). Finally, the lack of expression of this lectin in endothelial cells in contrast-enhancing tumors (as PILO and GBM) could be related to some alteration of the blood-brain barrier by the pathological vasculature.

Conclusion This chapter attempts to outline some of the steps involved in different genomic strategies, based on studies conducted by an interdisciplinary consortium comprised by clinicians, surgeons, pathologists, and molecular biologists. Starting from a limited number of samples, genes with aberrant expression in tumors can be identified with a combined use of high throughput analyses such as SAGE and DNA microarrays. Data can be further validated by quantitative real-time PCR and immunohistochemistry in additional tumor specimens, allowing the identification of potential markers of astrocytomas with distinct levels of malignancy. Besides refining diagnostic classification of brain cancers, this genomic-based approach may improve prognostic assessment and definition of therapeutic strategies, bringing useful knowledge into clinical decision-making routine.

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In: Trends in Brain Cancer Research Editor: Andrew V. Yang, pp. 119-144

ISBN 1-59454-972-9 ©2006 Nova Science Publishers, Inc.

Chapter III

Perspectives in Astrocytic Tumor Molecular Research Sergio Comincini∗ Department ofdi Genetics and Microbiology, University of Pavia, Italy

Abstract Astrocytomas are fairly common tumors of neuroectodermal origin that typically show a high degree of tumor malignancy. Specific pathological features of astrocytomas comprise a high degree of neoplastic cell proliferation and invasivity within the brain peritumoral tissues and, in addition, a prominent angiogenesis in the neoplastic tissue. The modern clinical practice, based on surgical interventions and on radio-chemicals approaches, need an accurate anatomical tumor localization and a well defined tumor grade classification. The classification of astrocytomas is based on morphological and immunohistochemical methods aimed at defining the predominant neoplastic cellular typology. Thus, glial tumors can be composed of astrocytes (giving rise to astrocytomas), oligodendrocytes (oligodendrogliomas) as well as of other different glial cells such as oligoastrocytes (oligoastrocytomas) and ependimal cells (ependimomas). The histological tumor classification is necessary associated with the histopathological inspection that states the malignancy grades according to suggested guidelines. The tumor classification system that is mainly in use is that proposed by the World Health Organization (WHO). According to this classification, astrocytomas are divided into four grades: pilocytic astrocytoma (WHO grade I), low-grade astrocytoma (grade II), anaplastic astrocytoma (grade III) and glioblastoma multiforme (grade IV). An important issue in the classification of astrocytoma is to assess whether the tumor was originated de novo (primary astrocytoma) or arose from the tumor progression of an existing lower-grade astrocytoma (secondary astrocytoma). Astrocytoma present a great heterogeneity of neoplastic cells involved, which makes their classification with regards to tumor progression rather difficult. This is an important ∗

Correspondence concerning this article should be addressed to Dr. Sergio Comincini (Ph.D.), Department of Genetics and Microbiology, University of Pavia, Italy.

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Sergio Comincini matter since the prognosis of patients does correlate with age, tumor type and, mostly, with the malignancy grade. Although several studies have improved the tumor classification with suitable histopathological criteria, recent data suggest that morphologically indistinguishable astrocytomas have distinct classes of causal oncogene activation, and that these subclasses may be targetable by oncogene/signaling pathway specific therapies. Recent technical advances such as the RNA and protein microarrays and the gene expression profiling can be very informative in terms of defining the global biological profiling of the different cancers, in identifying molecular tumor subsets and to develop predictive and prognostic tumor markers. In conclusion, the integration of these molecular data networks can be used to improve the knowledge on the genetic processes that regulate the tumor progression, to address to novel therapies, likely to result in significant improvement in the survival of astrocytomas patients.

Keywords: Astrocytoma; glia; molecular markers.

Introduction The nervous system is composed of two broad categories of cells, neurons and glial cells. In the central nervous system the main glial cell types are astrocytes and oligodendrocytes, while the peripheral nervous system is composed of Schwann, enteric and satellite cells. At the embryonic stage development, glial cells constitute a cellular framework that is fundamental for the development of the entire nervous system and it regulates the neuronal survival and differentiation (Jessen & Richardson, 2001). In the adult, the best characterized function of the glial cells is the formation of myelin sheaths around the axons of the neurons that allows the fast conduction of signaling essential for the nervous system function. Glia cells are also the major regulators of the neuronal repair and they are largely responsible for the difference in the regeneration capacity between the central and peripheral nervous system (Jessen, 2004). Furthermore, glial cells contribute to maintain the appropriate concentration of ions and neurotransmitters in the neuronal environment; these cells are also essential regulators of the formation, maintenance and function of synapses, essential structures for the functionality of the entire nervous system (Porter & McCarthy, 1997). The astrocytes are the more numerous types of glial cells: they present many radiating processes that interweave in complex and intimate ways between neuronal cell bodies and fibers. Some astrocytes processes contact blood vessels endothelial cells, contributing to control the blood-brain barrier, which protects the central nervous system from unwanted substances and biological agents. Astrocytes are also involved in high affinity uptake for the major brain neurotransmitters and in the control of the level of potassium ions in the extracellular space (Walz, 2000). Oligodendroastrocytes form one of the most highly specialized cellular structures in the organism, the myelin sheath. These structures form electrical insulations around nerve fibers, thereby making possible the rapid transmission of electrical signals in the nervous system (Nave & Trapp, 2000). The glial cells have also important pathological relevance. Multiple sclerosis, a progressive disease with a significant immune involvement that primary affect

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oligodendrocytes, is perhaps the most widely recognized pathology associated with glial cells (Lucchinetti & Lassmann, 2001). Noticeably, the majority of the malignant brain tumors are derived from glial cells or their progenitors (Louis et al., 2002). This is not surprising since after development ceases, neurons become post-mitotic and only a small compartment of stem cells remain, whereas glial cells retain the ability to proliferate throughout life (Sehgal, 1998). Most of the glial tumors have a prevalent astrocytic component, but a high degree of heterogeneity often makes it difficult to determine the cell-of-origin. In particular, it is not clear to what extent the frequency of glial tumors relates to the ongoing proliferative potential of adult cells, or whether the multipotent neural progenitors are significant targets of malignant mutations (Zhu & Panada, 2002).

Astrocytic Tumor Classification Tumors of glial origins are termed gliomas, and include tumors that are composed predominantly of astrocytes (astrocytomas), oligodendrocytes (oligodendrogliomas), mixtures of various glial cells (for example, oligoastrocytomas) and ependimal cells (ependimomas). In the case of the peripheral nervous system, the neurofibroma and schwannoma are the two most common glial tumors. Astrocytic tumors comprise a wide range of neoplasm that differ in their location within the central nervous system, age and gender distribution, growth potential, extent of invasiveness, morphological features, tendency for progression and clinical course. Most of the current glioma classifications are derived from the seminal system of Bailey and Cushing (1928), based on the relationships between the histological appearances of glial tumors and putative developmental stages of glia. The most widely used current classification of human gliomas is that of the World Health Organization (WHO), revised in 2000 (Kleihues & Cavenee, 2000). This system divides gliomas into astrocytic tumors, oligodendrogliomas, and oligoastrocytomas. These are then graded into histological degrees of malignancy. In particular, the WHO grading system classifies astrocytomas into four grades (I-IV) based on the degree of malignancy, as determined by histopathological criteria: I-Pilocytic astrocytomas are the most frequent brain tumors of children between the age of 8 and 13 years. It is a slowly growing lesion that rarely undergoes progression to neoplasm. Most of these tumors arise in the cerebellum, the region of the third ventricle, chiasm and optic nerves, thalamus, and, less frequently, the cerebrum (Burger et al., 1991). Histologically, the archetype pilocytic astrocytoma exhibits a biphasic growth patterns, one consisting of stellate cells with a sparsely fibrillary cytoarchitecture resembling “protoplasmic” astrocytes, the other of elongate, bipolar, highly fibrillated astrocytes. Vessels are generally abundant and are often hyalinized. Degenerative atypia and multinucleated cells are common features, as well as glomeruloid microvascular atypia and proliferation. Mitoses are absent or rare. Altogether, none of these features indicates anaplasia or malignant clinical behavior. II-Low-grade astrocytomas are slow growing astrocytic neoplasms with a high degree of cellular differentiation that diffusely infiltrate nearby brain. They generally affect young

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adults and have a tendency to progress to higher grade astrocytomas. The low-grade astrocytomas account for 15% of gliomas in adult and 25% of all gliomas of the cerebral hemispheres in children (Guthrie & Laws, 1990). These tumors may develop in any region of the central nervous system but most commonly in the cerebrum of both children and adults (Walker & Kaye, 2003). A number of different astrocytoma subtypes are recognized (fibrillary, protoplasmic and gemistocytic). In the commonest subtype, the fibrillary astrocytoma, the tumor cells resemble astrocytes morphologically, show little nuclear atypia, have limited perinuclear cytoplasm and they have multiple extensions producing a loosely textured matrix. III-Anaplastic malignant astrocytomas are highly malignant gliomas with increased cellularity, pleomorphism and nuclear atypia. Anaplastic astrocytomas show an increased tendency to progress to glioblastoma and are generally thought to develop from low-grade astrocytomas. These tumors are generally found in both young and old patients and they exhibit a high morphologic heterogeneity, pleomorphism and nuclear atypia, usually in association with a high cellularity. The tumor tissue may contain multiple microcysts and the tumor cells diffusely infiltrate into adjacent brain tissue. No evidence of spontaneous tumor necrosis or abnormal microvascular proliferation is generally present in anaplastic astrocytomas. The overall morphology is intermediate between low-grade astrocytomas and glioblastoma. They may develop from low-grade astrocytomas or de novo (Ichimura et al., 2004). IV-Glioblastoma multiforme is a highly malignant brain tumor and typically affects adults between 45 and 60 years of age. Glioblastoma multiforme is composed of poorly differentiated, fusiform round or pleomorphic cells. The mitotic activity of these cells is usually very high. Increased necrosis and vascular endothelial proliferation are two major histological markers for the diagnosis of glioblastoma multiforme. The poor prognosis of patients with glioblastoma multiforme is largely due to the spread of tumor cells to other regions of the brain. Two distinct sub-variants of glioblastomas are recognized in the WHO classification. The first, giant cell glioblastoma, is characterized by a predominance of enormous, bizarre, multinucleated giant cells. The second, gliosarcoma, represents a variant of glioblastoma in which cellular elements of the vasculature are presumed to undergo a sarcomatous transformation. Two major pathways for the occurrence of glioblastoma multiforme have been proposed, consisting of a primary origin, with a de novo arising, without any precursor lesion. Patients with primary glioblastoma have a short clinical history (less than 3 months in the majority of cases) and typically present with large tumors that on magnetic resonance imaging show central necrosis, ring enhancement and perifocal edema. Other glioblastoma multiforme may develop slowly by progression from a less malignant lower-grade glioma, generally over a period of 5-10 years. They are termed secondary glioblastomas. The rapid advances in genomic technology, coupled with the exploitation of relevant mouse models, might allow the tumor type to be defined and classified based on specific gene-expression profiles. In particular, efforts are producing in order to incorporate molecular genetic information into clinical classification and grading schemes (Pomeroy et al., 2002). This “new” molecular grading system, combined with the traditional histopathological approaches, should allow more accurate and reproducible diagnoses. In addition, improved

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imaging techniques will probably provide clinicians with opportunities to make ever-earlier diagnoses of these neoplasms. Although important genetic pathways that are involved in the initiation of secondary gliomas have been identified, it remains unknown whether alterations of these pathways are sufficient to induce tumor formation. Downstream effectors of these genetic pathways also need to be identified, which will provide additional therapeutic targets.

The Genetic Basis of Glioma Tumorigenesis In order to understand the pathological changes during the genesis of human gliomas, it is imperative to understand changes that take place at the cellular and molecular levels. It is estimated that the human genome has approximately 60,000-70,000 genes, and about 30,000 of these are expressed in the brain (Fields et al., 1994). The specific role of a number of genes and their protein products in the healthy brain have been widely documented (Adams et al., 1994): in particular, the most abundant genes are those involved in structural and regulatory functions, like transcription and signal transduction regulation. In the context of tumorigenesis, genes are divided into two main categories, oncogenes and tumor suppressor genes, many of them have been isolated and characterized in different kind of tumors (Klein, 1987). Oncogenes are those genes that when mutated and/or overexpressed in normal cells cause the cell to become tumorigenic (Hunter, 1997). Tumor suppressor genes, on the other hand, are genes that play an important role in normal cell growth, differentiation and progression through the cell cycle. In particular, it is well documented that the loss of tumor suppressor genes function can be responsible for aberrant cell proliferation. In addition, the insertion and expression of tumor suppressor genes in tumor cells causes them to become non-tumorigenic. Recent advances in cell and molecular biology techniques have led to the identification not only of chromosomal aberrations but also of oncogenes and tumor suppressor genes associated with these abnormalities and the initiation or progression of brain tumorigenesis. Brain tumors, like other well studied neoplasms, arise as a result of gradual accumulation of several genetic aberrations in differentiated or precursor cells (Funari et al., 1995). Genetic changes during brain tumorigenesis can occur either at the chromosomal level or at the gene expression level. Furthermore, changes at the genetic level can be either due to the loss of a major portion of a chromosome or due to point mutations within a single gene (Sehgal, 1998). Several investigations into the genetic bases of gliomas have yielded large amounts of information about specific genetic events that underlie the formation and progression of human gliomas (Zhu & Panada, 2002). Specific molecular alterations are associated with astrocytic gliomas, and other genetic changes with oligodendrogliomas. Significantly, however, particular genetic changes may occur in some astrocytomas and not in others, or in only some oligodendrogliomas, hinting that there may be molecular subtypes of histologically defined astrocytoma or oligodendroglioma. Given the likely biological differences occasioned by such genetic variety, it would be not surprising to learn that each glioma subtype should require a specific and unique set of treatments. Genetic pathways that are involved in the initiation and progression of astrocytomas have been partially identified (Ohgaki, 2005). Initially, the cytogenetics, allelic loss, and gene amplification events, reported for a specific

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tumor type and grade, were quite variable. As more data were acquired, patterns of specific abnormalities identified within the tumor type and grade became evident. Thus, specific genetic markers are now beginning to define pathways of evolution, diagnosis and, potentially, for therapeutic treatments. Two major pathways for the occurrence of gliomas have been proposed (Kleihues et al., 1995; von Diemling et al., 1995; Kleihues & Oghaki, 1999): first, in Type I or secondary gliomas, the tumor may develop slowly by progression from a less malignant precursor lesion, and second, in Type II or primary gliomas, the tumor may develop rapidly without any precursor lesion in a de novo fashion. The Type I pathway is a progressive step-wise evolution from a low-grade astrocytoma, to anaplastic astrocytoma, to glioblastoma multiforme. Patients diagnosed with this progressive glioma are generally younger (mean age, 39 years) and have a history of a less malignant tumor at the time of diagnosis (Kleihues et al., 1995; Kleihues & Ohgaki, 2000). In Type I or secondary gliomas, loss of p53 and activation of the growth-factor-receptortyrosine kinase signaling pathway initiates tumor formation, whereas disruption of the retinoblastoma (RB) pathway contributes to the progression of tumor development. The RB gene (mapped on human chromosome 13q14) coded for the corresponding protein that controls the progression through G1 into S phase of the cell cycle. The CDK4/cyclin D1 complex phosphorylates the RB protein, thereby inducing release of the E2F transcript factor that activates genes involved in the G1 to S transition. Promoter methylation of the RB gene was found significantly more frequent in secondary (43%) than in primary gliomas (14%). This molecular alteration was not detected in low-grade and in anaplastic astrocytomas, indicating that it is a late event during astrocytoma progression (Nakamura et al., 2001). Cytogenetic analysis of astrocytomas reveals the gain of chromosome 7 along with the loss of a single sex chromosome, as the most common chromosome numerical aberrations. Structural abnormalities are rare, but when they occur they generally involve chromosomes 1p and 9p (Shapiro, 2001). The most common findings involves a mutation or allelic loss of chromosome 17p, the target gene is the TP53 gene (chromosome location: 17p13.1), in which more than 200 mutations have been described in different human tumors (Hollstein et al., 1991). The TP53 gene was discovered in 1979, showing that it could transform cells in culture and could elicit tumor formation in transgenic animals: this indicated that TP53 is a dominantly acting oncogene (Eliyahu et al., 1984). The genetic lesion is a missense mutation that inactivates the TP53 gene that usually occurs in correspondence of specific “hot spots”, such as the protein codons 175, 248 and 273. The most current assessment of TP53 mutations in progressive gliomas is now placed at greater than 65% (Watanabe et al., 1997). A similar observation has been made with immunohistochemical analysis of the corresponding p53 protein. These investigations have shown that the large majority of glioma patients have an abnormal accumulation of p53 protein. This increase in accumulation of p53 protein is expected because it can occur as a result from a mutation in TP53, as well as aberrations on other genes controlling the expression of the TP53 gene. A second family of genes appears to be important to secondary astrocytoma evolution as well. These genes involve a growth factor and its receptor. The platelet-derived growth factor (PDGF) family consists of an A and B chain proteins (PDGF-A and PDGF-B) with two kinds of receptors, PDGFR-alpha and PDGFR-beta (Kirsch et al., 1997). The PDGF-A and –B chains dimerize to form AA, BB or AB homo- or heterodimers. The receptor PDGFR-alpha binds with AA and AB, whereas

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PDGFR-beta binds with only the BB homodimer. In astrocytomas, the A chain and the alphareceptor are predominantly over-expressed (Nister et al., 1991). This observation is interesting because the most common chromosomal abnormality identified in astrocytomas involved the aneuploidy of chromosome 7, the chromosomal location of the PDGF-A chain. This fact may also have some importance in therapy-related issues, because in vitro and in vivo treatment with 1,3-bis-chloroethyl)-1-nitrosourea (BCNU) selects for a minor subpopulation of cells containing amplified PDGF-A and –B chains (Scheck et al., 1993). Changes in the expression of growth factors and their receptors may also initiate local environmental changes that begin to stimulate angiogenesis. Over-expression of PDGFRalpha also correlates with the loss of heterozygosity for chromosome 17p, although the exact nature of these events is unknown (Cavenee et al., 2000). On about 30% of the secondary astrocytomas, allelic loss has been identified on chromosome 22q, and a likely gene candidate for this genetic loss was the NF-2 gene. However, extensive analysis of this gene in all grades of gliomas has failed to detect a consistent abnormality (Hoang-Xhuan et al., 1995). Furthermore, the allelic loss reported for chromosome 22q occurs more telomeric to the NF-2 gene, and thus a candidate gene or genes to explain this observation awaits further genetic analysis. Other chromosomes exhibiting allelic loss in astrocytomas include chromosomes 1, 3 and 13. Each allelic loss identified in these studies represents a probable site for tumor suppressor genes, but these analyses need further confirmation. O6Methylguanine-DNA methyltransferase (MGMT) is a repair protein that specifically removes pro-mutagenic alkyl groups from the O6 position of guanine in DNA. Therefore, MGMT protects cells against carcinogenesis induced by alkylating agents, and an inverse correlation has been reported between MGMT activity and tissue-specific tumorigenesis. Loss of MGMT expression may be caused by methylation of CpG islands around the gene promoter sequence and has been observed in a variety of human cancers, including gliomas (Esteller et al., 1999). MGMT promoter methylation was detected in 75% of secondary glioblastomas, significantly more than in primary glioblastomas (36%) (Nakamura et al., 2001). Although no difference in prognosis was observed for the MGMT gene expression, it has been demonstrated that immunohistochemical expression of MGMT in malignant gliomas was a strong predictor of survival in patients treated with bis-chloro-ethylnitrosourea (CENU) (Jaeckle et al., 1998). In contrast to Type I gliomas, the other pathway for glioma progression appears to arise de novo or very rapidly from a pre-existing tumor cell(s), although they cannot be histopathologically distinguished from Type I gliomas. Type II or primary gliomas appear to have not evolutionary component. This tumor is usually associated with older patients, who have not had a previous history of a lower grade of tumor (Watanabe et al., 1996; Cavenee et al., 2000). In contrast to Type I, the most common genetic aberration in primary Type II gliomas is the amplification of the EGFR gene mapped to human chromosome 7p13-p11. This gene is amplified and over-expressed in the majority of these tumors (Cavenee et al., 2000). More than half of these gliomas with amplification of the EGFR also have a rearrangement of the gene, generally in the form of an internal deletion (Ekstrand et al., 1991). This mutated form of the EGFR has a high level of tyrosine kinase activity in the absence of the EGF ligand, which essentially keeps this receptor in a “turned on” autocrine mode. Thus, the amplification of EGFR can potentially override the normal negative

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regulation of the PTEN gene product. In addition to the EGFR aberrations, the allelic loss on chromosome 10 was almost entirely restricted to the primary gliomas (Tohma et al., 1998). It has been suggested that the loss of chromosome 10 is a major factor in the evolution of the highly malignant glioblastoma multiforme. It is postulated that it is the loss of chromosome 10 that permits the abrupt change from a low-grade to the high-grade malignant mass. Thus, the major difference between primary and secondary gliomas is that the former tends to lose the entire chromosome 10 as opposed to the latter, which demonstrates only a loss of the chromosomal region 10q. The MDM2 gene product also appears to be restricted to the primary de novo pathway. In addition, the amplification of the MDM2 protein will essentially inactivate the p53 protein, by means of a direct protein binding. Thus, this is an alternative mechanism that allows a tumor cell to be removed from the cell cycle control of the p53 protein. Furthermore, most of the tumors that over-express MDM2 lack a mutation or allelic loss in the TP53 gene (Reifenberger et al., 1993). Genes important in the cell cycle also appear to be associated with the primary glioma type. The loss of genes such as the CDKN2 locus that codes for p16INK4A and p14ARF proteins, as well as the amplification or overexpression of the CDK kinase proteins, or the amplification, allelic loss or mutation of RB1 gene are all capable of deregulating the cell cycle that contributes to the uncontrolled proliferation associated with the gliomas. PTEN is a tumor suppressor gene located on chromosome 10q23.3. It encodes a protein that has homology to the catalytic domain of tyrosine phosphatases and to tensin and auxilin, which are cytoplasmic proteins involved in interactions with actin filaments at focal adhesions, and uncoating of clathrin-coated vesicles, respectively (Li et al., 1997). PTEN functions as a tumor suppressor gene by inhibiting P13K (phosphatydilinositol 3-kinase) and Akt proteins, which provides strong survival signals. PTEN is mutated or deleted in approximately 30-44% of high-grade astrocytomas, particularly in the secondary forms of gliomas (Duerr et al., 1998). Controversial data on the prognostic significance of PTEN mutations have been reported. Although some previous studies found no association between PTEN point mutations and the survival of patients with high-grade astrocytomas, many investigations have revealed that deletions of the PTEN locus, low levels of PTEN mRNA and low PTEN protein expression correlates with unfavorable clinical outcomes in patients with high-grade gliomas (Sano et al.,, 1999). Regardless of the classification of primary or secondary gl,iomas, an increasing body of genetic alterations are described, many with relevant prognostic significance (Fox, 1997). In particular, it is well known that tumor growth is dependent on angiogenesis. In keeping with this general trend, malignant progression in astrocytomas is associated with vascular productive changes, including some endothelial and microvascular proliferations (Plate, 1999). Brem and collaborators created an angiogenesis grading system based on vessel density, endothelial cell number and their cytological features (Brem et al., 1972). In these studies the authors documented that gliomas are the most vascularized human neoplasm. Tumor neovascularization is mediated by a number of angiogenic factors, although VEGF (vascular endothelial growth factor) is regarded as the central mediator in glioma angiogenesis (Plate et al., 1992). Patients with VEGF-immunopositive glioblastomas have a significantly shorter mean overall survival that those with negative tumors (Yao et al., 2001).

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Cell-of-Origin of Gliomas In contrast to the common epithelial human malignancies, the cell-of-origin for most primary brain tumors remains enigmatic. Their elusive nature has thwarted brain tumor research, in that it has prevented precise comparisons between such normal precursor cells and their neoplastic counterparts. Furthermore, without knowledge of the originally transformed cells, it is difficult to dissect out the tumorigenic events (Louis et al., 2001). Traditional neuro-oncology has suggested that tumors with an astrocytic phenotype arise from astrocytes or their immediate precursors. Although gliomas are thought to be derived from astrocytomas, oligodendrocytes or ependimal cells, they display a broad spectrum of histopathological features. The variation in the phenotype and biological behavior of gliomas reflect the type of transformation genes operative in the neoplastic development. For oncogenic events to occur and undergo selection, however, tumorigenic cells must be proliferative. Problematically, there is no evidence to suggest that most brain cells are undergoing division normally during adult life. Glial cells could undergo neoplastic events during reactive proliferation, but no epidemiological evidence convincingly links processes likely to evoke reactive proliferation, with the development of glial brain tumors (Davis & Preston-Martin, 1998). Major scientific advances suggested that diffuse gliomas could arise from neuroectodermal stem cells that are present throughout life. The observation that neuroectodermal stem cells reside in adult human brains (Gage, 2000) raises the logical possibility that this cell population could give rise to gliomas. These stem cells have a proliferative potential, are highly migratory, and can pursue remarkably diverse pathways of differentiation, all features intrinsic to glioma cells and likely characteristics for neoplastic cells-of-origins. The further observation that systemic precursor cells, such as those of the bone marrow, can differentiate along neuroectodermal lines (Mezey et al., 2000), creates the additional possibility that such stem cells could arise or even undergo oncogenic events elsewhere and then proliferate in the apparent immunological safety or nutritive environment of the brain (Louis et al., 2001). Although this theory would be highly speculative at the present time for primary neuroectodermal tumors, the presence of adult neuroectodermal stem cells provides the first endogenous population that would be likely cells-of-origin for primary brain tumors.

Gene Expression Profiling Gene expression is a critical determinant of protein expression and thus of biological functions. Cellular behavior is dictated in large part by which of a large number of possible genes is being expressed. Therefore, identifying patterns of gene expression may provide enhanced information about the biology of a tumor and may help to identify subsets of tumor types that might potentially respond to specific targeted therapies (Comincini, 2001). Genomic methods such as cDNA or oligonucleotide arrays coupled to analytical methods that correlate expression patterns with external parameters such as survival or response to therapy (supervised approaches) or that identify unique transcriptional patterns without any a priori knowledge of types, groups or outcomes (unsupervised approaches), allow for the detection

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of complex patterns of gene expression; these patterns are useful to distinguish previously unknown molecular subsets and identify clinically important gene expression signatures (Staudt, 2003). Based on the relatively density of arrayed sequences, and hence the number of genes that can be analyzed in a single hybridization experiment, expression arrays can be roughly separated into two main categories: low-density and high-density. Low-density arrays typically contain hundreds to a few thousand genes, whereas high-density arrays usually contains between a few thousand to tens of thousand genes, arrayed on a single microscopic slide. Glioma genomic research employing expression array profiling, followed by tissue microarray immunohistochemical confirmation, has already identified glioma progression- and survival-associated markers, like insulin-like growth factor binding protein 2 (IGFBP2), which is uniformly and differentially over-expressed in glioblastoma multiforme specimens (Fuller et al., 1999; Sallinen et al., 2000). More recently, a study analyzing the expression of 1176 cancer-associated genes in 11 grade II astrocytoma specimens, uncovered six genes, including TIMP3, EGFR, and GDNPF, that were expressed in the majority of grade II tumors examined and were not expressed in three non-tumor tissue samples derived from different brain regions (Huang et al., 2000). In the same experimental study, it was also reported that seven genes including PDGFR-alpha, PTN, LRP, and SPARC, were upregulated by at least 2-fold in 20-60% of the grade II tumors, compared with the non-tumor brain tissue samples. In another study, Rickman and colleagues (2001) used oligonucleotide microarrays to compare the expression pattern of about 6800 genes between grades IV and grade I astrocytic tumors, identifying a group of 360 genes that distinguish the two astrocytoma grades. Gene expression clustering enabled to define clusters of tumor samples that conform to their clinico-pathological classification as well as outlier tumors that clustered with tumors of a different grade. Further validation experiments clearly identify a restricted set of novel differentially expressed genes, consisted of ZYX, SDC1, FLN1, FOXG1B, and FOXM1, within the different astrocytic grades (Rickman et al., 2001). In a more recent contribution, Godard and collaborators (2003) performed cDNA-array analysis of 53 patient biopsies, comprising low-grade, secondary and newly diagnosed primary astrocytomas, combining both supervised and unsupervised statistical analysis methods. The derived characterization of astrocytic gliomas by their gene expression profiles revealed consistent inherent differences between the tumor grades. Thus, the previous histological and clinical recognition of these gliomas entities can be strongly supported by gene expression data. Of note, the low-grade samples were found to be the most closely related group, while primary astrocytomas display the most heterogeneous expression profiles. Analysis of the expression profiles for correlated genes separating tumor subtypes allowed identification of sets of genes implicating in particular biological features that are associated with tumorspecific subtypes. In the same study, more strikingly, a cluster of correlated genes suggests that inherent angiogenic activity, manifested in over-expression of known angiogenic factors, including VEGF, FLT1 and PTN, distinguished primary astrocytomas from the other tumor grades. In fact, this feature alone was sufficient to correctly classify most astrocytic gliomas (92%), according to their subtype (Godard et al., 2003). In addition, patterns of transcriptional activation may be more informative than individual genes, for identifying molecular subsets and developing predictive and prognostic “biomarkers” (Mischel et al., 2003a). Many of the genes expression studies of cancer to date have demonstrated that

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morphologically different tumors have distinct transcriptional profiles, and that there are patterns of gene expression that correlates with increasing grade of malignancy, including in gliomas (Fuller et al., 2002). In particular, low-grade astrocytomas, oligodendrogliomas and glioblastomas have distinctive global gene expression profiles, which are clearly separable from each other and from normal brain tissues. These different types and grades of gliomas can be accurately distinguished from each other by a relatively small number of genes, which are heavily weighted towards genes encoding proteins involved in such critical processes as cellular proliferation, proteosomal function, energy metabolism and signal transduction (Mischel et al., 2003b; Shai et al., 2003). A search for alternative pathways must be based on identification of genes whose expression profiles differs significantly between the various glioma tumor classes. More detailed knowledge of underlying mechanisms and their relevance for the cancer process will allow treating cancer specifically by targeting deregulated pathways, leading to rational design of future treatment modalities, tailored according to the biology of the individual tumors (Shawver et al., 2002). An essential initial step toward this goal is the establishment of a taxonomy of tumors on the basis of their expression profiles.

Proteomics Patterns The biological features of astrocytomas, which are characterized by highly heterogeneous biological aggressiveness even in the same histological category, would be precisely described by global gene expression data at the protein level. The rapid development of novel proteomic technologies has brought with it great hope in speeding up the rate at which new biomarkers for conditions such as cancer can be discovered. Because of the multifactorial nature of cancer, it is very likely that a combination of several markers will be necessary to effectively detect and diagnose cancer. To look for such “fingerprints” of cancer, it will require not only high-throughput genomic or proteomic profiling, but also sophisticated bioinformatics tools for complex data analysis and pattern recognition (Zhukov et al., 2003). A biomarker-discovery approach might use conventional shotgun proteomics, where tumor samples are digested into peptides and then individually fractionated and analyzed by liquid chromatography, coupled directly on-line with mass spectrometry. Tandem mass spectrometry is then used to identify the peptides within each of the mixtures. Subtractive algorithms can be applied to the datasets of peptides identified in the samples to recognize peptides that are either unique to, or more highly abundant in the cancer-affected patients. Alternatively, a widely used method to identify aberrantly regulated proteins is fractionation by two-dimensional polyacrylamide gel electrophoresis before tandem mass spectrometry identification of differentially abundant protein spots (Celis et al., 1996). In a recent contribution, Iwadate and collaborators (2004), examined several human glioma specimens, using the above mentioned proteomics approaches, to compare proteome profiling patterns between low- and high-grade tumors. These approaches enabled the authors to show that proteome-based clustering of gliomas significantly correlated with patient survival; in particular, the results of hierarchical clustering analysis showed that the tumor classification

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based on proteome profiling patterns could generate an accurate patient stratification that is more clinically relevant than the conventional histological classification (Iwadate et al., 2004). In a different study, Hiratsuka and coworkers (2003) employed a proteomics-based approach to identify possible gene products involved in glioma tumorigenesis which may serve as potential diagnostic molecular markers for this type of cancer. By comparing protein spots from gliomas and non-tumor tissues, using two-dimensional (2D)-gel electrophoresis, they identified 11 up-regulated and 4 down-regulated proteins in gliomas. Interestingly, they also discovered that a group of cytoskeleton-related proteins are differentially regulated in gliomas, suggesting the involvement of cytoskeleton modulation in glioma pathogenesis. Other recent studies, using profiling proteomic approaches in cell models of primary astrocytes and glioblastomas with amplified EGFR gene, permitted the identification of several proteins not previously described in gliomas, providing new information on pathways potentially involved in glial cell malignancy (Zhang et al., 2003). In a more sophisticate approach, using a diagnostic proteomics approach, tumor samples from healthy and cancer-affected individuals are processed using a protein chip modified with a specific chromatographic surface. During the hybridization step, the immobilized peptides forming the chip matrix specifically react with the protein counterparts and the resulting proteomic pattern of each sample is then computer-acquired. Through the use of complex bioinformatics algorithms, the source of the sample can be classified as being obtained from a normal patient, cancer-affected patient, or from neither (Veenstra et al., 2004). In particular, very recently Liu and colleagues (2005) used surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to detect proteins affinity- bound to a protein-chip array, to screen and evaluate protein biomarkers for the detection of gliomas (astrocytoma grades I to IV). The authors highlighted 22 novel biomarkers, whose expression profiles discriminates with high sensitivity and specificity the different glioma tumor grades. Although the proteome analysis in cancer research, and in particular in the astrocytic tumors, is still in its infancy, the proteomics could help in discovering novel cancerassociated markers, that can be used to diagnose diseases, predict susceptibility, monitor the tumor progression, identify novel therapeutic targets.

Glioma Models There are two basic approaches to address the complexity of cancer. One is to integrate large data sets, to yield a model for tumor development and behavior. Another is to reduce complexity through the analysis of experimental models, such as cell lines or animal models, to characterize the fundamental process of tumor growth and to elucidate the effects of single genes (Hanash, 2004). A major recent advance in neuro-oncology has been the ability to model glioma in mice using transgenic technologies (Holland, 2001). Such models faithfully replicate the cardinal histological features of glioma, including white and grey matter invasion, vascular proliferation, and necrosis with tumor cell palisades. Such glioma models could be valuable for understanding the pathways responsible for glia tumorigenesis and for identifying novel

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therapeutic targets. One way of determining which genetic alterations identified in human gliomas are the actual etiological events, is to model the formation of gliomas by mimicking those genetic alterations in mice. Moreover, accurate animal models are powerful tools to investigate important aspects of glioma biology that can not be studied in cell culture systems, such as angiogenesis, invasion, and metastasis. Animal models that accurately duplicate human gliomas will hopefully provide excellent opportunities for identifying the most promising therapeutic targets for this disease and for testing any potential therapeutic strategies in vivo (Dai & Holland, 2001). To date, four major strategies have been successfully employed to generate glioma models in mice: chemical mutagen-induced, xenoor allograft transplantation-induced, germline genetic modification-induced, and somatic genetic modification-induced models. Modeling gliomas with undefined genetics can be induced by treating animals with DNA alkylating agents, such as nitrosourea derivates that generate point mutations (Barth, 1998). The histology of these lesions bears similarities to the equivalent tumors in human. Because these changes are point mutations they are difficult to identify and the total number of mutations in any tumor can not easily be determined. Furthermore, identification of which mutation is difficult and the cell-of-origin for these tumors is also not determinable from such experiments. A different approach for generating animal models for gliomas that has been used most for preclinical trials is xeno- or allograft transplantation (Palma et al., 2000). In these models, human and rodent glioma cell lines, that are maintained in culture, are injected into the flank or brain of nude mice. These cells grow into a mass at relatively predictable rates and the mice develop tumors with high incidence. However, the histology of these xenografts is dissimilar to that in human tumors and the genetic alterations are not known. In spite of their limitations, these models have provided reproducible tumors and have been used extensively in testing therapies in vivo. Unfortunately, they have not been good predictors of response in human (Dai & Holland, 2001). To address the functions of certain genetic loci in glioma formation, gain-of-function or loss-of-function germline modifications can be achieved by transgenic or gene targeting techniques (Aguzzi et al., 1995). Gain-of-function mutations expressed in specific cell populations can be achieved by driving expression of oncogenes from tissue-specific promoters. For loss-of-function mutations, conventional gene targeting techniques cause the deletion of a target gene in all cells and biologic effect in those cells that normally expressed the deleted gene. Combining transgenic and gene-targeted mutations by breeding of mouse lines allows determination of the cooperative effects between oncogenes and tumor suppressor genes. The last strategy for modeling human gliomas is somatic cell genetic modification using retroviral vectors. Two systems have been reported, one using a MMLV (moloney murine leukemia virus)-based replication competent vector that spread throughout the infected tissues. The second system uses an ALV (avian leucosis virus)-based replication incompetent system that limits infection to specific cell types and does not spread throughout the infected tissues (Dai & Holland, 2001). There are several reasons why modeling human cancer is important. First, genetically accurate animal modeling are useful to demonstrate the causal relationship between mutations found in human tumors and the formation of those tumors. Second, many of the biological pathways involved in cancer biology have been studied extensively in cell cultures. Finally, once the targets are identified and animal models are generated by activation of these targets, such preclinical models will be excellent test

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animals for therapeutics intervention. In human gliomas, most of the genetic alterations identified to date ultimately result in either abnormal activation of signal transduction pathways, downstream of receptor tyrosine kinases or disruption of cell cycle arrest pathways. These genetic alterations require accurate animal model studies in order to establish unequivocal correlation between genetic alterations and gliomagenesis (Dai & Holland, 2001).

Cellular Changes During Glioma Progression Tumorigenesis is a dynamic process that occurs through a series of events that confer increasing in vivo growth advantage to affected cells. Among these, the microvascular proliferation is one of the histopathological hallmarks of gliomas. Blood supply is necessary for tumor growth and metastasis, since it plays the nutritive role for neoplastic tissue and provides the way of dissemination (Weidner, 2000). Angiogenesis is a complex process regulated by growth factors and inhibitors released from neoplastic cells, endothelial cells and macrophages. The most important angiogenic factors in gliomas are VEGF (Vascular Endothelial Growth Factor) with its receptors Flk1 and Flt1, angiopoetin 1 and 2 with their receptors, PDGF (Platelet Derived Growth Factor B), and b-FGF (basic Fibroblast Growth Factor) (Plate, 1999). Angiogenesis grading systems based on microvascular density, endothelial cell number and their cytological features, have showed that gliomas are the most vascularized human neoplasm (Brem, 1999). The vascular stroma of gliomas consists of many forms of blood vessels. There are vessels with usual structure, incorporated or formed de novo, and pathological vessels with proliferative and degenerative changes; in addition, glomeruloid, telangiectases, haemangioma-like and sinusoidal structures are present in glioma pathological tissues (Plate, 1999; Novacki & Kojder, 2001). In gliomas, florid neovascularisation causes proliferation that exceeds the migration, remodeling and maturation of new vessels (Wesseling et al., 1994). The distribution of blood vessels and the angioarchitecture within the gliomas is heterogeneous, generally showing a more intense microvascular proliferation in older glioma patients, suggesting that some angiogenetic features are age-related. These areas of proliferation are mostly located around necrotic cells (Izycka et al.,, 2003). High vascular density in most types of tumors is an unfavorable prognostic factor, mainly because it is related to the increased risk of neoplastic dissemination. Gliomas, however, very rarely gives metastases outside the central nervous system. Apoptosis (programmed or suicidal cell death) occurs in numerous normal and pathological conditions, including neoplastic processes (Langlois et al., 2000). Immunohistochemical technique for apoptosis identification has been demonstrated in several series of various tumors of the central nervous system, including astrocytic tumors (Yew et al., 1998). According to different studies, the apoptotic index in gliomas was found to be relatively low (Korshunov et al.,, 2002). The prognostic significance of the apoptotic rates has been already considered for various human neoplasms, although the predictive value of apoptosis intensity in relation to clinical outcome of astrocytic tumors may be estimated as controversial (Korshunov et al.,, 2002).

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It has long been observed that after glioma surgical removal, tumors recur predominantly within 1 cm of the resection cavity. This is mainly due to the fact that, at the time of surgery, cells from the bulk tumor have already invaded normal brain tissue. Therefore one of the most important hallmarks of malignant gliomas is their invasive behavior. Invasion of tumor cells into normal tissue is thought to be a multi-factorial process, consisting of cell interactions with the extra-cellular matrix and with adjacent cells, as well as accompanying biochemical processes supportive of active cell movement (Demuth & Berens, 2004). To migrate, the cell body must modify its shape and stiffness to interact with the surrounding the extra-cellular matrix, which may act both as a mechanical barrier to the cells, and as a permissive substratum for traction for the migrating cells. Cell movement necessitates a change in cell morphology: the cell becomes polarized and membrane protrusions develop, including the extensions at the leading edge of pseudopodia, lamellipodia, filopodia, and invadopodia. These extensions contain filamentous actin and various structural and signaling proteins necessary for attachment to the extra-cellular matrix. The formation of membrane anchors allows cytoskeletal contraction, which finally advances a cell forward. The extracellular matrix protein components (fibronectin, laminin, collagen, vitronectin and tenascin) play an important role in the progression of gliomas: in detail, studies have shown that the extracellular matrix composition is changed by invading tumors (Rutka et al., 1988; Giese et al., 1994). Furthermore, these components appear to stimulate glioma cell migration in vitro (Onhishi et al., 1997), while laminin stimulates radial migration from glioma spheroids (Berens et al., 1994). A growing body of literature deals with the influence of matrixmetalloproteinases on glioma migration and invasion. Matrix-metalloproteinases degrade extracellular matrix proteins, conceptually creating space for invading glioma cells. Several studies address the fact that matrix-metalloproteinases are over-expressed in glioma cells, compared to the normal brain tissue. In addition, several matrix-metalloproteinases-inhibitors have effectively down-regulated glioma invasion (Tonn et al., 2003).

Novel Therapeutic Approaches Patients harboring glioma tumors usually present with common central nervous system abnormalities including focal paresis, headaches, seizures, and language disturbance (Holland, 2001). Upon the diagnosis of an astrocytoma, based on imaging studies and biopsy, surgical resection is usually recommended, if considered feasible (DeVita & Bleickardt, 2001). However, due to the highly infiltrative nature of astrocytomas and the lack of clear tumor margin, it is nearly impossible to remove the entire tumor. These problems, in spite of a tremendous progress in tumor researches in the past two decades, have led to increasing interest in alternative treatment strategies, including immunotherapy. Previous studies indicated that immunotherapy for glioma tumors was quite uniformly ineffective (Proescholdt et al., 2001). One of the formulated explanations of such failures was the special immunological environment of the brain, with its low expression of MHC molecules and the limited access to inflammatory cells and humoral immune effectors within the brain: these characteristics are attributed in part to the effectiveness of the blood-brain barrier. Immune responses in the brain are considered to exhibit special features, consistent

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with the peculiarity of this unique location. Indeed, the brain is frequently described as an immune-privileged site (Streilein et al., 1997). More precisely, immune reactions can do occur in the central nervous system, but the rules governing them are not the same as for other tissues (Walker & Dietrich, 2001). Naive T-cells are excluded from the healthy brain (Hickey, 1999), although they may infiltrate the parenchyma following inflammation (Krakowski & Owens, 2000). Local antigen-presenting cells are important either for capturing and transporting antigens to secondary lymphoid organs or for activation/reactivation of recruited T-cells. However, constitutive MHC expression in the brain is very low and conventional professional antigen-presenting cells such as the dendritic cells are rarely found (Walker & Dietrich, 2001). There are several brain cell types that may contribute to antigen presentation in the central nervous system, including normal astrocytes, endothelial cells, pericytes and microglial cells. A prerequisite for antigen presentation is the expression of MHC molecules. Interestingly, MHC class I and II molecules are either undetectable or weakly expressed in normal astrocytes in vivo (Lampson & Hickey, 1986), whereas cultured astrocytes can express high level of MHC class I and II molecules, particularly after incubation with interferon-gamma (Weber et al., 1994). Furthermore, primary cultured cells can present foreign antigens to class I and class II restricted T-cells, but may be unable to trigger a complete T-cell activation program (Aloisi et al., 1998). Other antigen-presenting cells candidates in the brain are endothelial cells and capillary pericytes. The perivascular location of pericytes seems to be particularly appropriate for antigenpresenting cells function, being accessible to resident central nervous system and blood derived immune cells. Specialized macrophages are also found in the perivascular space, although their function can be fully assessed. Recent literature data suggests that microglial cells may be attractive antigen-presenting cells candidates (Abbas & Sharpe, 1999; Aloisi et al., 2000). They correspond to 5-15% of the total cellular composition of brain tissue and are distributed throughout the brain (Davis et al., 1994). When activated they possess certain characteristics of dendritic cells, with expression of CD1a, MHC class I and II molecules, as well as adhesion and co-stimulatory molecules such as B7.1 (CD80), LFA3 (CD58), and ICAM-1 (CD54) (Satoh et al., 1995). In recent years, it has become increasingly clear that the immune defense functions of microglial cells against glioma are compromised and that deficient antigen presentation may be an underlying cause (Flugel et al., 1999; Badie & Schartner, 2001). Since the description of a melanoma antigen, MAGE (van der Bruggen et al., 1991), numerous other tumor antigens have been characterized, providing cumulative evidence for the antigenicity of at least some tumor types. MAGE comprises a gene family of several members which are silent in normal cells, with the exception of testis, but which are expressed as peptides in a significant proportion of melanoma and other neoplasms (e.g. breast, lung tumors, head and neck carcinomas). To date, few results have been published concerning glioma antigens able to elicit an immune response (Walker & Dietrich, 2001). MAGE family members are expressed by some glioblastoma cell lines, which can be recognized by antigen specific T-cells, confirming the presence of MHC-MAGE peptides complexes at the surface of tumor cells; however, there is no MAGE expression in uncultured tumors (Scarcella et al., 1999). This discrepancy between in vitro and in vivo data could be due to a different level of DNA methylation within the promoters of the different genes,

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producing differences in transcription regulation (De Smet et al., 1995). More recently, other different melanoma-associated cancer-testis genes GAGE family members were shown to be expressed in a high proportion of malignant astrocytoma biopsies (Scarcella et al., 1999). However, the recognition and the lysis of tumor cells by specific T-cells needs a certain expression threshold of MHC-peptides complexes (Lethe et al., 1997), a critical point that remains unsatisfied in the case of tumor astrocytic GAGE expression. The failure of conventional approaches of immunotherapies for gliomas has led to increasing interest in alternative approaches, particularly dendritic cell-based immunotherapy (Steinman & Dhodapkar, 2001). Dendritic cells are the most potent antigen-presenting cells and are central to the regulation, maturation, and maintenance of a cellular immune response to cancer (Yamanaka et al., 2003; Parajuli & Sloan, 2004). The dendritic cells can mobilize several immune resistance mechanisms including CD8+ cytotoxic T lymphocytes, CD4+ helper T cells, Natural Killer (NK), and NK-T cells. Each of these cell types recognizes targets through a distinct mechanism and has the capacity to kill tumor cells and release protective cytokines such as gamma-interferon. Generally, dendritic cells used in vaccine protocols have been derived from monocytes of from monocyte precursors (CD34+ bone marrow cells), following stimulation with cytokines such as GM-CSF and IL-4. There are several different strategies to load dendritic cells with multiple tumor antigens, such as the use of apoptotic (irradiated) tumor cells, the transfection with acid-eluted peptides or tumorderived RNA or cDNA, the fusions between dendritic cells and tumor cells, and the use of exosomes. Several experimental studies in glioma-bearing mice, adopting the above mentioned strategies for delivering tumor antigens to dendritic cells, showed significant increase in survival, comparing with control animals that received untreated normal dendritic cells (Gilboa, 1999; Liau et al., 1999; Akasaki et al., 2001; Ni et al., 2001). Differently, clinical studies on dendritic cells-based active immunotherapy in gliomas began only very recently. The first phase I clinical trial using dendritic cells-based vaccines for glioma patients was reported by Yu and collaborators (2001): the median survival for the vaccinated group was significantly higher than the controls. In a different study, it was reported a phase I trial of vaccination of glioma patients with fusion of dendritic and glioma cells, showing two partial responses without any serious adverse effects (Kikuchi et al., 2001). Yamanaka and coworkers (2003) have recently reported results of a phase I/II study where they vaccinated glioma patients with autologous tumor lysate-pulsed dendritic cells: in this case, some patients showed increased responses after vaccination. Nevertheless, these preliminary results, obtained so far, indicated that malignant gliomas can be responsive to vaccination with tumor antigen-pulsed dendritic cells and that no adverse side effects, toxicity, or evidence of autoimmunity were observed in studies in rodents or human clinical trials. Immunization strategies are likely to be most beneficial when applied to patients after surgery with minimal residual disease, as is done in the case of glioma. In most clinical studies performed thus far, the patients has undergone and failed conventional radiotherapy and cytotoxic chemotherapy. They, therefore, likely had some resultant immunosuppression. Immunotherapy may be more effective if performed with minimal cytotoxic chemotherapy and if possible, with only localized radiation therapy (Parajuli & Sloan, 2004).

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Conclusion Every year in the United States, approximately 30,000 people are diagnosed with gliomas, making this type of tumor the most common primary brain tumor (Konopka & Bonni, 2003). Astrocytomas have the propensity to infiltrate throughout the brain. This feature is present even in the low-grade tumors, making complete surgical resection impossible. Unfortunately, these tumors are largely resistant to radiation and chemotherapy. In a meta-analysis of 12 randomized clinical trials, the overall survival rate of patients with high-grade gliomas (glioblastoma multiforme and anaplastic astrocytoma) was 40% at one year and only slightly higher (46%) after combined radio- and chemotherapy (Stewart, 2002). In a recent population-based study in Switzerland, survival of patients with glioblastoma multiforme was extremely poor, with observed survival rates of 42% at six months, 18% at one year, and 3% at two years. Several therapy trials and hospital-based studies have shown that younger glioma patients (with less than 50 years of age) have a better prognosis than older patients (Ohgaki et al.,, 2004). Since the introduction of computerized tomography and magnetic resonance imaging, the incidence rates of brain tumors have been rather stable, with a tendency of higher rates in highly developed, industrialized countries. With the exception of pilocytic astrocytomas, the prognosis of glioma patients is still poor. Less than 3% of glioblastoma patients are still alive at 5 years after diagnosis, higher age being the most significant predictor of poor outcome. Several occupations, environmental carcinogens, and diet have been reported to be associated with an elevated glioma risk, but the only environmental factor unequivocally associated with an increase risk in gliomas is therapeutic X-irradiation (Ohgaki & Kleihues, 2005). Several chemotherapeutic agents have been tested for the treatment of astrocytomas, but no single agent has proven to be any more effective than the typical regimen of surgery plus radiation treatment (Konopka & Bonni, 2003). The clinical management of patients with astrocytomas has remained essentially unchanged for decades. However, a view that is widely held by neurologists, neurosurgeons, and neurobiologists is that effective treatments can be developed once the genetic and molecular factors that cause these tumors are elucidated. Not surprisingly, there has been an intense recent effort aimed at ascertain the molecular mechanisms underlying gliomagenesis. These investigations are beginning to improve our understanding of the biology of astrocytomas, in order to provide a rational framework for the development of effective interventions.

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In: Trends in Brain Cancer Research Editor: Andrew V. Yang, pp. 145-165

ISBN 1-59454-972-9 ©2006 Nova Science Publishers, Inc.

Chapter IV

Targeting the Renin-Angiotensin System and the Endothelin Axis in Human Brain Cancer Lucienne Juillerat-Jeanneret∗ University Institute of Pathology, Lausanne, Switzerland

Abstract The renin-angiotensin system (RAS) and the endothelin (ET) axis, in addition to controlling blood pressure, may be involved in cell growth and/or death in the brain. In order to question these issues in glioblastoma, we have compared the expression of the components of the RAS and ET axis in surgical specimens of human brain tumors and adjacent tissue. Human brain tumor cells or rat brain cells in culture were used to evaluate the functions of the RAS and ET axis. From these experiments, we have demonstrated that the RAS is involved in maintaining the functions of the cerebral vasculature (the blood-brain-barrier) by controlling the ratio between angiotensin (Ang) II/Ang III production, and the enzyme renin more directly in the survival of glioblastoma cells, whereas the ET axis is mainly involved in the survival of tumor cells.

Keywords: glioblastoma – renin – angiotensin – aminopeptidase A – endothelia – endothelia converting enzyme - tumor vasculature - antagonists

∗

Correspondence concerning this article should be addressed to Lucienne Juillerat-Jeanneret, University Institute of Pathology, Bugnon 25, CH1011 Lausanne, Switzerland. Phone: + 41 21 314 7173; Fax: +41 21 314 7115; e-mail: [email protected].

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Abbreviations AT1/2: Ang: AGT: RAS: ACE: APA: ET: ECE: ETA/B:

angiotensin receptors type 1 or 2 angiotensin angiotensinogen renin-angiotensin system angiotensin converting enzyme aminopeptidase A endothelin endothelin converting enzyme endothelin receptors type A or B

Introduction Glioblastoma is the most frequent malignant brain tumor in adults with an incidence of 35 new cases per 100’000 population per year. It is highly malignant and characterized by its rapid and diffusely infiltrative growth into the adjacent brain tissue, preferentially along long fiber tracts and the perivascular/subpial space, thus displaying a high migratory potential in the central nervous system. However, it does not invade the subarachnoid space and hematogenous metastasis is exceptional. Tumor cells, mostly of astrocytic origin, are surrounded by activated astrocytes, the reactive astrogliosis, which may facilitate tumor progression. Cancer progression, including in glioblastoma, requires the development of a tumor-associated vascular system, either neovascularization or co-optation of existing vessels. In many cancers tumor vasculature presents defects of vascular maturation, a loss of contractile capacity and an inadequate number of perivascular cells, resulting in increased permeability and intratumoral hydrostatic pressure, promoting further tumor proliferation, vascular dysfunction, and a poor perfusion and distribution of chemotherapeutic agents. Glioblastoma are highly vascularized brain tumors with highly abnormal vasculature presenting with glomeruloid appearance and increased permeability which are induced in the tumor by co-optation of pre-existing vessels. Therefore, glioblastoma cells are responsible for the development of this defective tumor-associated vasculature. These effects depend, in part, of the VEGF/VPF system, which is also involved in increased vascular permeability. Very recent information has suggested that anti-angiogenic treatments in glioblastoma increase tumor cell mobility and brain invasion, a major problem of these cancers, suggesting that normalization of tumor vasculature would be a more efficient therapeutic approach than antiangiogeneic approaches. The molecular mechanisms underlying the defects observed in the vasculature of tumors, in particular the loss of constrictor response to vasoactive peptides resulting in poor perfusion of the tumor and increased permeability, are presently only poorly understood. The renin-angiotensin system (RAS) and the endothelin (ET) axis are main regulators of vascular functions, and in addition may promote vascular cell migration, proliferation, differentiation and/or growth. The expression of selected RAS and ET components is either induced or decreased in cancer, according to a cell-specific and cancer-specific pattern.

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Inhibitors for the enzymes or antagonists for the receptors of the RAS and the ET axis have anti-tumor effects in human and in animal models, which have been attributed to their direct effects on tumor cells and/or on the tumor-associated vasculature. In glioblastoma our published results have mainly associated the RAS and the enzymes metabolizing angiotensins with defects of the tumor vasculature [50-54], and the ET axis with favoring survival of tumor cells and human glioblastoma progression [21, 22].

The Renin-Angiotensin System in Human Glioblastoma The functions of the renin-angiotensin system (RAS) are well-characterized in vasoconstriction, the regulation of the hydric and electrolyte balance, edema and the thickening of the vascular wall. The RAS also exerts a regulatory role in vascular cell migration, proliferation, death and/or survival functions. In cardiovascular diseases, clinical use of drugs targeting the RAS improves the conditions. These properties of the RAS make it a potential target to control the inappropriate functions of the glioblastoma-associated vasculature which include defective perfusion, edema and high tumor interstitial pressure and proliferation and recruitment of endothelial and mural cells of the tumor-associated angiogenic vessels. The human RAS, including in the brain is composed of a precursor protein, the angiotensinogen (AGT) [36, 53, 70] from which the proteases renin (EC 3.4.23.15), angiotensin converting enzyme (ACE, EC 3.4.15.1) [53], and several aminopeptidases (aminopeptidase A (APA, EC 3.4.11.7) [27, 51, 52, 53] and aminopeptidases B/N [53]) and carboxypeptidase (prolyl-carboxypeptidase (EC 3.4.21.26) [95]) sequentally release families of peptides, the angiotensins (Ang) I, II, III, IV [110, 111] and (1-7) [95], with subtle differences in biological functions, and acting on the 7-transmembrane G-protein coupled receptors (GPCR) AT1 et AT2. The RAS is expressed independently of the circulating RAS in many tissues, including the central nervous system (CNS) [34, 36, 39, 70, 105]. In the CNS, RAS function includes the regulation of cardiovascular functions [73], and also cell growth and death [55, 56]. The RAS functions are endocrine in the blood via AT1, and paracrine/autocrine in tissues other than the blood via AT1 and/or AT2 binding of angiotensin peptides locally formed depending on the relative expression of the proteases involved in their metabolism and on the relative expression of their cellular receptors. These tissue fonctions include the control of cell proliferation and death [2, 55, 56, 91]. In the normal central nervous system (CNS), the expression of renin, AGT, ACE and AT1 had been demonstrated mainly in rodents [29, 38, 90, 104, 120] (Figure 1). In normal human brain it had been shown that AGT was mainly expressed by astrocytes [36, 70], renin by some astrocytes and neurons [73]. Our results also demonstrated renin in brain macrophages [51, 53]. It is however, not clear whether renin is secreted in the brain [51, 53] whereas the secretion of’AGT by astrocytes [53] is accepted and may control angiogenesis [15]. Inhibitors/antagonists of renin, ACE, AT1 and AT2 receptors have been developed and are in clinical use or under clinical evaluation in the context of cardiovascular disorders. In

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cancer, the role of the RAS has been mainly evaluated in the context of the contractile functions of tumor vasculature, however, some information exists suggesting a role for the RAS in cancer progression and angiogenesis. The existing information concerning a potential role for the different components of the RAS in the development of cancer will be briefly reviewed. ___________________________________________________________________________ angiotensinogen (astrocytes some neurons) ⇓ renin (astrocytes and neurons) angiotensin I Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu ⇓ ACE (vasculature) angiotensin II ⇒ AT1/2 Asp-Arg-Val-Tyr-Ile-His-Pro-Phe Pro-carboxypeptidases ⇒ Asp-Arg-Val-Tyr-Ile-His-Pro (angiotensin(1-7) ⇓ aminopeptidase A (vasculature) angiotensin III Arg-Val-Tyr-Ile-His-Pro-Phe

⇒ AT1/2

⇓ aminopeptidase B (vasculature) angiotensin IV Val-Tyr-Ile-His-Pro-Phe

⇒ AT4

⇓ aminopeptidases N (vasculature) inactive fragments ___________________________________________________________________________ Figure 1: Expression of the enzymes and peptides of the RAS in normal human and rodent brain.

Angiotensinogen (AGT) In the context of the vascular system, AGT is the only known substrate of the enzyme renin, and the obligatory precursor of the angiotensin peptides. AGT belongs to the superfamily of the non-inhibitory serpins (serine protease inhibitors), which have the potential to decrease angiogenesis in several cancer models. In human cancer AGT inhibits VEGF-induced or FGF-induced angiogenesis [15]. In glioblastoma AGT is involved in the maintenance of the normal function of the cerebral vasculature [55] and in AGT-ko mice some angiotensin peptides have the potential to restore the functions of the cerebral

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vasculature [55]. We have demonstrated that human glioblastom cell lines express AGT mRNA, that AGT did not modify glioblastoma cell proliferation, and that AGT is released by tumor cells in vivo in humans [53]. Thus AGT is expressed and secreted by glioblastoma and is involved in vascular functions of brain tumors.

Renin (EC 3.4.23.15) and Renin Inhibitors In the context of the vascular system, renin has only one known substrate, AGT, of which renin releases Ang I. In human cancers renin-secreting tumors of several non-renal origins have been described, and the expression of renin has been detected in different human cancers, including glioblastoma and glioblastoma cells [5, 51, 53], however tumor cells do not secrete renin. In human glioblastoma cells, renin inhibition directly blocked glioblastoma cell proliferation, independently of extracellular production of angiotensin peptides [53]. This effect of renin was dependent on inhibition of serum-induced ERK phosphorylation [Juillerat-Jeanneret L, unpublished experiments, Figure 2] Thus the cell-proliferation inhibitory effects of renin may be mediated by ERK phosporylation.

-

+

+

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-

+

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-

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+

+ +

+ Renin inhibitor

+ 0.5% FCS p44 p42

0

5

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Figure 2: Renin inhibitors inhibit the phosphorylation of ERK induced by serum proteins in human glioblastoma cells.

Angiotensin Converting Enzyme (ACE, EC 3.4.15.1), ACE-Inhibitors and Angiotensin II (Ang II) In cancer, ACE-inhibitors are not anti-proliferative for cancer cells, but inhibit VEGF expression by cancer cells [102]. Several studies have been published linking ACE expression and polymorphism, or the use of ACE inhibitors, to cancer development or control [1, 28, 51, 63, 69, 79, 89, 117, 118]. However, no clear picture can be obtained from these studies. ACE inhibitors may slow cancer progression [63, 79, 89, 118], mainly acting via their anti-angiogenic potential and as more general zinc metalloprotease inhibitors, independently of the RAS and of ACE inhibition. ACE is highly expressed in the abnormal

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vessels of human glioblastoma, however, our own in vitro and in vivo results did not convincingly demonstrate an advantage to ACE inhibition in glioblastoma [51, 53]. In cancer, Ang II, is pro-angiogenic and promotes vascular growth. AT1 antagonists inhibit tumor growth, mediated by the blockade of Ang II-dependent tumor cells production of VEGF [91]. Ang II can also stimulate the migration of pericytes via TGF-β and PDGF receptors [77] and the expression of VEGF receptor by these cells [84, 115]. In glioblastoma, we have shown that Ang peptides do not directly induce proliferation, apoptosis and/or DNA synthesis in glioblastoma cells [53]. Therefore, Ang II seems mainly involved in promoting angiogenesis.

Aminopeptidase A (APA, EC 3.4.11.7) and Angiotensin III (Ang III) Aminopeptidase A (APA) releases Ang III from Ang II in the brain [27]. APA can control angiogenesis and tumor growth in mice [68, 102]. In glioblastoma, we have shown that APA activity is upregulated in the abnormal vasculature of gliobastoma depending on the secretion by tumor cells of a not yet identified soluble factor [51, 52]. Using in vitro 3dimensional models of cell cultures, of brain vessels and cells, we have shown that the increased APA and decreased Aminopeptidase B/aminopeptidase N activities in brain tumor vasculature is induced by glioblastoma cell-secreted factors which are not dependent on tumor cell hypoxia [52]. APA increase is inhibited by glucocorticoid or TGF-β exposure of activated brain-derived endothelial cells [51] with a concomitant induction of ACE [48, 49, 50]. Thus, glioblastoma-derived factors increase APA expression in glioblastoma vascular cells, and TGF-β decreases this expression. We have shown that APA is not involved in the proliferation of brain-derived endothelial cells and have postulated that it is related to increased vascular permeability and tumor oedema [52]. Therefore in glioblastoma, Ang III is the main peptide of the angiotensin family produced in the tumor-associated cerebral vasculature, and is involved in the edema and the increase of glioblastoma-associated vascular permeability [51], in accordance with the obseravtion that in AGT-ko mice bloodbrain barrier function is lost and can be restored by Ang II or Ang IV, but not Ang III [55, 56].

Angiotensin Receptors (AT1 and AT2) AT1 is the receptor involved in the control of vascular tone, while the role of AT2 is less well understood, and may be vasorelaxant antagonizing AT1 effects [10, 18]. In cancer, AT1 may exert growth stimulatory effects and AT1 antagonists decrease tumor proliferation [108] inhibiting VEGF [103]. In a C6-cells glioblastoma rat model, losartan, an AT1 antagonist, reduced tumor growth, vascular density, cell proliferation and mitotic index [91]. We have shown the selective expression of AT1 and AT2 in human glioblastoma [53]. AT1 blockade had no effect on glioblastoma growth, while AT2 blockade decreased growth. Therefore,

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AT1 favors glioblastoma development by increasing tumor angiogenesis, while the function of AT2 is not known. In summary, in glioblastoma, renin and AGT are expressed in tumor cells, AGT but not renin, being secreted by gliobastoma and glioblastoma cells [5, 53, 70]. AGT expression is possibly lower in tumoral than in normal astrocytes [53]. Renin expression is inhomogeneous in glioblastoma cells but generally high, as is ACE expression in glioblastoma-associated vasculature. Aminopeptidase A activity is highly increased in glioblastoma vasculature whereas Aminopeptidase B/Aminopeptidase N activity is almost suppressed [51, 52] (Figure 3). AGT or angiotensin peptides did not modify glioblastoma cell growth, however exposure of human glioblastoma cells to piperidine-type renin inhibitors [67, 110] resulted in a marked inhibition of cell growth and induction of apoptosis was observed, [53] compatible with an AT2-mediated effect [16, 66]. ___________________________________________________________________________ normal tumoral differences ___________________________________________________________________________ angiotensinogen ++ + ÈÆ ↓ renin

+

++

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angiotensin I ↓ ACE

Ð

angiotensin II ↓ aminopeptidase A

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angiotensin III ↓ aminopeptidase B

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Ï

angiotensin IV ↓ aminopeptidases N

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inactive fragments __________________________________________________________________________ È : decreased expression ; Ç : increased expression ; Æ : unmodified expression + : expressed ; - not expressed Figure 3: Comparison of the expression of the RAS in human normal brain and glioblastoma.

Therefore our results and other published information demonstrate that the RAS is involved in the survival of normal and tumor cells of the CNS not necessarily involving the binding of angiotensin peptides on their receptors, and as a regulator of the functions of cerebral vasculature including angiogenesis, in normal situation and in tumors, mediated by the overproduction of Ang III. Ang II binding to AT1 receptors on glioblastoma cells will

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result in the production of angiogenic factors, which will induce the proliferation/death, migration of vascular cells, vascular permeability defects and increased intratumoral hydrostatic pressure which are associated with brain tumors, including glioblastoma. These defects in the differentiation of tumor-associated vascular cells result in a poor intratumoral distribution of anti-cancer drugs and are associated with resistance to treatment. Increase in aminopeptidase A and decrease in aminopeptidase N activities are either causative or reactive responses to Ang II-induced secretion of glioblastoma-derived factors and may be used as reporter molecules of the tumor-associated vasculature for development of these permeability disorders whereas angiotensinogen may be beneficial.

The Endothelin System in Human Glioblastoma Endothelins (ETs) 1, 2 and 3 constitute a family of 21 amino-acid peptides, proteolytically released from precursor polypeptides (ppETs) by at least two endothelin converting enzyme (ECE) isozymes [6, 75, 98, 109, 114], ECE-1, active at neutral pH and expressed as four isoforms, ECE-1a-d, and ECE-2, active at acidic pH. ET peptides act on two distinct high-affinity receptor subtypes, ETA and ETB, presenting 55% homology and located on target cell membrane. These receptors belong to the family of seven transmembrane Gprotein-coupled receptors (GPCR). At physiological concentrations ET-1 and ET-2, but not ET-3, bind to ETA receptors (KD ET-1, ET-2 = 20-60 pM, ET-3 = 6500 pM), whereas all three ET ligands bind ETB receptors with a similar affinity (KD = 15 pM). ET-1 is ubiquitously expressed, while ET-3 is mainly restricted to the nervous system. Both ETA and ETB receptors share common signal-transduction pathways through phosphatidylinositol hydrolysis and an increase in cytosolic calcium. The two receptors can also signal through different G proteins and stimulate a variety of other effector systems such as phospholipase D and A2, PKC or the protein tyrosine kinase-MAP-kinase/ERK pathways [60, 61, 96]. The transactivation of EGF receptor by ET-1 has also been shown [13]. In addition to its potent vasoconstrictor activity, ET-1 is an autocrine/paracrine (co-)mitogen in many cell types, involving several intracellular signaling pathways (Figure 4). stroma cells (paracrine) or tumor cells (autocrine/intracrine) Ð ECE-1a-d bigET-1/2/3 Î

ET-1/2/3

NEP + others Î

Ð

inactive fragments

intracellular signaling :PKC,

ERK ETA/B

Î

tumor cell survival and growth anti-apoptotic signaling

Figure 4: The components of the endothelin axis. ET: endothelin; NEP: neutral endopeptidase 24.11; ECE: endothelin converting enzyme; PKC: protein kinase C; ERK: extracellularly regulated kinase.

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Splice variants of both receptors have been described [12, 24, 37, 62, 64, 71, 80, 86, 99, 107, 119] and mutagenesis and chimeric receptor studies have indicated that splice variants of ET receptors may have altered signal-transduction properties and binding characteristics, resulting in either increased or decreased binding affinities for receptor agonists and antagonists [62]. These variants may express conformational changes that may either allow the binding of ligands other than endothelins, or the binding of cellular proteins able to modify the biological functions of the receptors resulting in either increased or decreased binding affinities for receptor agonists and antagonists and changes in signalling pathways. Several studies have shown that the loss of ETB receptor functions plays an important role in ET-induced proliferation and cell death, as well as the activation of intracellular signal transduction pathways that regulate ET production. These effects of ETB receptor functions may be important in tumor development, both in cancer cells and cancer-associated stromal cells. The mechanisms allowing glioblastoma to progress include the potential for tumor cells to resist apoptosis and to invade the brain by displaying migratory potential. ET axis has been inlvoved in both processes, migratory potential and facilitating role in tumor progression which has been shown by our group and others in several human tumors. ET-1 is a migrationpromoting factor of vascular cells, either endothelial or smooth muscle cells, in response to stress, involving the activation of the transcription factor ets-1 and matrix collagenases [4, 42, 78, 85, 88, 93, 96]. In cancer, ET-1 was not a growth factor by itself, but proved to potentiate tumor cell growth induced by growth factors such as bFGF, EGF, IGF-I and IGF-II, through a PKC-mediated pathway, involving several intracellular signaling pathways. Several studies, including ours, have evaluated the expression (Figure 5) and functions (see below) of the endothelin system in normal and tumoral astrocytes [7, 9, 21, 23, 31, 32, 33, 40, 41, 44, 59, 60, 61, 72, 83, 92, 96, 97, 100, 101, 106, 113]. ppET-1

ECE-1

ETA

ETB

In situ histochemisty

vascular

vascular

vascular

tumor

Immunohistochemistry

vascular (+ tumor)

vascular (+ tumor)

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vascular

<

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Figure 5: Expression of the components of the endothelin axis in human glioblastoma.

In human cancers, depending on ETA/ETB expression the endothelin system may act as growth-promoting or apoptosis-promoting effector. Using ex vivo, in vivo and in vitro approaches, we have determined the expression of endothelin-1 (ET-1), endothelin converting enzyme (ECE-1a-d) and endothelin receptors (ETA/ETB) in human glioblastoma and tumor cells in culture, and have shown that endothelins are necessary survival factors, protecting tumor cells against apoptosis. In glioblastoma, the main source of endothelins was

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the tumor-associated vasculature. Functional studies have demonstrated that these these peptides were involved in the control of FasL-induced apoptosis, implying the regulation of the caspase-8 inhibitory protein FLIP, the PKC and the extracellular signal-regulated kinase ERK1/2 [21, 87] pathways. We have also observed an important induction of the ETB-type receptor in tumor stroma, mainly in reactive astrocytes and myofibroblasts ensheathing tumor neovessels [21, 22]. These results indicated that the endothelin system is important in the control of tumor cell death or survival, and in the tumor-stroma interactions. Our results also suggested that the receptors may not be expressed at the cell surface but intracellularly, and displayed modified ligand binding properties when compared to ETB-receptor expressed in non-tumoral astrocytes (Figure 6). From these informations, endothelin (ET)-1, ETA and/or ETB-receptors have been involved in proliferation and activation of normal and tumoral astrocytes. Antagonists to endothelin receptors have been developed for the treatment of cardiovascular diseases and have reached clinical use [40, 45, 46, 65, 74, 81, 82]. Unexpectedly they may also show promising effects in the context of cancer, as molecules with the potential to control tumor growth and potentiate apoptosis. Therefore we have evaluated this possibility. In vitro the concentration of the antagonists necessary to induce apoptosis or potentiate FasLigand-induced apoptosis in cancer cells were about 10-50 times higher [21, 87] (Figure 7) than the concentration effective in the context of treatment of cardiovascular diseases, ressembling the effects observed when modifications, using either mutagenesis or chimeric receptors, were introduced in the transmembrane domain 3 and cytoplasmic domain 2 of ET-receptors [62]. Therefore it may be possible that the target(s) of endothelin receptor antagonists in cancer cells are different when compared to the effects of these antagonists in vascular cells. These modifications may include splice variants, mutated receptors or additional endothelin receptors. We have therefore evaluated that possibility using RT-PCR and primers spanning the whole translated domain of ETB (Figure 8), but no ETB variant was detected.. s t r o m a l c e lls (p a r a c r in e ) b ig E T -1 E C E -1

tu m o r c e lls (a u t o c r in e /in t r a c r in e ) b ig E T - 1 E C E -1

a -d

E T -1

2 a -d

E T -1

E T A /B E T A /B

E T A /B

Figure 6: Potential paracrine, autocrine or intracrine effects of endothelin-1.

1

apoptosis index

Targeting the Renin-Angiotensin System and the Endothelin Axis… 7 6

LN18yy

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5 4 3 2 1 0 0

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bosentan [µM]

bosentan [µM]

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bosentan [µM]

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bosentan [µM]

Figure 7: Dual ETA/B antagonisms induces apoptosis in human glioblastoma cells. Translated 1

2

1

3

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3

423 bp Primers 9 and 10 1

2

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517 bp Primers 15 and 16

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458 bp Primers 17 and 18

451 bp Primers 19 and 20

1 = LN18 2 = LNZ308 3 = CHO-ET B

Figure 8: Expression of the mRNA of ETB in human glioblastoma cells using primers spanning the different domains of the receptor.

Human glioblastoma as well as human glioblastoma cells have higher expression of ECE-1 than ET-1 [11]. Therefore, another approach to achieve the blockade of ET-1 effect in cancer might be to control its activation by inhibiting ECE-1 (EC 3.4.24.71), which is widely expressed in the CNS [8, 13] and overexpressed in glioblastoma [11, 21]. ECE-1 inhibitors have been developed and evaluated in the context of cardiovascular disorders [3, 14, 17, 19, 25, 26, 30, 43, 57, 58, 76, 94, 112, 116]. To achieve ECE-1 inhibition in glioblastoma, we prepared and characterized a series of very potent, low nanomolar, non-peptidic ECE-1 inhibitors whose effects were evaluated on the proliferation of human glioblastoma cells in culture [11]. Depending on the particular ECE-1 isoform expressed or on external factors, ECE-1 may be secreted or expressed at the cell surface (ECE-1a and ECE-1c) and thus be directly accessible to inhibitors. Alternatively, ECE-1 may be intracellular (ECE-1b and ECE1d), thus necessitating that inhibitors traverse the cell membrane. The growth-inhibitory effect of ECE-1-inhibitors in human glioblastoma cells was rapid and accessible to the less hydrophilic inhibitors or the hydrophobic pro-drugs of these molecules, suggesting that inhibition of bigET-1 activation by cell-surface ECE-1 may not be the main pathway targeted by ECE-1 inhibitors in the context of cell growth. Exogenous addition of ET-1 to human glioblastoma cells did not induce cell proliferation [21], while antagonists to receptors, but only at high concentration, induced apoptosis. Addition of exogenous ET-1 together with the ECE-1 inhibitors did not reverse the growth inhibitory effect of these inhibitors in human glioblastoma cells. Therefore, our results suggest that the

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growth-regulatory role of ET-1 is intracellular, and that only cell-permeable inhibitors can achieve this role. Alternatively, other enzymes or other peptides substrates of ECE-1 [20, 35, 47] may be involved, and some of the effects of ECE inhibition may be mediated by Ang II [76]. Therefore, thiol ECE-1 inhibitors displayed growth inhibitory properties for human glioblastoma cells, involving a rapid mechanism. This effect was not linked to a deficit in extracellular production of ET-1 and a decrease of ET-1 binding to its membrane receptor(s), but may depend on either alternate substrates for ECE-1 or an intracellular role of ECE-1 and/or ET-1 in the control of cell growth. In summary, data from our groups as well as from others, indicate that the main endothelin receptor implicated in human glioblastoma cells is the ETB-receptor, possibly mutated ETB receptor(s). This receptor may selectively control the apoptosis of tumor cells or of tumor-associated cells. Results from our group and from other groups suggest that the endothelin system is a potential target to control glioblastoma progression. ETB rather than ETA, is mainly involved in the survival functions of ET-1 in glioblastoma cells [21]. However, the detailed mechanisms, in particular the characteristics of the endothelin receptor(s) involved, by which endothelins promote tumor cell survival and resistance to apoptosis are not completely defined. We can thus postulate that in cancer, ETA or/and ETB receptors display less favourable affinity constants for the antagonists because either a splice variant, a truncated or a mutated receptor may be expressed or because a new endothelin binding protein, different from the ETA and ETB receptors, may be involved, implying an intracrine function (Figure 9). Consequently, a better knowledge of the mechanisms and receptors involved in the cancer-specific functions of endothelins is necessary. In glioblastoma the functions of the ET axis for cell survival and proliferation seems both intracrine and paracrine and mediated by ETB in glioblastoma cells, the tumor vascular system being the source of ET-1, and aucrine for the tumor vascular system, mediated by ETA. Therefore dual ETA/B antagonism would be a better option for these tumors than single antagonism.

tumor cells (autocrine/intracrine) bigET-1 ECE-1 a-d

2

ET-1

ETA/B

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growth inhibition

Apoptosis induction or senstization to apoptosis

ETA/B Figure 9: Intracrine/paracrine hypothetic endothelin-1 functions in human glioblastoma cell survival.

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Conclusions In the last years, the selective expression of the components of the RAS and ET axis in human glioblastoma has been demonstrated, and some information has suggested that specific components of this system, i.e renin, angiotensinogen, Ang II, AT1 and AT2, aminopeptidase A, for the RAS, and and ECE-1 and ETB for the ET axis in glioblastoma may be important for cancer progression and for the development of disorders of cancerassociated vasculature. The treatment of human cancers, and in particular brain cancers, is limited by the systemic toxicity of chemostatic or chemotoxic anti-cancer agents and by the existence of resistance mechanisms, such as the mdr/P-glycoprotein system, that limit the possibilities to reach efficient local concentrations of the agents. The use in this context of molecules which have shown efficiency in cancer-unrelated human disorders, and which have also shown an unexpected efficiency in cancer cells and cancer-associated stromal cells, may be a way to improve cancer treatment and overcome resistance mechanisms. The therapeutic agents developed in the context of cardiovascular disorders and which target the reninangiotensin system (RAS) and the endothelin (ET) axis have shown such a potential and deserve evaluation in the field of cancer. Drugs targeting the RAS and ET axis are currently used in clinics or are at the late stage of clinical evaluation. Therefore this approach offers the advantages of getting immediate access to drugs already tested in humans, without long chemical and biological development. Patients under ACE inhibtion have a statistically diminihed risk of cancer and the blockade of the ET axis via ETA antagonism (Atrazentan) is under clinical trials for prostate cancer, and of melanoma with dual ETA/B antagonism (Bosentan) is under early clinical evaluation. However, before designing means to treat cancer by targeting the RAS and/or ET axis, it is necessary to better define the characteristics of the cell populations expressing it and to understand the exact molecules of the RAS and ET axis and the biological mechanisms behind these effects.

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[65] Liu G. Recent advances in endothelin antagonism. Ann Rev Med Chem, 8: 73-82, 2000. [66] Lucius R, Gallinat S, Rosenstiel P, Herdegen T, Sievers J, Unger T. The angiotensin II tape 2 (AT2) receptor promotes axonal regeneration in the optic nerve of adult rats. J Exp Med, 88:661-670, 1998. [67] Maerki HP, Binggeli A, Bittner B, Bohner-Lang V, Breu V, Bur D, Coassolo PH, Clozel JP, D’Arcy A, Doebeli H, Fischli W, Funk CH, Foricher J, Giller T, Gruninger F, Guenzi A, Guller R, Hartung T, Hirth G, Jenny CH, Kansy M, Klinkhammer U, Lave T, Lohri B, Luft FC, Mervaala EM, Muller DN, Muller M, Montavon F, Oefner CH, Qiu C, Reichel A, Sanwald-Ducray P, Scalone M, Schleimer M, Schmid R, Stadler H., Treiber A, Valdenaire O, Vieira E, Waldmeier P, Wiegand-Chou R, Wilhelm M, Wostl W, Zell M, Zell R. Piperidine renin inhibitors:from leads to drug candidates. Il Farmaco, 56: 21-27, 2001. [68] Marchio S, Lahdenranta J, Schlingenmann RO, Valdembri D, Wesseling P, Arap MA, Hajitou A, Ozawa MG, Trepel M, Giordano RJ, Nanus DM, Dijkman HBPM, Oosterwijk E, Sidman RL, Cooper MD, Bussolino F, Pasqualini R, Arap W. Aminopeptidase A is a functional target in angiogenic blood vessels. Cancer Cell, 5: 151-162, 2004. [69] Medeiros R, Vasconcelos A, Costa S, Pinto D, Lobo F, Morais A, Oliveira J, Lopes C. Linkage of angiotensin I-converting enzyme gene insertion/deletion polymorphism to the progression of human prostate cancer. J Pathol, 202:330-335, 2004. [70] Milsted A, Barna BP, Ransohoff RM, Brosnihan KB, Ferrario CM. - Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA. Proc Nat Acad Sci, USA, 87: 5720-5723, 1990. [71] Miyamoto Y, Yoshimasa T, Arai H, Takaya K, Ogawa Y, Itoh H, Nakao K. Alternative RNA splicing of the human endothelin-A receptor generates multiple transcripts. Biochem J, 313:795-801, 1996. [72] Morga E, Faber C, Heuschling P. Stimulation of endothelin B receptor modulates the inflammatory activation of rat astrocytes. J Neurochem, 74:603-612, 2000. [73] Morimoto S, Cassell MD, Sigmund CD. Glia- and neuron-specific expression of the renin-angiotensin system in brain alters blood pressure, water intake, and salt preference. J Biol Chem, 277: 33235-33241, 2002. [74] Morris CD,. Rose A, Curwen J, Hughes AM, Wilson DJ, Webb DJ. Specific inhibition of the endothelin A receptor with ZD4054: clinical and pre-clinical evidence. Br J Cancer. 92:2148-52, 2005. [75] Muller L, Barrett A, Etienne E, Meidan R, Valdenaire O, Corvol P, Tougard C. Heterodimerization of endothelin–converting-enzyme-1 isoforms regulates the subcellular distribution of this metalloprotease. J Biol Chem, 278: 545-555, 2003. [76] Muller DN, Mullally A, Dechend R, Park JK, Fiebeler A, Pilz B, Löffler BM, BlumKaelin D, Masur S, Dehmlow H, Aebi JD, Haller H, Luft FC. Endothelin-converting enzyme inhibition ameliorates angiotensin II-induced cardiac damage. Hypertension, 40: 840-846, 2002

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[94] Sansom CE, Hoang MV, Turner AJ. Molecular modeling and site-directed mutagenesis of the active site of endothelin converting enzyme. Prot Engeen, 11: 1235-1241, 1998. [95] Santos RA, Campagnole-Santos MJ, Andrade ESP. Angiotensin-(1-7): an update. Reg Peptides, 91: 45-62, 2000. [96] Sasaki Y, Hori S, Oda K, Okada T, Takimoto M. Both ETA and ETB receptors are involved in mitogen-activated protein kinase activation and DNA synthesis of astrocytes: study using ETB receptor-deficient rats (aganglionosis rats). Eur J Neurosci, 10:2984-2993, 1998. [97] Schmitt-Ott KM, Xu AD, Tuschick S, Liefeldt L, Kresse W, Verkhratsky A, Kettenmann H, Paul M. Hypoxia reverses dibutyryl-cAMP-induced stellation of cultured astrocytes via activation of the endothelin system. FASEB J, 15:1227-1229, 2001. [98] Schweizer A, Valdenaire O, Nelböck P, Deuschle U, Dumas M, Edwards JB,; Stumpf JG. Löffler BM. Human endothelin-converting enzyme (ECE-1): three isoforms with distinct subcellular localizations. Biochem J, 328: 871-878, 1997. [99] Shyamala V, Moulthrop TH, Stratton-Thomas J, Tekamp-Olsson P. Two distinct human endothelin B receptors generated by alternative splicing form a single gene. Cell Mol Biol Res, 40:285-296, 1994. [100] Sone M, Takahashi K, Totsune K, Murakami O, Arihara Z, Satoh F, Mouri T, Shibahara S. Expression of endothelin-1 and endothelin receptors in cultured human glioblastoma cells. J Cardiovasc Pharm, 36:S390-392, 2000. [101] Stiles JD, Ostrow PT, Balos LL, Greenberg SJ, Plunkett R, Grand W, Heffner RR. Correlation of endothelin-1 and transforming growth factor beta with malignancy and vascularity in human gliomas. J Neuropath Exp Neurol, 56:435-439, 1997. [102] Suganuma T, Ino K, Shibata K, Nomura S, Kajiyama H, Kikkawa F, Tsuruoka N, Mizutani S. Regulation of aminopeptidase A expression in cervical carcinoma: role of tumor-stromal interaction and vascular endothelial growth factor. Lab Invest 84:639648, 2004. [103] Suganuma T, Ino K, Shibata K, Kajiyama H, Nagasaka T, Mizutani S, Kikkawa F. Functional expression of the angiotensin II type 1 receptor in human ovarian carcinoma cells and its blockade therapy resulting in suppression of tumor invasion, angiogenesis, and peritoneal dissemination. Clin Cancer Res, 11:2686-2694, 2005 [104] Sumners C, Myers LM Kalberg CJ, Raizada MK. Physiological and pharmacological comparisons of angiotensin II receptors in neuronal and astrocyte glial cultures. Progress Neurobiol, 34:355-385, 1990. [105] Tahmasebi M, Puddefoot JR, Inwang ER, Goode AW, Carpenter R, Vinson GP. Transcription of the prorenin gene in normal and diseased breast. Eur J Cancer 34:1777-1782, 1998. [106] Teixeira A, Chaverot N, Strosberg AD, Cazaubon S. Differential regulation of cyclin D1 and D3 expression in the control of astrocyte proliferation induced by endothelin-1. J Neurochem, 74:1034-1040, 2000. [107] Tsutsumi M, Liang G, Jones PA. Novel endothelin B receptor transcripts with the potential of generating a new receptor. Gene, 228:43-49, 1999.

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In: Trends in Brain Cancer Research Editor: Andrew V. Yang, pp. 167-191

ISBN 1-59454-972-9 ©2006 Nova Science Publishers, Inc.

Chapter V

Central Nervous System Lymphoma Andrew Lister1, Lauren E. Abrey2 and John T. Sandlund3 1

St. Bartholomew’s Hospital, West Smithfield, London, United Kingdom, 2 Memorial Sloan-Kettering Cancer Center, New York, NY 3 St. Jude Children’s Research Hospital, Memphis, TN

Abstract Central nervous system involvement with malignant lymphoma whether primary or secondary is an uncommon but not rare complication observed in the management of patients with hematological malignancy. Its importance lies in the considerable morbidity and mortality with which it is associated and the inadequacy of therapy. In Section I, Dr. Lauren Abrey addresses the totality of the problem of primary central nervous system lymphoma, with emphasis on strategies increasingly dependent on systemic chemotherapy. In Section II, Dr. John Sandlund reviews the success of sequential clinical trials of overall therapy for acute lymphoblastic leukemia in child-hood, identifying those patients at high risk of central nervous system leukemia and the development of a rational therapeutic strategy for prevention. In Section III, Dr. Andrew Lister discusses the issue of secondary central nervous system involvement with lymphoma and the indications for prophylaxis.

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I. Primary Central Nervous System Lymphoma Lauren E. Abrey ∗ Primary central nervous system lymphoma (PCNSL) is a rare form of non-Hodgkin’s lymphoma (NHL) arising within and confined to the CNS. It was first described by Bailey[1] in 1929 as a perithelial sarcoma. Subsequent classifications have included reticulum cell sarcoma and microglioma. Improvements in histopathology and immunohistochemical techniques definitively established the lymphoid nature of PCNSL. PCNSL is of particular interest for several reasons. First, this tumor has increased in incidence over the past several decades. Therefore, although it remains relatively rare, it is an increasingly important differential diagnosis of intracranial mass lesions. Second, unlike many primary brain tumors, PCNSL is very responsive to treatment, and aggressive management may lead to prolonged remission or cure. Finally, the long-term consequences of aggressive therapy may result in significant neurologic dysfunction.

Epidemiology PCNSL accounts for approximately 1% of all primary brain tumors in large autopsybased series. More recent data suggest that the incidence among immunocompetent patients in the United States is increasing. Data from the National Cancer Institute Surveillance, Epidemiology, and End Result (SEER) database found a threefold increase in PCNSL between 1973-1975 and 1982-1984. Further analysis found a tenfold or greater increase between 1973 and 1992. The incidence of ocular lymphoma has similarly in-creased by 1 .5-fold. There has been a parallel rise in the incidence of all extranodal lymphomas, but the increase has been disproportionate in the brain and eye. This in-creased incidence is not explained by advances in neuroimaging or tumor diagnosis. As PCNSL primarily affects individuals age 60 and older, one possible explanation would be the general aging of the population; however, the data indicate an increase across all age groups. PCNSL is diagnosed in 1.6% to 9.0% of the human immunodeficiency virus (HIV)infected population[2, 3] and is the second most common intracranial mass lesion. Prior to the introduction of highly active antiretroviral therapy (HAART), the incidence of PCNSL in the HIV-infected population was continuing to rise. However, the impact of these new drug regimens on the CD4 count may result in a decline in PCNSL, as the susceptibility to PCNSL is inversely proportional to the CD4 count.[4]

∗

Correspondence concerning this section should be addressed to Dr. Lauren E. Abrey, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021

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Pathology Grossly, PCNSL is a soft, granular, ill-defined lesion. Associated necrosis, hemorrhage, and neovascularity are uncommon except in AIDS-related PCNSL. Microscopically, PCNSL is a diffuse lesion with an angiocentric growth pattern; some tumors may even invade the blood vessel wall. In addition to malignant lymphocytes, there are varying numbers of small, benign, reactive T lymphocytes infiltrating the tumor, and reactive astrocytes are common. Malignant lymphocytes freely invade normal surrounding brain, and autopsy studies have demonstrated widespread infiltration of normal brain. Immunohistochemical stains are extremely useful in differentiating PCNSL from highgrade glioma and metastatic carcinoma. Leukocyte common antigen clearly identifies the malignant cells as white blood cells but may be negative in a small number of PCNSLs. Ninety percent or more are B-cell lymphomas (CD20+), usually of diffuse large-cell, largecell immunoblastic, or lymphoblastic subtype. These tumors can be identified by the immunohistochemical B-cell marker L26. The reactive infiltrating cells are typically T lymphocytes, although primary T-cell lymphomas (CD3+, CD45RO+) are reported. Histologically, PCNSL is indistinguishable from systemic NHL. Biologically, PCNSL behaves in an aggressive fashion, and it should be considered a high-grade tumor. Genetically, PCNSL has been found to demonstrate clonal abnormalities of chromosomes 1, 6, 7, and 14, identical to those detected in systemic NHL.[5] Analysis of cell surface markers including NCAM and integrins is also identical to that of systemic lymphoma. Kumanishi et al found p15 and p16 deletions in 4 out of 5 PCNSL tumors.[6]

Pathogenesis The pathogenesis of PCNSL in immunocompetent patients is unknown. T lymphocytes normally traffic in and out of the CNS; however, there is no normal traffic of B lymphocytes. Therefore, several different hypotheses have been proposed. There are no data to support or disprove any of these potential mechanisms. PCNSL may arise from a systemic lymphoma that seeds multiple organs, including the brain. The immune system has the capacity to find and eliminate the systemic tumor, but the brain, an immunologically privileged site, gives sanctuary to the malignant lymphocytes, thereby allowing tumor development. This seems unlikely, as there is no evidence of concomitant lymphoma in other immunologically privileged sites, such as the testes, concomitant with PCNSL. Another theory is that lymphocytes become trapped in the CNS after an inflammatory process and then undergo malignant transformation. However, inflammatory diseases almost exclusively attract T lymphocytes, and PCNSL is usually of B-cell origin. Also, the incidence of PCNSL is not increased in patients with inflammatory CNS diseases.

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Clinical Features and Evaluation (Table 1) The typical patient is between 55 and 70 years old; most have had symptoms for only a few weeks prior to seeking medical attention. Cognitive and personality changes are the most common initial symptoms, reflecting the predilection of PCNSL to involve the frontal lobes, corpus callosum, and deep periventricular structures. PCNSL is multifocal in approximately one third of patients and may present with any focal neurologic finding, such as hemiparesis or aphasia. Seizures are a presenting complaint in about 10% of patients, less frequent than glioma or brain metastasis. Age less than 60 and an excellent performance status are the most important prognostic factors. In AIDS-related PCNSL, the typical patient is younger (30-40 years old), and seizures are more common (25%). The median latency from HIV diagnosis is approximately 5 years. Some studies have found a higher incidence of multiple lesions in AIDS-related PCNSL; however, multifocal lesions in AIDS patients may have different etiologies. More than 40% of patients have evidence of leptomeningeal dissemination, but concomitant clinical findings are uncommon. Primary leptomeningeal lymphoma is rare and typically presents increased intracranial pressure, multifocal cranial neuropathies, or multilevel root involvement. Cerebrospinal fluid (CSF) should be obtained in all newly diagnosed patients. CSF evidence of PCNSL may also be a poor prognostic indicator. Tumor markers, including lactate dehydrogenase isoenzymes, â-glucuronidase, and â2microglobulin, may provide circumstantial evidence of leptomeningeal lymphoma. Immunocytochemical analysis and detection of immunoglobulin gene rearrangements by polymerase chain reaction have been used in the diagnosis of lymphomatous meningitis when routine cytologic evaluation is in-conclusive. Table 1. Initial evaluation for primary central nervous system lymphoma (PCNSL). Gadolinium-enhanced cranial MRI scan CSF cytology Ophthalmologic examination, including slit lamp HIV serology CT scan of chest, abdomen, and pelvis Bone marrow biopsy Gadolinium-enhanced spinal MRI, if spinal symptoms are present MRI, magnetic resonance imaging; CSF, cerebrospinal fluid; CT, computed tomography.

About 15% of patients with PCNSL have ocular disease at presentation, while 50% to 80% of patients with isolated ocular lymphoma go on to develop parenchymal brain lymphoma. Ocular symptoms include blurred, cloudy vision, decreased visual acuity, or “floaters,” but as many as half of affected patients are asymptomatic. Complete ophthalmologic evaluation, including slit lamp examination, is recommended in all patients. Diagnosis is often delayed in patients with isolated ocular lymphoma because of misdiagnosis as chronic vitreitis or uveitis.

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Systemic lymphoma is an uncommon finding in PCNSL, and there is disagreement as to whether a comprehensive systemic extent of disease evaluation is needed. In a series from the Mayo Clinic, approximately 2% to 3% of PCNSL patients were found to have systemic lymphoma on an abdominopelvic computed tomography (CT) scan or bone marrow biopsy. The optimal neuroimaging of PCNSL is gadolinium-enhanced magnetic resonance (MR) scanning. Most lesions are supratentorial and periventricular, often involving deep structures such as the corpus callosum and basal ganglia. Lesions may be hypo- or hyperintense on pre-contrast T1 imaging. Dense, homogeneous contrast enhancement is seen in immunocompetent patients but may be irregular and heterogeneous in AIDS-related PCNSL. Peritumoral edema and local mass effect are often less than expected with intracranial lesions of other etiologies. Calcification, hemorrhage, or cyst formation is rare.

Treatment Surgery The role of surgery is to establish a histopathologic diagnosis; therefore, a stereotactic needle biopsy is the procedure of choice. Aggressive resection does not improve survival and may result in neurologic deterioration. Corticosteroids Corticosteroids are used empirically in the treatment of vasogenic edema caused by any intracranial mass. In PCNSL, corticosteroids also have a potent oncolytic effect, causing tumor cell lysis and radiographic regression in up to 40% of patients.[7] The onset of action is quite rapid, with resolution of symptoms and marked reduction in tumor size within 24 to 48 hours. This can be problematic if a tissue diagnosis has not been obtained. Therefore, steroids should be withheld in any patient with a presumptive diagnosis of PCNSL until stereotactic biopsy has been performed. Radiotherapy PCNSL is a radiosensitive tumor, and whole-brain radiotherapy (RT) was the standard treatment for many years. Whole-brain RT is necessary because of the diffuse infiltrative nature of this neoplasm and results in median survivals ranging from 10 to 18 months.[8, 9] Craniospinal RT does not confer any additional survival benefit and is associated with significant morbidity, limiting the administration of subsequent chemotherapy. The optimal dose of whole-brain RT remains controversial, but the results of several studies suggest a dose between 40-50 Gy. The addition of a boost does not improve local tumor control or survival. In patients with evidence of ocular lymphoma, the posterior two thirds of the globe should be radiated to a dose of 36-40 Gy. Treatment planning should take into account both intracranial and ocular disease to eliminate overlapping fields and to minimize any toxicity to the optic nerve and retina.

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Chemotherapy The use of chemotherapy has significantly improved the treatment of PCNSL. However, the standard regimens (CHOP, MACOP-B) used in the treatment of systemic lymphoma are not effective in PCNSL because of their inability to penetrate the blood-brain barrier. High-dose methotrexate (MTX) is the single most active agent in the treatment of PCNSL. While standard-dose MTX does not cross the blood-brain barrier, doses ~ 1 g/m2 result in tumoricidal levels in the brain and doses ~ 3.5 g/m2 yield tumoricidal levels in the CSF. Therefore, most treatment regimens now incorporate high-dose MTX (1 to 8 g/m2) alone or in combination with other chemotherapeutic agents followed by whole-brain RT. This combined-modality approach has resulted in response rates approaching 100% and median survivals ranging between 30 and 60 months (Table 2).[10-14] Table 2. Chemotherapy regimens for PCNSL. Ref 35

Type Series

N 10

Regimen DHAP

RT +/–

36

Series

10

PCV

+

11

Series

13

+

10

Series

25

37

Series

74

38

Series

31

23

Phase II

14

39

19

40

Prospectiv e Series

MTX 3.5 g/m2 MTX 3.5 g/m2 MTX BBBD MTX 1 g/m2 MTV IT Ara-C BOMES

41

Phase II

102

14

Prospectiv e

52

19

MTX-based 3.5-8 g/m2 MPV Ara-C MPV Ara-C

+ – + – + – + +/–

Result 70% response 40% prolonged remission 100% response 30-mo median survival 92% response 9+- mo median survival 88% response 33-mo median survival 65% complete response 40.7-mo median survival 64% response 41-mo median survival 100% response 16.5-mo median PFS 84% response rate 6-mo median PFS 94% response rate 94% response rate 30+-mo median survival 60-mo median survival

Other 4 newly diagnosed, 6 recurrent Several did not receive RT PCV given post-RT 1 pt received carmustine Survival up to 54+ mos 59% relapse rate

68.8% alive at 54 mos 2 pts with severe leukoencephalopathy 5 pts with concurrent systemic lymphoma

22 older pts did not receive RT

PCNSL, primary central nervous system lymphoma; DHAP, dexamethasone, high-dose cytarabine, and cisplatin; PCV, procarbazine, CCNU, and vincristine; MTX, methotrexate; BBBD, blood-brainbarrier disruption; MTV, methotrexate, thiotepa, and vincristine; IT, intrathecal; Ara-C, cytarabine; BOMES, BCNU, vincristine, methotrexate, etoposide, and methylprednisolone; MPV, methotrexate, procarbazine, and vincristine; RT, whole-brain radiotherapy; mo, months; PFS, progression-free survival; pt, patient. Adapted with permission from Abrey LE, Primary central nervous system lymphoma.The Neurologist. 2000;6:245-254.

There has been increasing interest in using chemotherapy alone in order to minimize long-term effects of treatment. One approach has been to employ hyperosmolar agents to disrupt the blood-brain barrier, followed by intra-arterial MTX.[13] This technique results in

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similar overall response and survival rates as the combined-modality approach; however, the acute toxicities are more significant and include focal seizures, cerebral ischemia, cerebral edema, and local arterial trauma. Careful neuropsychological testing of this patient cohort has been performed and indicates that patients who remain in remission are not at increased risk for delayed neurotoxicity. In our experience, it is possible to treat older patients with MTX-based chemotherapy alone and achieve similar results as those achieved using combined-modality treatment in older patients. Both groups have a median survival of 32-33 months;[14] the difference is that patients treated with chemotherapy alone are more likely to relapse early, while patients treated with combined-modality therapy are more likely to develop delayed neurotoxicity. Importantly, older patients are able to tolerate aggressive chemotherapy without any excess acute morbidity. High-dose chemotherapy with autologous peripheral blood stem cell support has been used a strategy to dose intensify chemotherapy given to patients with primary CNS lymphoma. Theoretically the administration of high dose consolidation chemotherapy can be used in place of standard cranial radiotherapy in an effort to avoid treatment-related neurotoxicity. There have been two small trials for newly diagnosed patients and the preliminary results indicate that this strategy is feasible.[1, 2] Further studies will be needed to identify the optimal induction and high dose chemotherapy regimens.[15, 16] AIDS-Related The treatment of AIDS-related PCNSL is dictated in large part by the clinical condition of the patient. One of the most critical factors is making a definitive diagnosis early, as delay may result in significant neurologic deterioration, precluding the ability to tolerate aggressive treatment. Small series suggest that individual patients may benefit from aggressive combinedmodality therapy.[17-20] HAART was reported to cause a 26-month remission in 1 AIDS patient with PCNSL and may represent an important new treatment alternative.[21] Ocular Lymphoma There is no standard approach to isolated ocular lymphoma. Ocular lymphoma is exquisitely sensitive to corticosteroids (including topical ophthalmic preparations) and focal RT. Unfortunately, in most patients the disease will recur either in the eyes or in the brain, at which time the disease may be more refractory to therapeutic intervention. Systemic administration of MTX and cytarabine can yield therapeutic levels of drug in the intraocular fluids, and clinical responses have been documented; however, relapse is common.[22-24] Therefore, our current approach is to treat isolated ocular lymphoma with combined-modality therapy.[25, 26] Direct intravitreal administration of chemotherapy is being explored as a therapeutic alternative.

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Relapse The risk of relapse for patients treated with combined-modality therapy is about 50%. Most recurrences are observed within 2 years of completing initial therapy, but relapses have been seen as late as 5 years. Patients with ocular or leptomeningeal disease at diagnosis have a higher likelihood of recurrence. Relapse primarily occurs in the brain at either the original or distant sites; however, leptomeningeal and ocular relapses are seen, and systemic relapse has been reported to account for as much as 10%. The prognosis at relapse is generally poor, but further treatment often results in transient remission. Pro-longed survival is possible, and some patients continue to be sensitive to salvage therapy despite multiple re-lapses. Success has been reported using high-dose MTX (even in patients previously treated with MTX), high-dose cytarabine, PCV (procarbazine, lomustine, and vincristine), and high-dose cyclophosphamide. RT is particularly effective for ocular relapse. Intensive chemotherapy with autologous peripheral blood stem cell sup-port is standard therapy for patients with relapsed, chemosensitive, systemic NHL; this strategy has been used with some success for relapsed PCNSL.[3] How-ever, patients previously treated with whole brain radiotherapy have a higher risk of neurologic toxicity.[27]

Treatment-Related Neurotoxicity The most significant consequence of aggressive combined-modality therapy utilizing MTX followed by cranial RT is delayed neurologic toxicity. Older patients are at particularly high risk of developing a progressive neurological syndrome characterized by dementia, gait ataxia, and urinary dysfunction. Up to 90% of patients over 60 who survive 1 year after completion of treatment will be affected.[28] Patients usually become symptomatic within 1 year of treatment, with a significant decline in their performance status necessitating constant supervision and custodial care. Attempts to treat delayed neurotoxicity have been generally unrewarding, although a subset of patients may have transient improvement following placement of a ventriculoperitoneal shunt.[29, 30] Other agents, such as methylphenidate, have been utilized with success in individual patients. Delayed treatment-related cerebrovascular disease has been observed in younger patients 7-10 years after completion of therapy.[31, 32] This has been observed in isolation or in conjunction with a progressive leukoencephalopathy. Accelerated atherosclerosis is a known complication of cranial RT that typically develops 10 to 20 years after treatment.[33, 34] Stroke-like episodes have been re-ported acutely in children receiving high-dose MTX, but these typically occur days to weeks after chemotherapy and resolve spontaneously. It is also possible that PCNSL may predispose patients to cerebrovascular damage if lysis of angiocentric tumor cells damages neighboring endothelium.

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Lymphomatous Meningitis: The Acute Lymphoblastic Leukemia Model John T. Sandlund∗ CNS involvement among children with acute lymphoblastic leukemia (ALL) has historically been defined at most institutions by either the presence of at least 5 leukocytes per microliter of cerebrospinal fluid (CSF) associated with the presence of leukemic blasts (identified on a cytocentrifuged preparation) or the presence of a cranial nerve palsy on physical examination.[1] Therapeutic approaches for both CNS prophylaxis and therapy have included the following: (1) intrathecal administration of chemotherapy ranging from singleagent MTX to triple-agent intrathecal therapy consisting of MTX, hydrocortisone, and cytarabine; (2) cranial irradiation; and (3) the systemic administration of chemotherapeutic agents with good CNS penetration (e.g., high-dose MTX, high-dose cytarabine, and dexamethasone). De-spite these measures, there are patients who have been shown to still be at increased risk for CNS treatment failure.

CNS Status Refinement Mahmoud et al2 challenged the conventional definition by showing that the presence of leukemic blast cells in the CSF, regardless of cell count, increased the risk of CNS relapse. In that study, all 351 children with newly diagnosed ALL were entered on a randomized trial in which each patient received intrathecal therapy throughout the first year. Patients who were considered at in-creased risk for treatment failure because of their clinical or cytogenetic features also received 1 8-Gy cranial irradiation and intrathecal chemotherapy 1 year from the remission date. Those with CNS disease at diagnosis (as defined by at least 5 leukocytes/microliter of CSF with leukemic blasts on a cytocentrifuged prep or by the presence of cranial nerve palsy on physical examination) received 24-Gy cranial irradiation and additional intrathecal chemotherapy. Patients were classified retrospectively into 3 CNS groups based on the CSF findings: 291 patients had CNS-1 status (no blasts in the CSF), 42 had CNS-2 status (blasts present with fewer than 5 leukocytes/microliter), and 18 had CNS-3 status (5 or more leukocytes/microliter of CSF with leukemic blasts on a cytospin sample or cranial nerve palsy). The probability of an isolated CNS relapse in patients with CNS-2 status was higher than in those with CNS-1 status but was not different from that of patients with CNS-3 status. All CNS relapses occurred during the first year of treatment, before scheduled cranial irradiation. In a multivariate analysis, CNS-2 status was independently related to the risk of an isolated CNS relapse, suggesting that these patients require intensification of CNSdirected treatment early in the course of therapy. While a study of the former Pediatric ∗

Correspondence concerning this section should be addressed to Dr. John T. Sandlund, 3St. Jude Children’s Research Hospital, 332 N. Lauderdale, Box 318, Memphis, TN 38101-0318 Supported by grant CA2 1765 from the National Cancer Institute and by the American Lebanese Syrian Associated Charities (ALSAC).

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Oncology Group confirmed this result,[3] studies by the former Children’s Cancer Group and the Dutch Childhood Leukemia Study Group did not find a significant difference in outcome between patients with or without a lower number of blasts in the CSF.[4, 5] These seemingly conflicting results may reflect differences in therapy.

Traumatic Lumbar Punctures Gajjar et al.[6] performed a single-institution retrospective study of children with newly diagnosed ALL in which they demonstrated that a traumatic lumbar puncture (LP) at diagnosis adversely affected outcome. In this study, 546 children were treated on 2 consecutive St. Jude trials in which 2 sequential LPs were performed at presentation—the first for diagnosis and the second for instillation of the first intrathecal chemotherapy treatment, generally 1 to 2 days later. It was demonstrated that patients with 1 CSF sample contaminated with blast cells had an inferior event-free survival compared to those with CNS-1 status (P = 0.026). The prognosis for those with 2 consecutive contaminated CSF samples had a particularly poor treatment result (5-year event-free survival = 46% ± 9%); this feature was shown to be the strongest prognostic indicator in a Cox multiple regression analysis, with a hazard ratio of 2.39 (95% confidence interval, 1.36-4.20). It was concluded from this study that contamination of the CSF with circulating leukemic blasts adversely influences treatment outcome and is an indication for early intensification of intrathecal chemotherapy administration. This result was recently con-firmed by the investigators of the Berlin-Frankfurt-Münster group (BFM; M Schrappe, personal communication). A recent study by Howard et al (unpublished data) examined risk factors associated with the occurrence of traumatic (at least 10 red blood cells per microliter) and/ or bloody (at least 500 red blood cells per microliter) LPs. Risk factors associated with traumatic or bloody taps included the following: (1) age less than 1 year; (2) black race; (3) early treatment era during which sedation was used very seldom; (4) a platelet count less than 100 × 109/L; (5) a short (1 day) time interval since the previous LP; and (6) a less experienced practitioner. On the basis of these findings, the investigators recommended that diagnostic LPs in newly diagnosed patients with ALL should be performed by an experienced practitioner, in a dedicated procedure area with general anesthesia, following platelet transfusion if the platelet count is less than 100 × 109/L and circulating blasts are present. Using this approach, we have already substantially reduced the rate of traumatic LP with blasts from 11% to 4% to date.

Impact of Intensified CNS Therapy/Prophylaxis In a St. Jude Children’s Research Hospital study per-formed by Pui et al,[7] it was demonstrated that early intensification of intrathecal chemotherapy used in the context of the Total Therapy Study XIII virtually eliminates CNS relapse in children with ALL. Children with any amount of leukemic blasts in the CSF, whether or not the CSF blasts were introduced iatrogenically to the CSF because of a traumatic LP and regardless of the presence

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or absence of other high-risk clinical features, received additional doses of intrathecal chemotherapy (MTX, hydrocortisone, and cytarabine) during induction and throughout the first year of continuation therapy. Cranial irradiation at 18 Gy, given during weeks 56 to 59 of the continuation phase, was reserved for only those with certain high-risk features: B-cell progenitor phenotype with a leukocyte count of at least 100 × 109/L, T - cell phenotype with a leukocyte count of at least 50 × 109/L, or a karyotype with the Philadelphia chromosome. The 5-year cumulative risk of an isolated CNS relapse among the 165 patients studied was 1.2% (95% confidence interval, 0%-2.9%), whereas the risk of any CNS relapse was 3.2% (95% confidence interval, 0.4%-6.0%). It appears from this study that early intensification of intrathecal chemotherapy administration may reduce or eliminate the occurrence of CNS relapse associated with the above-mentioned risk factors (i.e., CNS-2 status at diagnosis, and traumatic or bloody LP at diagnosis). A similar result was obtained in a subsequent St. Jude clinical trial (XIIIB) (unpublished data).

Trend Toward Reducing Use of Radiotherapy for CNS Disease/Prophylaxis Most clinical trials limit the use of cranial irradiation to 5% to 10% of patients at high risk of CNS relapse, in large part because of the concern of late sequelae such as second cancer, endocrinopathy, and neuropsychologic defects. Moreover, in some protocols, cranial irradiation is given at a reduced dose. For example, the BFM has reduced the dose of prophylactic cranial irradiation to 12 Gy and the dose of therapeutic cranial irradiation for those with overt CNS disease to 18 Gy.[8] Other trials, which have eliminated cranial irradiation in all patients, have not observed an excessive rate of relapse.[9-11] The elimination of cranial irradiation is also being studied in our current St. Jude trial. Thus far, no CNS relapse has been observed among 150 patients treated with median follow-up of 2 years (unpublished data).

CNS Disease in Pediatric NHL Children with NHL are considered to have CNS involvement if lymphoma cells are identified in the CSF on a cytocentrifuged preparation or if a cranial nerve palsy is identified in a physical exam. [12] These criteria are similar to those used for children with ALL, although there are some differences. For example, children with Burkitt’s lymphoma who have any classic L3 blasts in the CSF would be considered to have CNS disease, even if there were fewer than 5 white cells per microliter in the unspun CSF. In a single-institution study of 445 children with newly diagnosed NHL, 36 (8%) were found to have CNS disease.[13] Among these, 23 had morphologically identifiable lymphoma cells in the CSF, 9 had cranial nerve palsies, and 4 had both features. CNS disease at diagnosis was identified in 13%, 7%, and 1% of Burkitt’s, lymphoblastic, and large-cell lymphoma cases, respectively. In a multivariate analysis of various risk factors, including CNS disease, stage, and LDH, only stage and serum LDH had prognostic significance.

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Among patients with Burkitt’s lymphoma, a multivariate analysis demonstrated that only serum LDH had independent prognostic significance. This review therefore suggested that CNS disease per se was not an independent risk factor. Other studies have made similar observations.[14, 15] How-ever, in a retrospective study performed by the CCG, [16] it was concluded that among patients with Burkitt’s lymphoma, the presence of meningeal disease or CNS parenchymal masses at diagnosis was associated with a nominally worse outcome independent of initial bone marrow status and LDH level, although this effect was not statistically significant. In the recently published result of the French LMB-89 study for children with B-cell lymphoma and L3 leukemia, CNS involvement was the only adverse prognostic factor identified among group C patients.[17] The modalities used for both CNS prophylaxis and treatment of overt CNS disease are similar to those used for children with ALL. They include high-dose systemic chemotherapy (e.g., MTX, cytarabine), intrathecal instillation of chemotherapy (e.g., single-agent MTX, triple-agent therapy [MTX, hydrocortisone, and cytarabine]), and, less frequently, cranial irradiation. The implementation of these approaches does vary with respect to histologic subtype. 1. Burkitt’s lymphoma. Most centers currently use systemic high-dose MTX, and cytarabine and intrathecal MTX, hydrocortisone, and cytarabine for both CNS prophylaxis and treatment. Two of the most successful treatment regimens are the French LMB-89 [17] regimen and the German BFM-90 protocol.[18] The LMB-89 regimen incorporated cranial irradiation for those with overt CNS disease at diagnosis; however, most current regimens have excluded cranial irradiation. In fact, the current international collaborative French study has excluded cranial irradiation. In the BFM-90 regimen,[18] cranial irradiation was not incorporated; however, an intraventricular access device was used for drug delivery to the spinal fluid. In this regard, St. Jude is currently piloting a regimen in which an intraventricular access device is used in the context of LMB-89 directed systemic therapy. 2. Lymphoblastic lymphoma. Systemic and intrathecal chemotherapy is used for CNS prophylaxis and treatment. For patients with overt CNS involvement at diagnosis, many centers would consider incorporating cranial irradiation. The role of cranial irradiation for CNS prophylaxis is more controversial, although, as in the case for ALL, there is a distinct trend to move away from it. For example, in the highly effective BFM-90 regimen,[19] patients with stage III or IV disease receive 12-Gy cranial irradiation as prophylaxis; however, a subsequent study is determining the safety of its omission. Among patients with CNS-2 status at diagnosis, a current St. Jude study incorporates intensified intrathecal treatment without cranial irradiation. 3. Large-cell lymphoma. Determining the optimal approach to CNS prophylaxis and treatment for this group is somewhat problematic, in part because the large-cell lymphomas are a more heterogeneous group. Those with a B-cell immunophenotype are often treated with the same regimen used for Burkitt’s lymphoma, as described above. The majority of non-B-cell cases are anaplastic large-cell lymphomas for which a spectrum of therapeutic approaches has been reported. The BFM has had great success using a regimen derived from a Burkitt’s lymphoma strategy.[20] In

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the United States, the APO regimen has also been shown to be effective; with this approach, CNS prophylaxis includes single-agent intrathecal MTX.[21] The optimal approach to managing overt CNS disease at diagnosis is controversial, primarily because of the low frequency of this clinical presentation. 4. Primary CNS lymphoma. PCNSLs in children are very rare. Also, there is little information with respect to clinical trial data in children to guide treatment. For children who are HIV negative, most pediatric oncologists would consider intensive systemic multiagent chemotherapy, featuring agents with good CNS penetration (e.g., high-dose MTX/ cytarabine, dexamethasone); cranial radiotherapy would also be a consideration in some cases. Strategies that have been shown to be effective in adults are often used on an individual basis in children. Patients who are HIV positive and develop a PCNSL are considered to have an extremely poor prognosis. In an attempt to provide a novel curative approach, Slobod et al,[22] treated 2 HIV-positive patients who presented with primary EBVpositive CNS lymphomas with hydroxyurea. This strategy was used based on in vitro studies of an EBV-positive Burkitt’s lymphoma cell line, in which expo-sure to hydroxyurea resulted in loss of cytoplasmic EBV episomes and subsequent loss of malignant phenotype. On the basis of this observation, hydroxyurea was given to HIV-positive patients who had EBV-positive PCNSLs with objective clinical and radiographic responses, suggesting that antiviral approaches may have a role in these malignancies.

CNS Prophylaxis in Adult ALL The approaches most commonly used for CNS prophylaxis in adults are similar to those that have been used in children: (1) intrathecal therapy (e.g., MTX, cytarabine, hydrocortisone); (2) high dose systemic therapy; and (3) cranial irradiation.23 These measures have reduced the rate of CNS relapse to < 5-10% from the > 30% rate reported when no prophylaxis is provided.[23] Gökbuget and Hoelzer reviewed the published data on CNS prophylaxis and found that a combination of all three of the above mentioned approaches resulted in the lowest incidence of isolated or combined CNS relapses (5%, range of 1-12%).[23, 24] Nevertheless, the use of cranial irradiation remains controversial. In the GMALL studies, a higher rate of CNS relapses was observed when cranial irradiation was either omitted or delayed.[23, 24] However, in Kantarjian et al’s study of the Hyper-CVAD regimen, which features high-dose systemic (MTX and cytarabine) and intrathecal therapy (no cranial irradiation) for CNS prophylaxis, the CNS relapse rate was very low (4%).[26]

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Secondary Central Nervous System Lymphoma: The Case for Prophylaxis Andrew Lister∗ “Secondary” lymphomatous involvement of the CNS was first recognized in the 19th century when Murchison[1] described a tumor encroaching on the foramen magnum infiltrating the dura mater at autopsy. The problem of extradural deposits was recognized later.[2-4] By the middle of the 20th century, secondary central nervous system lymphoma (SCNSL) had been the subject of many manuscripts,[5-7] representing as closely as possible the natural history, with Sparling et al6 in 1947 reporting an autopsy incidence of only 1 in 118 cases. As the natural history of the lymphomas has been superseded by the clinical course (induced by partially successful therapy not targeting the CNS), survival of some subtypes has been prolonged. In the 1970s, incidence of SCNSL increased to approximately 10%.[8, 9] A clear clinical picture, reflecting the outcome of therapy introduced in the late 1960s and early 1970s, emerges from a number of retrospective analyses from both single institutions and groups,[10-22] in which symptomatic disease occurred in 4-29%, depending on histology and extent of disease. The commonest features were headache, cranial nerve palsies, spinal cord compression, and altered mental state and affect. These problems usually arose within the context of poorly controlled lymphoma elsewhere, although the nervous system was occasionally an isolated site of recurrence. In the large majority of cases, the diagnosis was based on the history and the finding of abnormal cells on a cytospin of CSF. There was a strong association with bone marrow involvement; a correlation was also drawn between central nervous system lymphoma (CNSL) and involvement of the testis or paranasal sinuses. Likewise, close correlation was found between histological subtype and probability of the occurrence of CNSL; it was common with lymphoblastic lymphoma and Burkitt’s and “Burkitt’slike” lymphoma, to the extent that the next generation of treatment included CNS-targeted therapy.

The Problem Today: Incidence, Risk Factors Twenty years on, the demonstration of new prognostic factors and the introduction of the International Prognostic Index (IPI) have made it possible to identify more closely those patients for whom SCNSL is a high enough risk to warrant specific prophylactic therapy. At the M.D. Anderson Hospital,[23] 24 of 605 patients with ‘large-cell’ or immunoblastic lymphoma developed CNS recurrence, with an actuarial risk at 1 year of 4.5%. In 5 cases, the recurrence was concurrent with systemic progression (within 40 days); in 7 others, it preceded systemic progression up to 6 months later. Involvement of more than 1 extranodal site and elevated LDH at presentation were both independently predictive of ∗

Correspondence concerning this section should be addressed to Dr. Andrew Lister, MD 1St. Bartholomew’s Hospital, 45 Little Britain, West Smithfield, EC1A 7BE London, United Kingdom.

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CNS recurrence on multivariate analysis: if both were present, the actuarial risk was almost 20% at 1 year (Figure1). However, despite intervention, with some apparent early benefit, only 1 of 24 patients was alive a year after recurrence. The Hovon multicenter group[24] reviewed the risk of CNS recurrence in a trial testing the role of high-dose therapy with hematopoietic stem cell rescue, in patients responding “slowly” to 3 cycles of CHOP. One hundred ninety-three of 267 patients entered complete remission (CR). Ten patients (5%) developed SCNSL, 8 of them simultaneously with systemic progression. The risk was highest for patients with a high IPI score, but CNS recurrence occurred in all the risk groups. Survival data were not presented. Zinzani et al [25] reported an apparently higher incidence of isolated CNS recurrence in an unselected series (excluding Burkitt’s and lymphoblastic lymphoma) of patients with highgrade NHL (Kiel classification). One hundred seventy-five patients entered CR following therapy with MACOP-B or F MACHOP, both of which include modest doses of MTX intravenously but exclude intrathecal therapy. None had clinical evidence of CNS involvement at presentation. The minimum follow-up at the time of analysis was 3 years. Nine of 175 developed isolated CNS recurrence at a median of 3 months after CR had been documented. Multivariate analysis revealed advanced stage (III and IV) to be the only independent predictor of the likelihood of isolated CNS recurrence, although B symptoms, elevated LDH, and bone marrow involvement were all significant on univariate analysis. The outcome, whether the recurrence was leptomeningeal or parenchymal, was appalling, with all patients having died within 2 years because of CNS progression.

Figure 1 .The risk of central nervous system (CNS) recurrence according to the number of risk factors (age, lactate dehydrogenase, albumin, number of extranodal sites, retroperitoneal involvement) in 1220 patients with high-grade non-Hodgkin’s lymphoma (NHL). Reprinted with permission from Van Besien K, Ha CS, Murphy S, et al. Risk factors, treatment and outcome of central nervous system recurrence in adults with intermediate-grade and immunoblastic lymphoma. Blood. 1 998;91:1178-1184.

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In contrast, Haioun et al [26] reported the outcome for 1373 patients treated in a GELA study for patients with ‘aggressive’ NHL; lymphoblastic lymphoma and Burkitt’s lymphoma were excluded. CNS prophylaxis included intrathecal MTX with each cycle of systemic chemotherapy and 2 pulses of MTX 2 g/m2 with folinic acid res¬cue. There were 16 isolated CNS recurrences and a further 6 with progression at other sites. Initial multivariate analysis confirmed more than one extranodal site and elevated LDH to be independent risk factors predictive of CNS recurrence, each with a relative risk (RR) of 5. A further multivariate analysis (in¬corporating IPI score as a unique parameter, male gender, and B symptoms) was subse¬quently performed. IPI score remained the only parameter significantly associated with increased risk (low and low-intermediate ver¬sus high-intermediate and high, RR 7). Once again, the prognosis overall was poor, the median survival being 5 months and progres¬sive disease being the predominant cause of death. A further study from the GELA[27] adds support for the benefit of CNS prophylaxis for this group of patients. Seven hundred eight adults aged 6 1-69 years with at least 1 adverse prognostic factor (IPI) were entered onto a trial comparing a relatively intensive chemotherapy program incorporating both intrathecal MTX and consolidation with systemic MTX, ifosphamide, and cytosine arabino side, with standard CHOP. The CR rates were the same, despite a higher treatment-related mortality in the trial arm; overall survival, however, was better in the latter (P = .002). The frequency of CNS recurrence was also significantly lower in the trial arm (8 versus 25; P = .003). These results have been published in abstract form only to date. They are, however, supported by an earlier analysis from the M.D. Anderson Hospital in which out-come of patients receiving CNS prophylaxis in the form of intrathecal and intravenous MTX was better than that of matched historical controls.[16] The largest body of data defining the extent of the problem at the end of the 20th century comes from the Norwegian Radium Hospital, Oslo.[28] Twenty-five hundred fourteen adults were treated for NHL according to protocols of the day, based on the histological subtype (Kiel) and the extent of disease at presentation. CNS prophylaxis was given to < 1%, 11%, and 83% of patients with low-grade, high-grade, and Burkitt’s or lymphoblastic lymphoma, respectively. The analysis ad-dressed only the question of CNS progression, so 30 patients presenting with CNS involvement were excluded. Overall, the incidence reported for the histological groupings was very similar to that of other series. Less than 3% of those with “low-grade histology” developed SCNSL. Multivariate analysis confirmed B symptoms and involvement of bone marrow and skin as significant prognostic factors, with relative risks of 2.8, 2.8, and 3.7, respectively. The incidence for patients with Burkitt’s or lymphoblastic lymphoma was, in contrast, very high, being 24% overall, 78% in those not receiving prophylaxis, and 19% at 5 years in those that did. As in several other series, the SCNSL rate in ‘high-grade’ lymphoma was about 4%, the minority having received prophylaxis with intrathecal methotrexate about which no conclusions were drawn. Univariate analysis revealed a multitude of factors, including IPI and age-adjusted IPI, to predict for CNS recurrence. Testicular involvement in itself was not significant. Further analysis confirmed 5 factors to have an independent impact on CNS involvement: age, LDH, albumin, retroperitoneal nodes, and number of extranodal sites (Table 3).

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Although the hazard ratios are not identical, a general picture may be created by adding the risk factors and correlating increasing numbers with time-to-CNS involvement (Figure 2). Table 3. Risk of central nervous system involvement. Variable No. of extranodal sites (>1 vs ≤ 1) Age > 60 vs ≤ 60 yrs Album in < 3.5 g/L vs > 3.5 g/L LDH ~ 450 m/L vs < 450 m/L Retroperitoneal glands:Yes vs no

Relative Risk 3.0 2.8 2.5 2.1 1.9

(95% Confidence Interval) (1.7-5.4) (1.5-5.4) (1.3-4.6) (1.0-4.4) (1.0-3.5)

P Value

< 0.001 0.002 0.005 0.049 0.037

Figure 2. Incidence of central nervous system (CNS) recurrence in patients with increased lactate dehydrogenase (LDH) and involvement of more than 1 extranodal site (n = 93; dotted line) versus all other patients (n = 512; solid line). Reprinted with permission from Hollender A, Kvaloy S, Nome O, et al. Central nervous system involvement following diagnosis of non-Hodgkin’s lymphoma: a risk model. Ann Oncol. 2002;1 3:1099-1107.

The Challenge Today The elimination of CNS involvement with lymphoma is a very important goal, even if it affects only a relatively small proportion of patients, most of whose overall survival will be dictated by uncontrolled disease elsewhere. It is a highly distressing complication, with potentially extensive morbidity which, when established, is very difficult to eliminate. Theoretically, therefore, a prophylactic strategy, analogous to that employed so success-fully for ALL, is indicated. The risk of meningeal involvement in childhood lymphoblastic leukemia has been reduced from more than 50% to very low levels, after painstaking observations, identification of groups with different degrees of risk, and clinical trials to determine the most effective therapy with the lowest acceptable toxicity for each category.

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Most children now do not develop CNSL, nor do most have excessive long-term morbidity from the therapy. The first part of the process has been achieved for NHL. Follicular lymphoma and the other lymphocytic lymphomas have been shown to have a less than 1% probability of CNS infiltration, except when transformation has occurred: there can thus be no justification for prophylaxis. Burkitt’s lymphoma and lymphoblastic lymphoma (T and B) both have a high incidence of SCNSL: patients therefore now receive both intrathecal chemotherapy and high doses of MTX (and cytosine arabinoside in some instances) or cranial irradiation. As a consequence of this strategy, the incidence of CNS involvement is much reduced. For the remainder of the lymphomas, predominantly diffuse large B-cell lymphoma (DLBCL) and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), there is still no uniformity of practice, which reflects the complexity of the situation and the fact that the data are open to differing interpretation. However, the picture may be clearer than it was 20 years ago. There is a recurring theme throughout the recent publications. CNS lymphoma is uncommon but not rare, and when it occurs, devastating. Patients presenting with a high IPI score, particularly reflecting the presence of a high LDH or involvement of more than one extranodal site, are at much higher risk of CNS involvement than the rest. Notwithstanding less impressive statistical proof of their individual significance, patients with testicular and sinus involvement are also at high risk. Some of the data reported above suggest that prophylaxis, with intrathecal therapy and systemic MTX, may reduce the risk. It could therefore be concluded that all patients with these histological subtypes of lymphoma (DLBCL and PTCL-NOS) should have the CNS evaluated by history, examination, and LP, and that those with a high IPI score, or high LDH and more than one extranodal site, should proceed to prophylaxis. There is a superficial attraction to designing a randomized trial to test the hypothesis. It might be difficult to execute. If it is difficult to select the appropriate group to receive CNS prophylaxis, it is equally difficult to deter-mine what constitutes the best prophylaxis. Before the introduction of ‘highdose’ MTX [29] into combination chemotherapy, the only modalities available were intrathecal chemotherapy and irradiation. It may be clear from the above that intrathecal chemotherapy of short duration, while probably reducing the risk, does not eliminate it. Extrapolation from ALL makes this unsurprising: all treatments relying on intrathecal therapy alone demand much more prolonged treatment. Vital information about the efficacy of systemic MTX and the dose required in the absence of intrathecal therapy will come from the long follow-up analysis of the Southwestern Oncology Group-Eastern Oncology Group (SWOGECOG) study comparing CHOP with M-BACOD, MACOP-B, and PROMACECYTABOM, the trial arms including MTX and folinic acid rescue at a dose of 200 mg/m2, 400 mg/m2, and 1500 mg/m2, respectively. It may be anticipated that only the last dose might be effective. Further information accrued from clinical trials incorporating high-dose cytosine arabinoside may be helpful. Given at a dose of 2 g/m2, daily for 5 days, as part of the therapy for adults with ALL, cytosine arabinoside was as effective (compared with historical controls) as cranial irradiation in a small study.[30] It would be foolhardy in the extreme to make didactic statements about optimal CNS prophylaxis: in the light of all that has gone before, recommendations can be made only on

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the basis of circumstantial evidence and must be seen as part of the best treatment of the disease overall. While none of the third-generation treatments above compared favorably with CHOP, perhaps a treatment for those with a high IPI score incorporating high-dose MTX (> 3 g/m2) and cytosine arabinoside (> 1 g/ m2) might improve outcome. Were that perceived to be the case, a prospective evaluation of the strategy, particularly including longterm toxicity, would be required. Attention has been focused on reasons in favor of prophylaxis as opposed to against it. Emphasis has been placed on the unpleasant nature of the complication and the difficulty of eliminating it, once established. There are powerful clinical and economic reasons for not giving CNS-directed treatment if it can be avoided. The long-term toxicity of irradiation given for PCNSL has been reviewed above. Even though the long-term sequelae of prophylactic cranial irradiation are less worrying, there are enough data to suggest that highdose systemic chemotherapy may be as effective and less toxic. It is, however, not without morbidity and mortality, which increase with the dose. Conversely, intrathecal therapy is inconvenient and not to be desired, has well-known toxicity, and is costly for both the patient and the hospital. All this must be taken into account in devising the best way to improve therapy, and demonstrate the improvement, while offering the individual the best advice. For future consideration: What emphasis should be given to the risk at the time of recurrent or progressive lymphoma? Do the same risk factors apply? Should more or less attention be directed to the problem? Should it be considered for only those still being treated with curative intent?

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Bailey P. Intracranial sarcomatous tumors of leptomeningeal origin. Arch Surg. 1929;18:1359-1402. Rosenblum ML, Levy RM, Bredesen SYT, Wara W, Zeigler JL. Primary central nervous system lymphomas in patients with AIDS. Ann Neurol. 1988;23:S13-S16. Welch K, Finkbeiner W, Alpers CE, et al. Autopsy findings in the acquired immune deficiency syndrome. JAMA. 1984;252:1 152-1159. Sparano JA, Anand K, Desai J, Mitnick RJ, Kalkut GE, Hanau LH. Effect of highly active antiretroviral therapy on the incidence of HIV-associated malignancies at an urban medical center. J Acquir Immune Defic Syndr. 1999;21:S18-S22. Itoyama T, Sadamori N, Tsutsumi K, et al. Primary central nervous system lymphomas. Cancer. 1994;73:455-463. Kumanishi T, Zhang S, Ichikawa T, Endo S, Washiyama K. Primary malignant lymphoma of the brain: demonstration of frequent p16 and p15 gene deletions. Jpn J Cancer Res. 1996;87:691-695. Weller M. Glucocorticoid treatment of primary CNS lymphoma. J Neurol Oncol. 1999;43:237-239.

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Andrew Lister, Lauren E. Abrey and John T. Sandlund DeAngelis LM. Current management of primary central nervous system lymphoma. Oncology. 1995;9:63-71. Rampen FHJ, van Andel JG, Sizoo W, van Unnik JA. Radiation therapy in nonHodgkin’s lymphomas of the CNS. Eur J Cancer. 1980;16:177-184. Glass J, Gruber ML, Cher L, Hochberg FH. Preirradiation methotrexate chemotherapy of primary central nervous system lymphoma: long-term outcome. J Neurosurg. 1994;81:188-195. Gabbai AA, Hochberg FH, Linggood RM, Bashir R, Hotleman K. High-dose methotrexate for non-AIDS primary central nervous system lymphoma. J Neurosurg. 1989;70:190-194. Littman P, Wang CC. Reticulum cell sarcoma of the brain. Cancer. 1975;35:14121420. Dahlborg SA, Braziel R, Crossen JR, Tableman M, Petrillo A, Neuwelt EA. Non-AIDS primary CNS lymphoma: first example of a durable response in a primary brain tumor using enhanced chemotherapy delivery without cognitive loss and without radiotherapy. Cancer J Sci Am. 1996;2:166-174. Abrey LE, Yahalom J, DeAngelis LM. Treatment for primary central nervous system lymphoma (PCNSL): the next step. J Clin Oncol. 2000;18:3144-3150. Abrey LE, Moskowitz CH, Mason WP, et al. A phase II study of intensive methotrexate and cytarabine followed by high dose beam chemotherapy with autologous stem cell transplantation (ASCT) in patients with newly diagnosed primary central nervous system lymphoma (PCNSL) [abstract]. Proc ASCO. 2001 ;20:53a. Illerhaus G, Marks R, Derigs G, et al. High-dose-chemotherapy with autologous PBSCT and hyperfractionated radiotherapy as first-line treatment for primary CNS lymphoma (PCNSL) – Update of a multicenter Phase II study. Onkologie. 200 1;54 (Suppl 6): 14. Forsyth PA, Yahalom J, DeAngelis LM. Combined-modality therapy in the treatment of primary central nervous system lymphoma in AIDS. Neurology. 1994;44:1473-1479. Chamberlain MC, Kormanik PA. AIDS-related central nervous system lymphomas. J Neurol Oncol. 1999;43:269-276. Jacomet C, Girard P-M, Lebrette M-G, Farese VL, Monfort L, Rozenbaum W. Intravenous methotrexate for primary central nervous system non-Hodgkin’s lymphoma in AIDS. AIDS. 1997;1 1:1725-1730. Corn BW, Trock BJ, Curran WJ Jr. Management of primary central nervous system lymphoma for the patient with acquired immunodeficiency syndrome. Cancer. 1995;76(2):163-166. McGowan JP, Shah S. Long term remission of AIDS-related primary central nervous system lymphoma associated with highly active antiretroviral therapy. AIDS. 1998;12:952-953. Baumann MA, Ritch PS, Hande KR, Williams GA, Topping TM, Anderson T. Treatment of intraocular lymphoma with high-dose Ara-C. Cancer. 1986;57:12731275. Sandor V, Stark-Vancs V, Pearson D, et al. Phase II trial of chemotherapy alone for primary CNS and intraocular lymphoma. J Clin Oncol. 1998;16(9):3000-3006.

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[24] Strauchen JA, Dalton J, Friedman AH. Chemotherapy in the management of intraocular lymphoma. Cancer. 1989;63:1918-1921. [25] Peterson K, Gordon KB, Heinemann MH, DeAngelis LM. The clinical spectrum of ocular lymphoma. Cancer. 1993;72:843-849. [26] Valluri S, Moorthy RS, Khan A, Rao NA. Combination treatment of intraocular lymphoma. Retina. 1995;15:125-129. [27] Soussain C, Suzan F, Hoang-Xuan K, et al. Results of intensive chemotherapy followed by hematopoietic stem-cell rescue in 22 patients with refractory or recurrent primary CNS lymphoma or intraocular lymphoma. J Clin Oncol. 2001;19:742-9. [28] Abrey LE, Yahalom J, DeAngelis LM. Relapse and late neurotoxicity in primary central nervous system lymphoma [abstract]. Neurology. 1997;48:A18. [29] Abrey LE, Thiessen B, DeAngelis LM. Treatment related neurotoxicity in primary CNS lymphoma. Society for Neuro-Oncology Annual Meeting. 1997. [abstract] [30] Thiessen B, DeAngelis LM. Hydrocephalus in radiation leukoencephalopathy: results of ventriculoperitoneal shunting. Arch Neurol. 1998;55:705-710. [31] DeAngelis LM, Yahalom J, Thaler HT, Kher U. Combined modality therapy for primary CNS lymphoma. J Clin Oncol. 1992;10:635-643. [32] Abrey LE, DeAngelis LM, Yahalom J. Long-term survival in primary CNS lymphoma. J Clin Oncol. 1998;16:859-863. [33] Duffey P, Chari G, Cartlidge NEF, Shaw PJ. Progressive deterioration o f intellect and motor function occurring several decades after cranial irradiation. Arch Neurol. 1996;53:814-818. [34] McGuirt WF, Feehs RS, Strickland JL, McKinney WM. Irradiation induced atherosclerosis: a factor in therapeutic planning. Ann Otol Rhinol Laryngol. 1992;101:222-228. [35] McLaughlin P, Velasquez WS, Redman JR, et al. Chemotherapy with dexamethasone, high-dose cytarabine, and cisplatin for parenchymal brain lymphoma. J Natl Cancer Inst. 1988;80(17):1408-1412. [36] Chamberlain MC, Levin VA. Adjuvant chemotherapy for primary lymphoma of the central nervous system. Arch Neurol. 1990;47:1113-1116. [37] McAllister LD, Doolittle ND, Guastadisegni PE, et al. Cognitive outcomes and longterm follow-up after enhanced chemotherapy delivery for primary CNS lymphoma. Neurosurgery. In press. [38] Nelson DF, Martz KL, Bonner H, et al. Non-Hodgkin’s lymphoma of the brain: can high-dose, large-volume radiation therapy improve survival? Report on a prospective trial by the Radiation Therapy Oncology Group (RTOG): RTOG:8315. Int J Radiat Oncol Biol Phys. 1992;23:9-17. [39] Cheng AL, Yeh KH, Uen WC, Hung RL, Liu MY, Wang CH. Systemic chemotherapy alone for patients with non-acquired immunodeficiency syndrome-related central nervous system lymphoma: a pilot study of the BOMES protocol. Cancer. 1998;82:1946-1951. [40] Cher L, Glass J, Harsh GR, Hochberg FH. Therapy of primary CNS lymphoma with methotrexate-based chemotherapy and deferred radiotherapy: preliminary results. Neurology. 1996;46: 1757-1759.

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[41] DeAngelis LM, Seiferheld W, Schold SC, Fisher B, Schultz CJ. Combined modality treatment of primary central nervous system lymphoma (PCNSL) [abstract]. Proc ASCO. 1999;18:140a.

Lymphomatous Meningitis: The Acute Lymphoblastic Leukemia Model [1]

Pinkel D, Woo S. Prevention and treatment of meningeal leukemia in children. Blood. 1994;84:355-366. [2] Mahmoud HH, Rivera GK, Hancock ML, et al. Low leukocyte counts with blast cells in cerebrospinal fluid of children with newly diagnosed acute lymphoblastic leukemia. N Engl J Med. 1993;329:314-319. [3] Lauer S, Shuster J, Kirchner P, et al. Prognostic significance of cerebrospinal fluid (CSF) lymphoblasts (LB) at diagnosis (dx) in children with acute lymphoblastic leukemia (ALL). Proc ASCO. 1994;13:317. [4] Gilchrist GS, Tubergen DG, Sather HN, et al. Low numbers of CSF blasts at diagnosis do not predict for the development of CNS leukemia in children with intermediate-risk acute lymphoblastic leukemia: a children’s cancer group report. J Clin Oncol. 1994;12:2594-2600. [5] van den Berg H, Vet R, den Ouden E, Behrendt H. Significance of lymphoblasts in cerebrospinal fluid in newly diagnosed pediatric acute lymphoblastic malignancies with bone marrow involvement: possible benefit of dexamethasone. Med Pediatr Oncol. 1995;25:22-7. [6] Gajjar A, Harrison PL, Sandlund JT, et al. Traumatic lumbar puncture at diagnosis adversely affects outcome in childhood acute lymphoblastic leukemia. Blood. 2000;96:3381-3384. [7] Pui C-H, Mahmoud HH, Rivera GK, et al. Early intensification of intrathecal chemotherapy virtually eliminates central nervous system relapse in children with acute lymphoblastic leukemia. Blood. 1998;92:41 1-415. [8] Schrappe M, Reiter A, Zimmermann M, et al. Long-term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Leukemia. 2000;14:2205-2222. [9] Manera R, Ramirez I, Mullins J, Pinkel D. Pilot studies of species-specific chemotherapy of childhood acute lymphoblastic leukemia using genotype and immunophenotype. Leukemia. 2000; 14: 1354-1361. [10] Vilmer E, Suciu S, Ferster A, et al. Long-term results of three randomized trials (58831, 58832, 58881) in childhood acute lymphoblastic leukemia: a CLCG-EORTC report. Leukemia. 2000;14:2257-2266. [11] Kamps WA, Bökkerink JPM, Hakvoort-Cammel FGAJ, et al. BFM-oriented treatment for children with acute lymphoblastic leukemia without cranial irradiation and treatment reduction for standard risk patients: results of DCLSG protocol ALL-8 (1991-1996). Leukemia. 2002;16:1099-1111.

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[12] Murphy SB, Fairclough DL, Hutchison RE, et al. Non-Hodgkin’s lymphomas of childhood: an analysis of the histology, staging, and response to treatment of 338 cases at a single institution. J Clin Oncol. 1989;7:186-193. [13] Sandlund JT, Murphy SB, Santana VM, et al. CNS involvement in children with newly diagnosed non-Hodgkin’s lymphoma. J Clin Oncol. 2000;18:3018-24. [14] Haddy TB, Adde MA, Magrath IT. CNS involvement in small noncleaved-cell lymphoma: is CNS disease per se a poor prognostic sign? J Clin Oncol. 1991;9:19731982. [15] Bowman WP, Shuster JJ, Cook B, et al. Improved survival for children with B-cell acute lymphoblastic leukemia and stage IV small noncleaved-cell lymphoma: a pediatric oncology group study. J Clin Oncol. 1996;14:1252-1261. [16] Gururangan S, Sposto R, Cairo MS, Meadows AT, Finlay JL. Outcome of CNS disease at diagnosis in disseminated small noncleaved-cell lymphoma and B-cell leukemia: a children’s cancer group study. J Clin Oncol. 2000;18:2017-2025. [17] Patte C, Auperin A, Michon J, et al. The Société Française d’Oncologie Pédiatrique LMB89 protocol: highly effective multiagent chemotherapy tailored to the tumor burden and initial response in 561 unselected children with B-cell lymphomas and L3 leukemia. Blood. 2001;97:337-339. [18] Reiter A, Schrappe M, Tiemann M, et al. Improved treatment results in childhood Bcell neoplasms with tailored intensification of therapy: a report of the Berlin-FrankfurtMünster group trial NHL-BFM 90. Blood. 1999;94:3294-3306. [19] Reiter A, Schrappe M, Ludwig W-D, et al. Intensive ALL-type therapy without local radiotherapy provides a 90% event-free survival for children with T-cell lymphoblastic lymphoma: a BFM Group report. Blood. 2000;95:416-421. [20] Seidemann K, Tiemann M, Schrappe M, et al. Short-pulse B-non-Hodgkin lymphomatype chemotherapy is efficacious treatment for pediatric anaplastic large cell lymphoma: a report of the Berlin-Frankfurt-Münster Group Trial NHL-BFM 90. Blood. 2001;97:3699-3706. [21] Laver JH, Mahmoud H, Pick TE, et al. Results of a randomized phase III trial in children and adolescents with advanced stage diffuse large cell non-Hodgkin’s lymphoma: a pediatric oncology group study. Leukemia Lymphoma. 2001;42:399-405. [22] Slobod KS, Taylor GH, Sandlund JT, Furth P, Helton KJ, Sixbey JW. Epstein-Barr virus-targeted therapy for AIDS-related primary lymphoma of the central nervous system. Lancet. 2000;56: 1493-1494. [23] Gökbuget N, Hoelzer D. Recent approaches in acute lymphoblastic leukemia in adults. Rev Clin Exp Hematol. 2002;6:1 14-40. [24] Gökbuget N, Hoelzer D. Meingeosis leukaemica in adult acute lymphoblastic leukaemia. J Neuro-Oncol. 1998;38:167-180. [25] Gökbuget N, Aguion-Freire E, Diedrich H, et al. Characteristics and outcome of CNS relapse in patients with adult acute lymphoblastic leukemia (ALL). Abstract #1287 [26] Kantarjian HM, O’Brien S, Smith TL, et al. Results of treatment with hyper-CVAD, a dose-intensive regimen, in adult acute lympocytic Leukemia. J Clin Oncol. 2000;18:547-561.

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Secondary Central Nervous System Lymphoma: The Case for Prophylaxis [1] [2] [3]

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Murchison C. Case of ‘lymphadenoma’ of the lymphatic system, liver, lungs, heart and dura mater. Trans Pathol Soc Lond. 1870;21:372-389. Welch JE. Tumor of the neck showing unusual histologic features. Proc NY Pathol Soc. 1910;10:161. Guillan, Alajouanine, Perisson. Lymphosarcoma extradural metastique ayant determine une compression medullaire d’apparence primitive, d’evolution rapidement progressive; laminectomie; extirpation et radiotherapie; guerison. Bull Mem Soc Med Hop Paris. 1925;49:1057. Verda DJ. Malignant lymphomas of the spinal epidural space. Surg Clin N Am. 1944;24:1228-1244. Davison C, Michaels JJ. Lymphosarcoma with involvement of the central nervous system. Arch Intern Med. 1930;45:908-925. Sparling HJ, Adams RD, Parker F. Involvement of the central nervous system by malignant lymphoma. Medicine. 1947;26:285-332. Williams HM, Diamond DH, Craver LF, Parsons H. Neurological complications of lymphomas and leukaemias. Springfield, IL: Charles C. Thomas; 1959. Griffin JW, Thompson RW, Mitchinson MJ, de Kiewiet JC, Welland FH. Lymphomatous leptomeningitis. Am J Med. 1971;51:200-208. Law IP, Dick FR, Blom J, Bergevin PR. Involvement of the central nervous system in non-Hodgkin’s lymphoma. Cancer. 1975;36:225-231. Gendlemon S, Rizzo F, Moues RJ. Central nervous system complications of leukemic conversion of the lymphomas. Cancer. 1969;24:676-682. Olson ME, Chernik NL, Posner JB. Infiltration of the leptomeninges by systemic cancer: a clinical and pathological study. Arch Neurol. 1974;30:122. Herman TS, Hammond N, Jones SE, Butler JJ, Byrne GE, McKelvey EM. Involvement of the central nervous system by non-Hodgkin’s lymphoma. Cancer. 1979;43:390-397. Young RC, Howser JM, Fisher RI, Jaffe E, DeVita VT. Central nervous system complications of non-Hodgkin’s lymphoma. Am J Med. 1979;68:435-443. Levitt LJ, Dawson DM, Rosenthal DS, Moloney WC. CNS involvement in the non Hodgkin’s lymphomas. Cancer. 1980;45:545-552. Mackintosh FR, Colby TV, Podolsky WJ, et al. Central nervous system involvement in non-Hodgkin’s lymphoma: an analysis of 105 cases. Cancer. 1982;49:586-595. Perez-Soler R, Smith TL, Cabanillas F. Central nervous system prophylaxis with combined intravenous and intrathecal methotrexate in diffuse lymphoma of aggressive histologic type. Cancer. 1986;57:971-977. Recht L, Strauss DJ, Cirrincione C, Thaler HT, Posner JB. Central nervous system metastases from non-Hodgkin’s lymphoma: treatment and prophylaxis. Am J Med. 1988;84:425-435. Liang R, Chiu E, Loke SL. Secondary central nervous system involvement by non Hodgkin’s lymphoma: the risk factors. Hematol Oncol. 1990;8:141-145.

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[19] Bashir RM, Bierman PJ, Vose JM, Weisenburger DD, Armitage OJ. Central nervous system involvement in patients with diffuse aggressive non-Hodgkin’s lymphoma. Am J Clin Oncol. 1991;14:478-482. [20] Bunn PA, Schein PS, Banks PM, de Vita VT. Central nervous system complications in patients with diffuse histiocytic and undifferentiated lymphoma. Blood. 1976;47:3-10. [21] Litam JP, Cabanillas F, Smith TL, Bodey GP, Freireich EJ. Central nervous system relapse in malignant lymphomas: risk factors and implications for prophylaxis. Blood. 1979;54:1249-1257. [22] Bollen ELEM, Brouwer RE, Hamers S, et al. Central nervous system relapse in nonHodgkin’s lymphoma. Arch Neurol. 1997;54:854-859. [23] Van Besien K, Ha CS, Murphy S, et al. Risk factors, treatment and outcome of central nervous system recurrence in adults with intermediate-grade and immunoblastic lymphoma. Blood. 1998;91:1178-1184. [24] Bos GMJ, van Putten WLJ, van der Holt B, van den Bent M, Verdonck LF, Hagenbeek A. For which patients with aggressive non-Hodgkin’s lymphoma is prophylaxis for central nervous system disease mandatory? Ann Oncol. 1998;9:191-194. [25] Zinzani PL, Magagnoli M, Frezza G, et al. Isolated central nervous system relapse in aggressive non-Hodgkin’s lymphoma: the Bologna experience. Leuk Lymphoma. 1999;32:571-576. [26] Haioun C, Besson C, Lepage E, et al. Incidence and risk factors of central nervous system relapse in histologically aggressive non-Hodgkin’s lymphoma uniformly treated and receiving intrathecal central nervous system prophylaxis: a GELA study on 974 patients. Ann Oncol. 2000;11:685-690. [27] Tilly H, Coiffier B, Casasnovas O, et al. Survival advantage of ACVBP regimen over standard CHOP in the treatment of advanced aggressive non-Hodgkin’s lymphoma (NHL). The LNH 93-5 study [abstract]. Ann Oncol. 2002;13(suppl 2):082a. [28] Hollender A, Kvaloy S, Nome O, Skovlund E, Lote K, Holte H. Central nervous system involvement following diagnosis of non-Hodgkin’s lymphoma: a risk model. Ann Oncol. 2002;13:1099-1 107. [29] Canellos GP, Skarin AT, Ervin T, Weinstein H. A chemotherapeutic approach to CNS lymphoma and leukaemia by the systemic administration of high doses of antimetabolites. In: Whitehouse JMA, Kay HEM, eds. CNS Complications of Malignant Disease. Macmillan Press; 1979:142-148. [30] Rohatiner AZS, Bassan R, Battista R, et al. High dose cytosine arabinoside in the initial treatment of adults with acute lymphoblastic leukaemia. Br J Cancer. 1990;62:454.

In: Trends in Brain Cancer Research Editor: Andrew V. Yang, pp. 193-251

ISBN 1-59454-972-9 ©2006 Nova Science Publishers, Inc.

Chapter VI

Epigenetic Mechanisms in the Development of Malignancies of the Central Nervous System (CNS) Sabrina Schlosser and Michael C. Frühwald University Children’s Hospital Muenster, Department of Pediatric Hematology and Oncology, Muenster, Germany

Abstract Malignant tumors of the central nervous system represent a rather heterogeneous group of neoplasms originating from virtually any anatomical structure within the spine and skull. While in adult patients malignant gliomas predominate, it is the group of embryonal malignancies (i.e. medulloblastoma, supratentorial primitive neuroectodermal tumor [sPNET], atypical teratoid, rhabdoid tumor [AT/RT] and pineoblastoma) that is prevalent in childhood. Despite major improvements in the clinical management including timely diagnosis, advanced supportive care and refined multimodality treatment prognosis remains grim for a large group of patients. In adulthood the group of high-grade glioma bears a dismal prognosis. Some authors advocate that the diagnosis of a high-grade glioma is synonymous with a palliative situation and should be managed as such. Thus a change of focus has been introduced into adult neurooncology which is quality of life as an outcome measure rather than survival. In childhood major advances have been made in the treatment of embryonal tumors such as standard risk medulloblastoma, which is defined by the following factors: age above three years, neurosurgical complete resection with minimal residual tumor and absence of metastatis. Other factors such as desmoplastic histology, high level of TRKC mRNA are under discussion as prognostic factors. Consequently the diagnosis of medulloblastoma in small children, with metastasis at diagnosis, recurrent or large residual tumor constitutes an almost inevitably fatal condition. This is also true for

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Sabrina Schlosser and Michael C. Frühwald malignancies like AT/RT or sPNET. No consistently curative therapy exists for these conditions. Understanding the genetic and epigenetic basis of the origin and progression of these tumors shows great promise for the development of prognostic markers and eventually improved diagnosis and treatment. Certain genetic events such as mutation of the tumor suppressor genes TP53 and PTEN or amplification of the growth factor receptor EGRF are long-known hallmarks of genetic mutations in gliomas of adults. Mutations in members of the sonic hedgehog - patched pathway (SHH-PTCH) have been described in medulloblastomas. Likewise deletions and basepair mutations of the SMARCB1 gene have been found in AT/RT of childhood. No single genes have been identified in sPNET. Epigenetic events i.e. changes in gene transcription not due to base pair mutations have recently received major attention. Foremost aberrant DNA-methylation and histone deacetylation appear to contribute to the malignant potential of CNS tumors. Gene-bygene approaches and genome scanning techniques such as chip-based-analysis have identified a number of genetic loci with relevance in the development/formation of neoplasms of the CNS in adults and children. Examples include aberrant methylation of the tumor suppressor gene candidate RASSF1A in medulloblastoma and sPNET, which together constitute the most common malignant brain tumors of childhood. Aberrant methylation of the DNA-repair gene O6-MGMT appears to be an important predictor of response to therapy in malignant gliomas of adults. Additional examples of epigenetically inactivated genes have been described. Lesions of the epigenome hold great potential for the elucidation of the pathomechanisms of central nervous system tumors. As epigenetic lesions may be reversed by chemical manipulation epigenetic therapy holds great promise for the management of malignant CNS tumors in adults and children.

Epidemiologic and Prognostic Aspects of Central Nervous System Tumors in Adults and Children Brain tumors comprise a rather heterogeneous group of neoplasias ranging from benign to highly aggressive clinical behaviour. They comprise the most common solid malignancies in childhood and account for up to 3% of cancer associated deaths in adults. It is estimated that about 17.000 patients are diagnosed annually with a malignant brain tumor and that 13.000 of these will die of the disease [1]. While for malignancies in other anatomical locations such as tumors of the bone or lung the distinction between malignant and benign usually describes the clinical behaviour of the tumors, this is less clear for patients affected by brain tumors. For instance a child with a nonresectable diffuse WHO grade I pilocytic astrocytoma may have a worse long-term prognosis than a matched child with a WHO grade IV medulloblastoma. Standard of treatment for essentially all brain tumors includes above all neurosurgical resection, aimed at a complete removal of all tumor tissue without risking neurological impairment. In malignant tumors resection is essentially never complete due to spread of the tumor along white matter tracts, seeding via the CSF and invasion of functionally important structures within the

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central nervous system. Therefore adjuvant therapy such as chemotherapy and radiation therapy is employed in most patients with malignant brain tumors. Children suffering from medulloblastoma, the most common malignant brain tumor occurring in the age groups between 3-4 and 7-9 years, have a chance for a cure if the residual tumor is less than 4 cm in size. Children below the age of three years who have had an incomplete tumor removal or who have metastasis at the onset of disease have a rather grim prognosis. Adult patients with malignant brain tumors such as anaplastic astrocytoma or glioblastoma still have a very poor prognosis. For this group of patients success is not defined as the absence of tumor, but more likely the absence of symptoms impeding on daily life. As aggressive chemotherapeutic approaches only seem to add to toxicity but not to a cure alternatives are desperately sought. Since brain tumors are, just as most other types of cancer, due to an accumulation of genetic lesions it is hoped that through elucidation of the molecular pathways involved it may become possible to design targeted therapies. In recent years data have been collected demonstrating that not only genetic but also epigenetic lesions of the genome may contribute to the origin of brain tumors. The investigation of epigenetic lesions in CNS tumors is promising regarding elucidation of the pathogenesis of these tumors and may provide a way to new epigenetically-based anti-cancer strategies. Therefore the field of epigenetics has attracted enormous interest over the last years and is still extensively studied.

The Basic Molecular Biology of Epigenetics The term „epigenetics“ was coined by Conrad Waddington in the 1940s to describe mechanisms by which genes and their products produce the phenotype of a cell and/or organism [2]. Nowadays „epigenetics“ refers to heritable alterations in gene expression patterns during development and cell proliferation, mediated by mechanisms other than changes in the primary nucleotide sequence [3-5]. In eukaryotes DNA is packaged in the nucleus of cells and associated with proteins in a complex known as the chromatin that consists of regularly repeating units, the nucleosomes. Each nucleosome is made up of 146 bp of DNA tightly wrapped around the core of eight histone proteins, two units of each histone H2A, H2B, H3 and H4. Two states of chromatin can be found within the human genome, transcriptionally active euchromatin which is easily accessible to components of the transcriptional machinery like RNA polymerases, and silent heterochromatin with tightly arranged nucleosomes. Heterochromatin is among others found in centromeres, telomeres and the inactive X-chromosome [6]. The balance between euchromatin and heterochromatin guarantees the maintenance of gene expression patterns of a certain cell type in it´s daughter cells as heritable traits [7]. Furthermore in response to regulatory signals gene expression in most cases involves alterations of the chromatin structure [8]. It is thus difficult to define the epigenome of a given cell even though human cells contain the same sequence of base pairs. The epigenome of cells depends on cell cycle, developmental stage, sex, age and various other aspects [9]. Epigenetic regulation (see figure 1) of gene expression is mediated by diverse mechanisms, such as DNA-methylation and histone modifications (acetylation, methylation, phosphorylation, ubiquitination, ribosylation,

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sumoylation) leading to changes in chromatin structure [9, 10]. In addition to that small noncoding RNAs seem to contribute to the epigenetic regulation of gene expression [11]. All those mechanisms do not simply act independently, but rather interdigitate with each other.

Figure 1. Mechanisms of epigenetic regulation

a) Epigenetic Regulation and Histone Modification The state of chromatin and its accessibility to transcription regulating molecules is epigenetically controlled by special histone modifications both dynamically and in a stably heritable fashion [12, 13]. There are three main mechanisms to modify histones and thus the chromatin structure. The first mechanism involves restructuring of nucleosomes by chromatin remodeling complexes [8]. Currently three major chromatin remodeling complexes can be distinguished. One, the Swi/Snf complex, removes histones from the DNA or mediates the transfer of histones from one DNA strand to another [14]. Second a complex termed NuRD is associated with repressed gene expression [15]. A third group of chromatin remodellers is comprised of the polycomb proteins which are conserved from vertebrates to humans and limit the expression of homeotic genes that sculpt the organism [16].

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Another variation in chromatin structure that influences gene expression is the replacement of core histones with histone variants such as H2AZ, H2AX or H3.3 [8, 17]. The H2AZ variant for instance is associated with reduced nucleosome stability, whereas the histone variant H2AX is a target of phosphorylation accompanying repair of DNA breakage. The inclusion of H3.3 seems to be associated with transcriptionally active genes [8]. Another example is the substitution of histone H3 by the variant CENP-A in centromeric chromatin regions [18]. Besides modifying chromatin by chromatin remodeling complexes and inclusion of histone variants, specific amino acid residues in the N-terminal tails of core histones can be covalently modified by acetylation, methylation, phosphorylation, ubiquitination, sumoylation and ADP-ribosylation [10]. Together the combination of histone modifications that governs gene transcription is known as the „histone code“ [13]. One element of this code, histone methylation can mediate both the activation as well as the repression of genes. Histone proteins can be mono-, di-, or trimethylated at lysine residues as well as mono- or dimethylated at arginine residues facilitated by histone methyltransferases (HMT). Specific sites for histone methylation are the lysine residues nine and four of histone H3. Methylated H3Lys9, which is associated with transcriptionally repressed chromatin (figure 2) is a binding site for the heterochromatin protein 1 (HP1) which mediates DNA-methylation and gene silencing [19-21]. In contrast methylation of H3Lys4 triggers the recruitment of ATPdependent chromatin remodeling complexes to activate genes and at the same time it prevents the recruitment of repressing complexes [22, 23]. The methylation state of histones is further balanced by histone demethylases (HDMases) and can be determined by the local histone acetylation state [24, 25]. The latter is balanced by the function of histone acetylases (HAT) and histone deactylases (HDAC). Deacetylated histone tails are positively charged and strongly bind to the negative charged backbone of the DNA excluding molecules of the transcription machinery. Histone acetylation prevents this interaction between histones and DNA allowing for access of transcription factors and other transcription regulators [26]. (see figure 2) Just as the acetylation of histones, its phosphorylation is associated with transcriptional activation. The activation of immediate early-response genes during interphase is just one example [27]. Histone phosphorylation occurs at serine residues located within the highly conserved amino acid sequence Ala-Arg-Lys-Ser (ARKS), in which serine residues S10 and S28 of the core histone H3 are important phosphorylation sites [10]. Phosphorylation is mediated by members of the aurora kinase family [28], by downstream kinases of MAP kinases, such as kinases of the RSK and MSK families [29, 30] and is balanced by the action of type 1 phosphatases (PP1) [31, 32]. The activation of repressed genes through phosphorylation might be accomplished by the change of repressive modifications like histone methylation. It is possible that phosphorylation blocks the binding of repressive proteins that recognize methylated histones thus allowing other enzymes like histone acetylases (HAT) to access and acetylate the DNA to pass into an active chromatin state. Another possibility is that phosphorylation recruits enzymes that demethylate H3Lys9, which is associated with inactive genes [10]. For example phosphorylation of histone H3Ser10 facilitates acetylation of H3Lys14 and methylation of H3Lys4 leading to gene activation.

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Furthermore it facilitates the acetylation of H3Lys9 thus preventing the repressive methylation of this histone site which is associated with inactive genes [12, 33].

Figure 2. Component of the epigenetic silencing machine (A) involved in alteration between active (B) and inactive (C) chromatin

Another type of modification is the ubiquitination of histone tails, which can occur at histones H1, H3, H2A and H2B, whereas in most cases H2A and H2B are affected in vivo [34]. Ubiquitination targets lysine residues and therefore raises the possibility of interplay with methylation and acetylation that can also occur at lysine residues [35]. H2B ubiquitination for example appears to participate in the regulation of histone H3 methylation and preserves transcriptionally active euchromatin. It seems that levels of ubiquitinated H2B (uH2B) depend on ongoing transcription. Thus uH2B may impede on nucleosome refolding allowing subsequent rounds of transcription [36]. The ubiquitination of histone H2A rather appears to be associated with transcriptional repression. It has been shown to contribute to polycomb silencing, whereas the mechanism remains to be elucidated [37]. Thus ubiquitination is not simply a mechanism to mark proteins for degradation by the proteasome, it is also involved in modulating protein activities, protein-protein interactions and subcellular localization. A histone modification that seems to be generally linked with inactive chromatin is sumoylation, the attachment of SUMO (small ubiquitin-related modifier) to histone tails [35]. Histone sumoylation has been shown to occur at histone H4 and to be at least partially

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triggered by histone acetylation. It might serve as a signal for termination of gene expression that had been induced through acetylation. This seems to be facilitated through recruitment of histone deacetylases which deacetylate histones allowing subsequent methylation and the binding of the repressor protein HP1 [38]. Poly-ADP-ribosylation of histones is the stepwise attachment of ADP-ribose monomers from the donor NAD+ to generate poly-ADP-ribose (PAR) chains, a reaction catalyzed by PAR polymerases (PARPs) from which PARP1 is primarily in mammals [39]. Poly-ADPribosylation of histones is associated with transcriptional activation probably through loosening of DNA-histone interactions and binding of activating factors to the DNA [40, 41]. The induction of heat shock and immune system genes for example depends on PARP1 activity [10]. Further on opening of the chromatin structure by poly-ADP-ribosylation seems to be involved in DNA repair processes [42, 43].

b) Epigeneitc Regulation and Small Noncoding RNAs There is increasing evidence that noncoding RNAs play a role in epigenetic regulation, i.e. chromatin dynamics and gene silencing [44]. Although in the past heterochromatin was thought to be inert, it is now known to give rise to small RNAs that direct modification of proteins and DNA in heterochromatic repeats and transposable elements by means of RNA interference (RNAi) [45, 46]. In yeast for instance RNAs transcribed from both strands of centromeric regions (duplex RNA) are cleaved by the RNAi machinery into small interfering RNAs (siRNAs) which recruite silencing complexes to target the chromatin [46]. Whether RNAi plays a role in the control of structural and regulatory genes of higher eukaryotes remains to be determined [6]. The silencing of genes has also been shown to depend on microRNAs (miRNAs) which are endogenous ~22 nt RNAs that target mRNAs for cleavage or translational repression. In contrast to siRNAs which silence the same locus from which they originate, eg. viruses, transposons or heterochromatic outer repeats of centromeres, miRNAs specify inactivation of genes different from the locus from which they are derived [44].

c) Epigenetic Regulation through DNA-methylation DNA-methylation is a biochemical modification of postreplicative DNA that affects only cytosine residues in CpG dinucleotides in humans and other mammals [3, 4]. CpG dinucleotides should occur at a frequency of about 6% in the human genome, however the actual presence is only 5-10% of the predicted frequency. This reduction is known as „CpG repression“ and may be related to the hypermutability of methylated cytosine residues [47]. Small regions of the genome, the CpG islands display a greater than expected CpG density. These regions with a CG content of approximately 60 to 70% range from 0.5 to 5 kb and occur on average every 100 kb. In normal adult somatic tissues about 70-80% of all CpG dinucleotides are methylated [7]. About half of all human genes contain CpG rich promoter regions [47], which are normally not methylated in active genes so that the nucleosomes are

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widely and irregularly spaced allowing transcription activating protein complexes to interact with appropriate DNA regions [4]. Methylation of such promoter CpG islands results in a compact and regular arrangement of the nucleosomes and generally correlates with transcriptional inactivation [7, 48, 49]. As an exception silenced genes on the inactive Xchromosome of females as well as silenced alleles of „imprinted genes“ exhibit fully methylated CpG islands leading to stable repression of gene expression, so that only one allele of a particular gene is expressed in normal tissues [7]. In the case of genomic imprinting the expression status of a gene depends upon the parent from which it is derived. For example the IGF2 gene is expressed only from the paternal allele [8]. The mechanism involves in part DNA-methylation, which mediates the dissociation of the chromatin protein CTCF that blocks an enhancer protein, allowing the free enhancer to activate IGF2 expression from the paternal allele [50, 51]. Methylated CpG islands can also be detected in genes expressed in a germ-line specific or tissue dependent manner. For instance genes which are expressed only in the male or female germline, such as MAGE genes, are not expressed in any adult tissue [52]. Methylation of the genome may help retarding unwanted gene expression of DNA sequences such as repeat elements, integrated viral genes, imprinted genes or genes on the inactive X-chromosome. It can prevent the expression of transposon-encoded genes as well as transposon-mediated DNA rearrangements and the transcription from transposon promoters into neighboured host genes [53-55]. Furthermore DNA-methylation seems to play an important role in maintaining chromosome and thus genome stability [56]. In summary the primary function of methylation in vertebrates appears to be gene silencing, the stabilization of inactivation and the permanent repression of silent promoters (for summary see figure 3). It is assumed that DNA-methylation functions as a developmental memory. Genes that are active during embryonic development remain potentially active during development and adulthood, whereas non-active regions become methylated and transcriptionally repressed [7]. DNA-methylation is accomplished by a family of enzymes termed DNA methyltransferases (DNMT) which transfer methyl groups from S-adenosyl-methionine (SAM) to the 5´ carbon of cytosines in CpG dinucleotides [4]. The currently known DNMT are DNMT1, DNMT1b, DNMT1o, DNMT1p, DNMT2, DNMT3a, DNMT3b with its isoforms and DNMT3L [57]. It is suggested that DNMT1 is responsible for the maintenance of DNA-methylation after each round of replication in adult cells and might predominantly maintain methylation patterns established in early embryonic development. DNMT3a and DNMT3b are the main methyltransferases involved in de novo methylation and seem to be responsible for the bulk of DNA-methylation patterns established during early development [58, 59]. Several studies indicate that all three of those enzymes cooperate and have both de novo and maintenance methyltransferase activities [60-64]. The function of DNMT2 is still not clear, since it seems to be unable to methylate DNA [65]. The DNMT3L enzyme might have a central role in maternal genome imprinting, even though it is not catalytically active at all [66]. Mammalian DNA-methylation patterns are established early in embryogenesis and are precisely regulated during development. Therefore in addition to DNMT other activities are required including potential demethylases that actively remove methl groups from DNA,

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methylation centers triggering DNA-methylation and methylation protection centers that protect against de novo methylation [67].

Figure 3. DNA-methylation in normal and cancer cells

DNA-methylation exerts different effects on gene expression. Increased methylation of the promoter regions of genes entails reduced expression, whereas methylation in the transcribed region has variable effects on gene expression [68]. The information provided by methylation of CpG islands is functionally significant only in the context of chromatin, so that further mechanisms are necessary to translate DNA-methylation into transcriptionally silent chromatin [52]. One mechanism how DNA-methylation accounts for transcritpional repression is the direct inhibition of transcription factor binding to the promoter. Transcription factors, such as AP-2, E2F or NFkB bind to DNA sequences containing CpGs

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and have been shown to be inhibited by DNA-methylation [69]. The second mechanism involves the binding of methyl-CpG-binding proteins (MBPs) to methylated DNA sites that further recruit histone deacetylases and other factors facilitating the exclusion of the transcription complex [69, 70]. It is also possible that DNA-methylation directly leads to changes in nucleosomal positioning, for example to the formation of certain nucleosome structures that silence transcription more effectively than conventional chromatin [71]. To summarize it is important to have the correct ammount and pattern of DNA-methylation at each stage of life to warrant proper functioning of cells. DNA-methylation in Cancer While cells are in general hypermethylated in intergenic regions and hypomethylated at gene promoters, the situation deviates in cancer cells. Intergenic regions containing repetitive elements, transposons or endogeneous retroviruses may become hypomethylated and expressed, whereas gene promoters, e.g. those of tumor suppressor genes become hypermethylated and repressed [6]. According to Knudson“s two-hit hypothesis for oncogenic transformation, disruption of a tumor suppressor gene requires the complete loss of function of both copies of the involved gene [72]. In addition to mutations, chromosomal deletions and loss of heterozygosity, DNA hypermethylation and associated gene silencing has been proposed as one of the two hits in Knudson´s hypothesis [4]. Genes affected by DNA-methylation are those involved in cell cylce regulation (p16INK4a, p15INK4b, RB, p14ARF), associated with DNA repair (BRCA1, O6-MGMT), apoptosis (DAPK, TMS1), drug resistance, detoxification, differentiation, angiogenesis and metastasis [5]. Hypomethylation, another methylation defect is common in solid tumors. While hypermethylation of DNA mostly affects promoter associated CpG islands, multiple types of sequences can be affected by cancer-specific hypomethylation. Among these sequences are high-copy repeats (heterochromatic repeats, e.g. satellite repeats or interspersed repeats such as LINE-1 elements), moderate copy repeats (e.g. latent viruses) as well as unique sequences (e.g imprinted genes or testes-specific genes). DNA hypomethylation appears to have an independent role in tumorigenesis and may contribute to oncogenesis by three basic mechanisms. It may cause 1) chromosomal instability, 2) the reactivation of transposable elements and latent viruses or oncogenes, and 3) loss of imprinting. In several cancers, such as malignancies of the brain, global hypomethylation shows a progressive increase with the grade of malignancy [73]. It has been described that aging, chronic inflammation and viral infections promote the methylation of non-core regions of promoter CpG islands. There are different possibilities how a promoter can be methylated, e.g. „peripheral methylation“ close to the border of a promoter, interspersed „seeds of methylation“, methylation of only the core region and dense methylation. Nonetheless, not all of these block transcription. When a gene is silenced by hypermethylation of the promoterassociated CpG island it is usually densely methylated. Moreover it is possible that methylation of non-core regions of the promoter, „seeds of methylation“ and diminished transcription can trigger dense methylation of a promoter CpG island [74]. It seems that in cancer cells the compartimentalization of the genome into hetero- and euchromatin, and into methylated and unmethylated regions breaks down, thus allowing the spread of heterochromatic silenced chromatin [75]. Indeed it has been shown that promoter CpG island

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methylation can spread from a heavily methylated DNA region into the adjacent unmethylated CpG island. For some genes the spreading of methylation seems to occur during ageing, emphasizing the role of older age as a risk factor for cancer [76]. The methylation of cytosines in coding regions may also influence tumorigenicity by increasing mutation rates. One mechanism is the elevated spontaneous hydrolytic deamination of methylated cytosines leading to increased C-to-T transitions [77]. Moreover CpG-methylation can increase the rate at which mutations are induced by ultraviolet light by shifting the absorption wavelength of cytosine in the range of sunlight fostering increased CC-to-TT mutations as can be observed in skin cancers [78]. In addition to that methylated CpGs are preferred binding sites for benzo(a)pyrene diol epoxide and other carcinogens that can be found in tobacco smoke. As a consequence augmented DNA adducts and G-to-T transversion mutations can be observed which may be linked with aerodigestive tumors of smokers [79]. An overview of effects of DNA methylation in cancer cells is given in figure 3.

d) Cooperation of Epigenetic Mechanisms in Gene Regulation According to the “histone code” hypothesis histone modifications, either alone or in specific combination team up with chromatin associated proteins to activate or inhibit gene transcription [13]. It is further known that histone modifying mechanisms interdigitate with DNA-methylation. (see figure 2) In this context it is well established that DNA-methylation, histone deacetylation and H3K9 methylation collaborate in gene silencing. Together these mechanisms might comprise a self-reinforcing epigenetic cycle, that e.g. could play a role in the imprinting of genes or hypermethylation associated gene silencing in cancer. The interactions between these three epigenetic mechanisms are rather complex and at the present time it cannot be defined whether DNA-methylation or histone modification is the leadoff event [80]. In the case of hypermethylation associated gene silencing in cancer, CpGmethylation appears to be the predominant event [81]. Nevertheless studies exist suggesting that histone modifications facilitate DNA-methylation and CpG-methylation might be a secondary event in silencing of certain genes. It further seems possible that DNA-methylation and histone modifications act independently under certain conditions. Other histone modifications such as H3K27 and H4K20 methylation seem to communicate with DNAmethylation [80]. In conclusion there is not a common series of events leading to the activation or repression of genes, but for any gene the order of recruitment of chromatin modifying factors may be crucial for the appropriate timing of expression [8].

Epigenetic Lesions in Brain Tumors In the last years interest has been directed towards epigenetic events that might be involved in tumorigenesis except for mutations or other genetic events. In this context DNAmethylation has become a major focus of cancer research [82], with regard to silencing of tumor suppressor genes by promoter hypermethylation, a common event in human tumors.

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DNA-methylation is one of the most extensively studied mechanisms of epigenetic regulation. The following part will thus primarily deal with observations of aberrant DNAmethylation in brain tumors and approaches how DNA-methylation has been studied on an experimental level.

a) Screening Approaches Genome wide methylation scanning is performed to detect differentially methylated gene-associated CpG islands. Potentially this may allow the detection of novel genes involved in the origin and progression of certain brain tumors or may point to genomic regions comprising other genetic defects [83]. Several methods have been described to screen the genome for epigenetic alterations. A common screening method is RLGS (Restriction Landmark Genomic Scanning), a twodimensional gel-based method to screen the genome for aberrantly methylated CpG sites (figure 4) [84, 85]. RLGS does not only allow the identification of methylated sites within the genome, but also detection of events such as DNA amplification, hypomethylation [86, 87] and DNA polymorphisms [88]. The procedure includes cleavage of genomic DNA with a rare cutting methylation-sensitive landmark restriction enzyme such as NotI [84] or AscI [89], followed by further digestion, radioactive labeling of the resulting fragments in a fill-in reaction and separation of the fragments in the first dimension. Following an in-gel digestion with HinfI, a more frequently cleaving enzyme is performed. The resulting RLGS profile displays an array of more than 2.000 gene spots in a single assay, each representing a specific identifiable DNA fragment (figure 4) [90]. Loss of fragments from the RLGS profile is due to methylation or very rarely a mutation in the cleaving site of the landmark enzyme. Using this approach DNA-methylation has been studied in 98 primary tumors of seven different entities including medulloblastoma, sPNET and glioma. About 1.200 unselected CpG islands have been screened and revealed specific methylation patterns of the investigated tumor types. It has further been estimated that ~600 out of ~45.000 CpG islands in the genome are hypermethylated in cancer tissues compared to control tissues [84]. Methylation screening by RLGS in low-grade astrocytoma revealed an average of 1.544 CpG island-associated genes to be aberrantly methylated in each tumor [91]. Furthermore distinct methylation patterns were detected in medulloblastoma and sPNET. In these tumors up to 1% of all CpG islands appear to be methylated [83, 92]. As for medulloblastoma RLGS further allowed the detection of hypermethylation of specific CpG islands in the major chromosomal breakpoint cluster region in 17p11.2, a region commonly affected by genomic disruption in these tumors. Aberrant methylation in this genomic region might be linked to chromosomal instability and formation of an isochromosome 17q, which is detected in up to 50% of medulloblastoma [93]. Integrated genomic and epigenomic approaches using CGH (Comparative Genomic Hybridization) and RLGS (Restriction Landmark Genomic Scanning) to determine aberrant methylation and genetic alterations as well have been reported, but these methods assess only a fraction of the human genome [94].

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Figure 4. Screening the cancer genome for aberrant DNA-methylation

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Methods for global methylation screening MS-AP-PCR (MethylationSpecific Arbitrarily Primed PCR) RLGS (Restriction Landmark Genomic Scanning) Microarray Based Methylation Analysis MSO Microarray (Methylation-Specific Oligonucleotide)

MSRF (MethylationSensitive Restriction Fingerprint) MS-RDA (MethylationSensitive- Representational Difference Analysis) MS-AFLP (MethylationSensitive Amplified Length Polymorphism)

Application

Reference

− fingerprinting method to screen the genome for regions with altered methylation patterns

[110]

− scanning the cancer genome for aberrant methylation patterns − highly reproducible, specific evaluation of CpG islands − genome wide analysis of methylation status of many genes and quantification of methylation at each site − potential for genome wide rapid screening of multiple CpG sites in a lot of gene promoters − high throughput method for methylation measurement within specific target regions containing multiple CpG sites − variation of MS-AP-PCR

[84, 85, 89]

− genome wide search for methylated genes that allows the identification of differentially methylated fragments in two DNA sources such as tumor and normal tissue − comparative genome wide scanning of aberrant methylation at NotI restriction sites − approaches combining MS-AFLP and DNA microarrays − separation of methylated DNA without requiring prior knowledge of any sequences

[112]

MBD Column Chromatography(MethylCpG Binding Domain) MIRA (Methylated-CpG − rapid detection of methylated CpG islands in Island Recovery Assay) small amounts of DNA MCA (Methylated CpG − allows both methylation analysis and cloning of Island Amplification) differentially methylated genes ERMA (Enzymatic − quantitative method to assess CpG density of Regional Methylation Assay methylation of particular DNA regions in mammalian cell, eg. promoter regions of TSGs − advanced SssI Methyltransferase Assay DMH (Differential − array-based technique for detection of Methylation Hybridization) differentially methylated sequence tags

[95, 96]

[98]

[97] [111]

[113] [114] [114-118]

[119] [120] [121]

[122]

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Table 1. Methods to study DNA-methylation (Continued) Methods to determine 5mC content TLC (Thin Layer Chromatography) DHPLC (Denaturing High Performance Li- quid Chromatography) HPCE (High Performance Capillary Electrophoresis)

Application

− quantitative evaluation of the genomic methylation level − methylation analysis of multiple CpG sites allows simultaneous detection of CpG methylation and point mutations − easy, fast and inexpensive method to quantify the total methylation degree of DNA, but no data about methylation clustering or location 5mC Antibody − detection of methylation at cellular level − quantitative only when DNA is not basepaired Headloop Suppression PCR − amplification of trace amounts of methylated DNA in tissues or body fluids − potential for clinical applications MS-MPLA (Methylation− simultaneous detection of CpG methylation and Specific Multiplex Ligationcopy number changes in a large number of Dependent Probe genes within minimum amounts of DNA Amplification) SssI Methyltransferase − rapid indirect estimation of overall methylation Assay status of DNA through quantification of incorporated labeled methyl groups MALDI MS (Matrix− precise quantification of CpG-methylation Assisted Laser status Desorption/Ionisation Mass Spectrometry) Bisulfite Sequencing − quantitation of methylated C within a target sequence Southern Hybridization − quantitative detection of methylation at single gene loci, but limited to CpGs in restriction sites MSP (Methylation Specific − detection of low level methylation at specific PCR) CpG sites Fluorescent MSP − qualitative MSP method using fluorescently labeled primer and automatic gene sequencer for analysis of the PCR products Microchip Electrophoresis − rapid and accurate analysis of PCR products from methylation-specific PCR COBRA (COmbined − quantitative detection of methylation at specific Bisulfite Restriction sites, but not for high throughput studies Analysis)

Reference [123] [124]

[125, 126]

[127] [128]

[129]

[130]

[131]

[103] [132]

[104] [108, 133]

[105, 134] [105]

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Sabrina Schlosser and Michael C. Frühwald Table 1. Methods to study DNA-methylation (Continued)

Methods to study/ verify Application methylation MS-SnuPE (Methylation- − quantification of average methylation at a specific Sensitive Single site Nucleotide Primer Extension) MethylQuant − high throughput, methylation-specific real time PCR using SYBR Green I allowing analysis of the methylation status of a single specific C MethyLight − high throughput, quantitative methylation assay using fluorescent real-time PCR (TaqMan) to determine the relative amounts of a special methylation pattern ConLight-MSP − use of an additional fluorescent probe to detect unconverted DNA and avoid false positive results in MethyLight HeavyMethyl − real-time PCR assay using methylation- specific oligonucleotide blockers and sequence specific fluorescent probes − potential for analysis of low concentrations of methylated DNA in clinical samples Melting Curve Analysis − based on differences in melting temperatures using LightCycler between methylated and unmethylated alleles as melting temperature increases with increased GC content McMSP (Melting Curve − high throughput method for qualitative MSP) determination of methylation of a CpG site McCOBRA (Melting − quantitative estimation of methylation frequency CurveCOBRA) at a specific locus with potential for high throughput application IP RP HPLC (Ion Pair − methylation detection of specific CpG sites Reverse Phase HPLC) Chloracetaldehyd − fluorescent assay for detection of DNA Reaction methylation in any sequence context (CpNpG), not only in CpG dinucleotides MS-SSCP (Methylation- − rapid screening for methylated CpG sites in Sensitive Single-Strand known sequences Conformation − allows screening for methylation within large Polymorphism) DNA regions requiring only ng amounts of DNA

Reference [135]

[107]

[108]

[136]

[109]

[137]

[106] [106]

[138] [139]

[140]

Other methods that allow genome wide methylation scanning of gene associated CpG islands are microarray approaches [95-98]. Shi and colleagues developed a microarray system allowing the simultaneous detection of gene expression and the epigenetic phenomenons

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DNA-methylation and histone aceteylation [96]. A fingerprinting method to screen the DNA for methylation changes is MS-AP-PCR (Methylation-Sensitive Arbitrarily Primed PCR). Here the DNA is digested with methylation-sensitive and methylation-insensitive restriction enzymes such as HpaII and MspI, followed by arbitrarily primed PCR (AP-PCR). The primers associate randomly with genomic DNA and produce several PCR fragments which are separated by polyacrylamid electrophoresis. The result is a methylation-specific fingerprint with certain patterns of bands depending on the methylation status of DNA [99]. A variation of MS-AP-PCR is the so called Methylation-Sensitive Restriction Fingerprinting (MSRF) method. In addition to that Methylation-Sensitive Amplified Length Polymorphism (MS-AFLP), MS-RDA (Methylation-Sensitive-Representational Difference Analysis) or MCA (Methylated CpG Island Amplification) are some exmaples which can be used to screen the genome for aberrantly methylated CpG sites in cancer. Methods such as TLC (Thin Layer Chromatography), HPLC (High Performance Liquid Chromatography), HPCE (High Performance Capillary Electrophoresis) or the antibody detection of 5mC can be performed to determine methylation levels, i.e. the content of 5mC in DNA of cancer cells compared to normal cells. These approaches might be applied to detect DNA hypomethylation, which is a common variation of the cancer cell genome. A summary of various methods to study DNA-methylation is given in table 1.

Candidate Gene Approaches Candidate genes can either be revealed through methylation screening approaches, or by selective analysis of chromosomal regions that are frequently deleted in certain cancers. It is further possible to perform expression arrays to identify genes that are upregulated after treatment with demethylating agents and study appropriate genes with respect to aberrant methylation [100, 101]. To determine the methylation status of genes several methods can be used, some of which are briefly described below. A lot of studies are based on the chemical modification of DNA employing bisulfite treatment. After bisulfite treatment of DNA unmethylated cytosine residues are conversed to thymine, while methylated cytosines remain unchanged [102]. Thus methylation-dependent sequence differences are introduced into genomic DNA. Bisulfite sequencing can be used to determine methylated cytosine residues and their localization in the genomic sequence. Bisulfite treated DNA is amplified by PCR in which all uracil and thymine residues are amplified as thymine and only 5mC remains cytosine. The PCR product can either be sequenced directly or it can be cloned and sequenced to provide methylation maps of single DNA molecules [103]. For detection of methylated CpG sites within a CpG island, independent of methylation-sensitive restriction enzymes, MSP (Methylation-Specific PCR) can be used. After bisulfite treatment of DNA PCR is performed using two primer sets, specific for methylated and unmethylated DNA, respectively [104]. COBRA (COmbined Bisulfite Restriction Analysis) is a restriction based method using methylation-sensitive restriction enzymes, such as BstUI and TaqI. The restriction sites of these enzymes contain CpG dinucleotides and thus the presence or absence of restriction sites depends on the methylation status of the DNA [105]. McMSP and McCOBRA are the conventional methods MSP and COBRA improved by combination with

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melting curve analysis (MCA) allowing detection of the methylation status of CpG sites in a high throughput and gel-free manner. These approaches are based on differences in melting temperatures between methylated and unmethylated alleles, as melting temperature increases with increased GC content [106]. To quantifiy methylation of specific cytosines within the genome, several real-time based methods have been developed. MethylQuant uses SYBR Green I [107], whereas MethyLight is fluorescence-based real-time PCR (TaqMan) [108]. HeavyMethyl uses methylation-specific oligonucleotide blockers and sequence specific fluorescent probes to achieve methylation-specific amplification, through blocking the amplification of unmethylated sequences [109]. In addition to that there are numerous additional methods to verify methylation, some of which are summarized in table 1. After methylation of a gene has been confirmed it is of interest whether the expression of the affected gene is suppressed. To determine the expression status of genes methods like competitive RT-PCR, RT-PCR or one of the various Real-time PCR approaches to quantitatively assay gene expression can be used. To further outline a correlation between methylation and transcriptional silencing, re-expression experiments can be performed. For this purpose tumor cell lines are treated with inhibitors of DNMTases such as 5-aza2´deoxycytidine at different concentrations and for varying lengths of time and the expression after treatment is determined. Methylation and acetylation inhibitors like trichostatin A (TSA) may be combined as methylation and acetylation cooperate in gene silencing. Following the detection and confirmation of DNA-methylation, functional studies such as of introduction of appropriate genes and proteins into non-expressing cancer cells can be performed and their effect can be studied in in vivo models. A multitude of studies have been undertaken to determine DNA-methylation in a variety of human tumors. The following section comprises various genes that have been described to be aberrantly methylated in brain tumors. Besides promoter hypermethylation, genomic hypomethylation is a common event in cancer cells. Overall net losses of 5mC have been found in many human tumors including brain tumors [141]. Characteristic methylation profiles, including both hyper- and hypomethylation have been reported for glioma. In this context the MYOD1 locus has been found to be hypomethylated in pilocytic astrocytoma [142]. Several genes that are frequently changed in tumors participate in various cellular processes, such as cell cycle control, apoptosis, DNA repair, cell adhesion and angiogenesis. First of all uncontrolled cell proliferation is a hallmark of cancer. Tumor cells have typically acquired damages in genes whose products are directly involved in cell cycle control such as p14ARF, p16INK4, p15INK4b and the retinoblastoma (RB1) gene [143, 144]. The p14ARF and p16INK4a tumor suppressor genes belonging to the CDKN2A (cyclin-dependent kinase inhibitor 2A) locus in chromosome 9p21 are two genes whose expression is frequently lost in human cancers including brain tumors [145]. Both genes control the antiproliferative functions of TP53 and the retinoblastoma proteins. P14ARF leads to cell cycle arrest in a TP53 dependent manner. It stabilizes the TP53 protein by preventing its degradation through binding and promoting the degradation of MDM2, and through preventing the nuclear export of TP53 [146-148]. It has been shown that loss of p14ARF expression may be caused by homozygous deletion or hypermethylation of CpG islands in the promoter region. Hypermethylation of the p14ARF promoter has been detected in low grade diffuse

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astrocytoma, and seems to be an early event in a subset of astrocytomas that overcome malignant progression to secondary glioblastoma [145]. Inactivation of p14ARF in oligodendroglial tumors by either deletion or methylation seems to be the major mechanism for disruption of the TP53 signaling pathway, as TP53 mutations occurs rarely in these tumors. Analysis of oligodendroglioma revealed methylation as the only detectable change, while in anaplastic oligodendroglioma both promoter hypermethylation and homozygous deletion have been shown [149]. Another group has determined p14ARF promoter hypermethylation in glioma in connection with aberrant cytoplasmatic localization of MDM2 [150]. Methylation of p14ARF seems not to be influenced by the p16INK4a methylation status, even though both genes overlap with each other on chromosome 9p21 [143, 151]. Frequent and differential methylation of the p14ARF, p16INK4a and p15INK4b tumor supressor genes has further been reported for ependymal tumors [152]. A study of cell cycle control genes by Yin and colleagues has demonstrated methylation of p14ARF in brain tumors such as astrocytoma, ependymoma, oligodendroglioma. Methylation of p15INK4b, p16INK4a and RB1 have been found with a relative low frequency and only in some of the investigated tumor types [143]. P16INK4a and p15INK4b inhibit the exit from G1- to the S-phase of the cell cycle via the RB pathway. They inhibit phosphorylation of the retinoblastoma protein through binding to and inactivation of CDK4 and CKD6 [144, 153-156]. Promoter hypermethylation associated with loss of p16INK4a expression [157, 158] and inactivation of p15INK4b through methylation at multiple sites in a 5´CpG island has been reported in glioma. RB1 and p16INK4a seem to contribute to the development of subset of low-grade ependymoma, as both genes have been found to be hypermethylated in these tumors [159, 160]. The retinoblastoma protein 1 (RB1) located in chromosome 13q14.2, a cell cycle regulator whose inactivation is generally associated with malignant transformation and tumor progression, as well as TP73, a p53related gene in 1p36.3 have been found to be methylated in several brain tumors such as glioblastoma, anaplastic astrocytoma, mixed oligo-astrocytoma, ependymoma, medulloblastoma, oligodendoglial tumors, pituitary adenomas [161-164]. Not only the proliferation rate, but also the rate of cell death influences the growth of tumor cell populations. A major source of cell death is the programmed cell death, apoptosis [165]. A key initiator caspase in apoptosis is caspase 8, which is activated after ligation of death receptors such as Fas receptor and the TRAIL receptors DR4/DR5 [166]. One strategy of cancer cells to reach a state of uncontrolled proliferation is to escape apoptosis, whereas loss or inhibition of CASP8 may be a mechanism to circumvent programmed cell death. Reduction of CASP8 mRNA expression by methylation of the CASP8 gene located in chromosome 2q33-34 has been observed in cell lines of childhood medulloblastoma that are resistant to apoptosis induction by TNF-related apoptosis-inducing ligand (TRAIL). Thus loss of CASP8 gene expression is for at least a subset of childhood brain tumors a putative mechanism to evade TRAIL induced apoptosis [167]. Significant lower expression of the CASP8 gene in a subset of childhood medulloblastoma compared to normal brain tissue has also been reported by another group. Although aberrant methylation seems to be a common reason for loss of CASP8 expression in medulloblastoma cell lines, no correlation between reduced expression and methylation have been observed in primary sPNET or medulloblastoma tumor tissues [168]. Mühlisch and colleagues analysed methylation of the 5´-CpG rich region of CASP8 in sPNET and AT/RT. They found that methylation of CASP8

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at least in the analyzed region seems not to be relevant in these tumors [169]. In contrast diminished expression of CASP8 in connection with DNA-methylation has been detected in pediatric medulloblastoma by Harada et al. Interestingly the methylation of CASP8 and the tumor suppressor gene RASSF1A were highly correlated in the evaluated medulloblastoma [170]. RASSF1 is a tumor suppressor gene located in chromosome 3p21.3 encoding two isoforms, one of which is RASSF1A (ras-association domain family, isoform 1 A). RASSF1A is a 340-amino acid protein located in the cytoplasm with a 97% identity to the mouse protein [171]. RASSF1A is frequently inactivated by methylation rather than by mutational events. In fact it is silenced by DNA-methylation in more than 50% of solid tumors with different histological features [172, 173]. Methylation and inactivation of RASSF1A has been demonstrated in many adult and childhood cancers including primary brain tumors, especially medulloblastoma and glioma. It seems likely that the primary mechanism of RASSF1A inactivation in adult glioma and both, adult and pediatric medulloblastoma is biallelic promoter hypermethylation. Loss of RASSF1A expression appears to play a role in malignant brain tumors rather than benign neoplasms, as inactivation of the RASSF1A gene has been found to be more common in medulloblastoma, grade II and grade III glioma as well as glioblastoma than in pilocytic astrocytoma, schwannoma and meningeoma. Loss of RASSF1A further seems to be involved in the formation and progression of glioma, as a correlation of methylation and higher tumor grade has been noticed [170, 173-176]. The epigenetic inactivation of the RASSF1A tumor suppressor gene has also been reported in adult and pediatric ependymoma and represents the most common tumor-specific variation in these tumors that has been identified so far [177]. Methylation of the RASSF1A promoter region associated with repressed gene expression has also been reported for sPNET and AT/RT, two embryonal tumors of childhood [169]. Lack of RASSF1A occuring frequently in several tumors might block death receptor mediated apoptosis and provide a mechanism for tumor cells to resist this apoptotic pathway [178]. The RASSF1A protein further functions in regulation of cell proliferation. It has been shown that RASSF1A negatively regulates cell proliferation through inhibition of G1/S-phase progression by inhibition of cyclin D1. Furthermore RASSF1A regulates mitotic progression and plays a role in facilitating correct mitosis. In this context the protein has been reported to interact with Cdc20 resulting in inhibition of the anaphase-promoting complex and prevention of cyclin A and cyclin B degradation until the spindle checkpoint becomes fully functional, and chromosomes are correctly arranged in the cell [179]. RASSF1A also seems to contribute to mitotic arrest through interaction with and stabilization of microtubules [180, 181]. Disturbed function of these checkpoints may lead to genomic instability and transformation [182]. To summarize the RASSF1A protein exerts several tumor suppressive functions and its loss affects important mechanisms in tumorigenesis such as cell cycle progression, cell adhesion, cell migration, angiogenesis, and apoptosis [183]. Located upstream of RASSF1 in 3p21.3 is the ZMYND10 gene (zinc finger, MYND typecontaining protein 10; ZMYND10), which is also known as BLU. Silencing of BLU has been shown to occur to a similar extent in all grades of adult glioma [174]. Promoter hypermethylation of the BLU gene and downregulation of its expression has been observed in lung, breast, kidney, neuroblastoma and nasopharyngeal tumor cell lines suggesting a role for epigenetic inactivation of BLU in the pathogenesis of common human cancers. Structural

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features of the protein suggest involvement in important transcriptional regulation pathways, but its function remains to be determined [184]. A further apoptosis associated gene that has been shown to be aberrantly methylated in brain tumors is TMS1/ASC (target of methylationinduced silencing 1/apoptosis-associated speck-like protein containing a card), also known as PYCARD (PYD and CARD domain containing) which is located in chromosome 16p12-11.2. The gene encodes an intracellular signaling molecule with putative roles in apoptosis and in regulation of NFκB and cytokine maturation via caspase 1. It´s loss due to methylationmediated silencing could contribute to tumorigenesis by providing a mechanism for tumor cells to circumvent apoptosis, to evade anti-tumor immune responses and to allow uncontrolled NFκB mediated transcription of anti-apoptotic and proliferation genes. Aberrant methylation of TMS1/ASC has been reported in glioblastoma samples by Stone et al. [185, 186]. HIC-1 (hypermethylated in cancer) located in 17p13.3 is a candidate tumor suppressor gene that seems to play a role in several cancers including brain tumors. Various studies reported hypermethylation of this gene in human tumors such as breast tumors, colorectal cancer, hepatocellular carcinomas, lung cancer, acute lymphoblastic leukaemia and brain tumors [187-191]. Hypermethylation of HIC-1 was reported in medulloblastoma, the most common nonglial malignant brain tumor in children and correlated with poor prognosis [192194]. Although mutational analysis of the HIC-1 coding region revealed a single deletion in the second exon leading to in-frame deletion of four amino acids, strikingly reduced HIC-1 expression is caused by altered CpG island methylation in a subset of medulloblastoma [194]. The same group reported HIC-1 hypermethylation in human ependymoma, which predominantly occur in children and young adults. Elevated methylation of HIC-1 could be correlated to ependymoma with non-spinal location indicating that those tumors differ genetically from spinal ependymoma [195]. The HIC-1 gene encodes a transcriptional repressor containing five Krüppel-like C2H2 zinc finger motifs and a N-terminal autonomous transcriptional repression domain [196, 197]. HIC-1 also contains a TP53 binding site in the 5´flanking region and its expression is activated by wild-type TP53 [198]. HIC-1 deficient mice die perinatally, show reduction in overall size, developmental defects and mice that lack one copy of the HIC-1 gene develop many different spontaneous malignant tumors [199, 200]. In addition to that re-expression experiments in tumor cell lines have revealed growth suppressing abilities of HIC-1. A certain association of HIC-1 methylation and tumor aggressiveness has been found [193]. Deltour et al. demonstrated that HIC-1 mediates transcriptional repression by both HDAC-independent and HDAC-dependent mechanisms [201]. Another enzyme of interest in tumor biology of which epigenetic changes in brain tumors have been reported is the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Alkylation of DNA at the O6-position of guanine is an important step in the generation of mutations in cancer. On one hand O6-methylguanine tends to pair with thymine during replication resulting in a conversion of G:C to A:T pairs in the DNA, on the other hand O6-methylguanine may cross-link with opposite cytosine residues and block replication [202]. The O6-methylguanine-DNA methyltransferase removes cytotoxic chlorethyl and methyl adducts from the O6 position of guanine to its own cysteine residues leading to inactivation of one MGMT molecule for each repaired lesion [203]. In this way MGMT not

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only protects cells from accumulating mutations, but also exerts a protective effect to tumor cells and contributes to the resistance of tumors to alkylating chemotherapeutic agents such as BCNU (carmustine) and temozolomide [204, 205]. Several studies have been undertaken to determine the content and activity of MGMT as well as the regulation of its expression in human tumors. The MGMT gene located in chromosome 10q26 was found to be expressed in a large number of human tumors including brain tumors. Remarkably, MGMT activity is generally higher in malignant tissues compared to the corresponding normal tissue [206-208]. For example Bobola and colleagues studied 110 pediatric brain tumor samples and detected MGMT activity in 94% of the tumors. Only six of the investigated tumor samples, which were astrocytic glioma and ganglioglioma/DNT, lacked detectable MGMT activity. The MGMT content was age dependent, associated with tumor malignancy and appeared up to 9fold higher in tumors compared to the adjacent normal brain tissue [206]. Similar results were obtained by Hongeng et al., who found that medulloblastoma and ependymoma had the highest level of MGMT [207]. Thus tumorigenesis in pediatric brain tumors appears to be frequently accompanied with increased MGMT activity. Nevertheless epigenetic changes, i.e. hypermethylation of the MGMT gene promoter were observed in malignant astrocytomas, medulloblastoma, oligodendroglioma, ependymoma and in serum DNA of patients with glioblastoma multiforme [202, 209-214]. Methylation of the MGMT gene has also been found in low-grade and anaplastic ependymomas, suggesting inactivation of MGMT to be a key step in the formation of ependymoma [160]. MGMT methylation could be linked with gene silencing in some studies on brain tumors [202, 214], whereas others did not find a correlation between MGMT methylation and mRNA expression in pediatric medulloblastoma [210]. A recent study of anaplastic glioma did not reveal a statistical correlation between MGMT expression and MGMT promoter hypermethylation and between MGMT promoter methylation and survival, respectively, but a correlation between MGMT protein expression and survival of patients after chemotherapy. Taken together, these results indicate that epigenetic inactivation of the MGMT gene plays a role in human primary tumors including those of the central nervous system, but may be due to a more complex regulation of MGMT expression than regulation solely by promoter methylation [215]. In view of the controversial results regarding the relationship between promoter hypermethylation and lack of MGMT expression it is important to mention the possibility that cells carrying promoter hypermethylation may represent only a certain part of the tumor. Recent studies indicate that patients with glioblastoma and oligodendroglial tumors in which the MGMT promoter is methylated have a favorable outcome after treatment with alkylating agents (i.e. temozolomide) compared to patients who do not have a methylated MGMT promoter [214, 216]. Thus MGMT inhibitors such as the pseudosubstrate O-6-benzylguanine (BG) may have a potential role in therapy of patients with unmethylated MGMT promoter [205, 217]. It has further been reported that methylation of MGMT in serum and tumor DNA of patients with glioblastoma multiforme predicts response and time to progression in BCNU (carmustine)treated patients [212]. Lack of MGMT activity apparently has two consequences for cancer cells. On one hand it may lead to accumulation of mutations in cancer related genes such as the tumor suppressor gene TP53 or the oncogene KRAS [202, 218], on the other hand it’s loss makes tumors more sensitive to chemotherapy using alkylating drugs [204].

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Hallmarks of human cancer are invasion and dissemination, for which cellular processes such as migration and cell adhesion play major roles. CDH1 (ECAD) is a tumor/invasion suppressor gene located in chromosome 16q22.1. It encodes a calcium dependent epithelial cell-cell adhesion molecule that prevents invasive growth and metastasis. Downregulation of CDH1 expression in human cancers often correlates with strong invasive potential, poor prognosis and malignancy [219, 220]. Hypermethylation of CDH1 has been reported for astrocytoma, medulloblastoma and pituitary adenoma [83, 221-223]. PCDHGA11 (protocadherin-γ-subfamily A, 11) has recently been shown to be a new target gene hypermethylated and epigenetically silenced in glioma grade II, III and IV [224]. The PCDHGA11 gene is located in chromosome 5q31 and is part of the protocadherin gene cluster [225]. Protocadherins belong to the cadherin superfamily of calcium-dependent adhesion molecules which are highly expressed in the nervous system [226, 227]. Members of the protocadherin-γ-cluster are supposed to be involved in synaptic connections and cellcell interactions of neurons. PCDHGA11 might also play a role in cell-cell interactions of glial cells [224]. Therefore loss of PCDHG-A11 expression in astrocytoma may cause defective cell-cell junctions and release tumor cells to infiltrate surrounding normal brain tissue, which is a characteristic feature of invasive astrocytoma. SLIT2 (human homolog 2 of the drosophila slit gene) appears to participate in cell migration processes, which are crucial for normal neuronal development and tumor invasion [228]. Promoter hypermethylation and associated silencing of SLIT2 (4p15.2) has been detected in primary glioma and glioma cell lines. Previous reports also show epigenetic inactivation of SLIT2 in primary lung, breast and colorectal cancer [229-231]. In drosophila slit is a secreted glycoprotein that regulates axon guidance, branching and neuronal migration during development of the central nervous system via interaction with the roundabout (Robo) receptor [232]. Similar results have been obtained for vertebrates, where slit has been reported to be a repellent for olfactory bulb axons [228, 233]. The EMP3 (epithelial membrane protein 3) gene located in chromosome 19q13.3 encodes a protein that belongs to the peripheral myelin protein 22 (PMP2) gene family. Just as other members of this family the EMP3 gene product is thought to be involved in cell proliferation and cell-cell interactions [234]. Transcriptional silencing of EMP3 by promoter hypermethylation has been demonstrated in glioma. Indeed EMP3 seems to be a good candidate for the long-sought tumor suppressor gene in chromosome 19q13, a region frequently deleted in these tumors [235]. Recently a new putative tumor suppressor gene named DLC1 (deleted in liver cancer 1) was identified from hepatocellular carcinoma. It is located in chromosome 8p21.3-22 and encodes a protein that shows high homology to rat p122 RhoGAP [236]. RhoGAPs (Rho GTPase activating proteins) are one of the major classes of regulators of Rho GTPases. They stimulate the intrinsic GTPase activity of Rho to convert the active GTP-bound Rho protein into the inactive GDP-bound form. Rho GTPases are involved in various cellular functions such as cytoskeletal organization and migration, growth, differentiation, apoptosis, neuronal development as well as synaptic functions [237]. In addition to that Rho GTPases play a role in Ras-mediated transformation [238]. The tumor suppressing potential of the DLC1 protein has been shown in experiments with human breast and colon cancer cell lines, where cells were stably transfected with DLC1 and injected in nude mice. As a result tumorigenicity in

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the animals was significantly reduced. In addition DLC1 has been shown to inhibit cell growth in vitro and to induce apoptosis, likely through activation of caspase 3. Inhibition of migration by DLC1 in vitro indicates that DLC1 might be relevant for tumor cell dissemination, the major cause of cancer death [239]. This idea was supported by the discovery that DLC1 expression is diminished in metastatic human cancer cell lines compared to their non-metastatic counterpart. Restoration of DLC1 expression in metastatic breast cancer cells in vitro resulted in inhibition of migration and invasion and to a reduced ability of these cells to form metastases in athymic mice [240]. Actually reduction and loss of DLC1 expression has been reported in several human primary tumors and tumor cell lines and is associated with epigenetic changes of the DLC1 gene in some tumors [241-243]. Recently the DLC1 gene has been shown to be epigenetically inactivated by promoter hypermethylation in one case of a supratentorial primitive neuroectodermal tumor (sPNET). In medulloblastoma (MB), which are histologically similar to stPNET, DLC1 expression was found to be reduced as well, but not due to transcriptional silencing by promoter hypermethylation. Histone deacetylation and other mechanisms except for promoter hypermethylation, genomic deletion and mutation might contribute to reduced expression of DLC1 in medulloblastoma. Further evidence has to be gathered, as loss of DLC1 expression due to promoter hypermethylation has been shown in only one of the investigated sPNET samples [244]. The TIMP3 (tissue inhibitor of metalloproteinase-3) gene in chromosome 22q12.1-13.2 is silenced by promoter methylation in some brain tumors [245]. Recently promoter hypermethylation of TIMP3 was detected in a subset of astrocytic tumors. According to the findings of other groups a connection between promoter hypermethylation and gene silencing is proposed. It seems that loss of TIMP3 expression in these tumors might play a role in the progression to secondary glioblastoma [246]. TIMP3 belongs to the family of TIMP proteins that inhibit the proteolytic activity of matrix metalloproteinases (MMPs). It is the only member of this protein family that is exclusively found in the extracellular matrix (ECM) [247]. The local balance between TIMPs and MMPs plays a crucial role in ECM remodeling during development and in diseases such as cancer [248]. Recently a new function for TIMP3 which seems to be independent of its MMP-inhibitory activity has been discovered. TIMP3 may inhibit vascular endothelial growth factor (VEGF)-mediated angiogenesis by blocking the binding of VEGF to VEGF receptor-2, thus inhibiting downstream signaling. This could be one mechanism for TIMP3-mediated suppression of tumor growth [249]. Loss of TIMP3 expression may provide advantages to the tumor by allowing expansion of tumor cells due to increased action of MMPs, through the induction of growth factor release or by favoring angiogenesis [248]. Thrombospondin-1 (THBS1) is an angiogenesis inhibitor that might be important in tumor biology, as neovascularization is a common feature of human cancers. Methylation of THBS1 (15q15) and associated silencing of expression has been reported in various human tumors including adult and pediatric ependymoma and glioblastoma multiforme [160, 250]. A tumor suppressor gene that affects multiple cellular processes is PTEN (phosphatase and tensin homologue). The gene is located in chromosome 10q23.3 and encodes a protein that negatively regulates the Akt signaling pathway. Aberrant methylation of PTEN has been observed and seems to be associated with focal loss of PTEN expression in glioblastoma

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[251]. PTEN is mutated in 20-40% of primary glioblastoma and loss of heterozygosity (LOH) on chromosome 10 occurs in about 70% of these tumors. Inactivation of PTEN and overexpression of VEGF appear to be the two most common events observed in high-grade malignant glioma. Therefore PTEN is a major contributing factor to the tumorigenesis of these malignancies. In glioblastoma cell lines PTEN induces growth suppression by blocking cell cycle progression in the G1-phase, probably through negative regulation of the PI3-kinase/Akt signaling pathway [252]. As also shown in glioblastoma cell lines, PTEN blocks the expression of the hypoxia inducible genes VEGF, COX1, PGK1 and PFK and prevents the stabilization of HIF1 by hypoxia. Therefore loss of PTEN is supposed to contribute to tumor expansion by deregulation of Akt activity and HIF1-regulated gene expression [253]. Furthermore PTEN may be a regulator of tumor cell invasion and metastasis by influencing focal adhesions. This seems to be likely as the PTEN protein exhibits extensive homology to tensin, that interacts with actin filaments at focal adhesions [254]. Another factor that is associated with the Akt signaling pathway is CTMP (carboxylterminal modulator protein) or THEM4 (thioesterase superfamily member 4), which is located in chromosome 1q21.3. CTMP negatively regulates the protein kinase B/Akt by binding directly to the C-terminal regulatory domain of PKBα and preventing its phosphorylation [255]. Hypermethylation and transcriptional repression of CTMP has been shown in glioblastoma [256], whereas loss of CTMP might allow uncontrolled Akt-mediated signaling, a mechanism which has frequently been observed in these tumors [257, 258]. MCJ (DNAJC15, DnaJ [Hsp40] homolog, subfamily C, member 15) has recently been shown to be methylated and epigenetically silenced in malignant brain tumors in children (MB, sPNET, ependymoma). No methylation was detected in normal brain tissue indicating that MCJ methylation is a tumorspecific event [259]. The MCJ gene is located in chromosome 13q14.1 and has been reported as a new member of the DNAJ-protein family [260]. J-family domain proteins, also called Hsp40 chaperone family proteins, contain a 70amino-acid functional J-domain and act as co-chaperones, recruite Hsp70 chaperone partners and accelerate the ATP-hydrolysis step of the chaperone cycle [261]. The role of downregulated MCJ expression in malignant pediatric brain tumors and possibly in other brain tumors has to be further analyzed. Studies of ovarian cancer indicate that loss of MCJ expression is in part due to methylation [260] and linked with increased chemotherapeutic drug resistance and poor overall survival in these malignancies [262]. The SLC5A8 gene (solute carrier family 5, member 8) located in chromosome 12q23.1 has been reported to be frequently methylated in human astrocytoma and oligodendroglioma. Indeed in low-grade astrocytoma and oligodendroglioma aberrant CpG island methylation seems to be the most common mechanism for inactivation of SLC5A8 [94]. Recently SLC5A8 has been identified as a potential tumor suppressor gene in colon cancer and seems to have a similar role in glioma [263]. Thus far it is known that SLC5A8 encodes a transporter of the Na+/glucose cotransporter gene family that transports short chain fatty acids and other monocarboxylic acids such as pyruvate or butyrate in a Na+-dependent manner [264, 265]. Detailed functional identity and the mechanism how SLC5A8 facilitates growth suppression remain to be detected [94].

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Table 2. Aberrantly methylated genes in CNS tumors Gene (Location) RASSF1A (3p21.3)

Tumor Type Glioma

Astrocytoma Ependymoma Medulloblastoma

HIC1 (17p13.3)

MGMT (10q26)

sPNET AT/RT Schwannoma Meningeoma Glioblastoma multiforme Ependymoma Medulloblastoma

Glioma Oligodendroglial tumors Asrocytoma Astrocytic tumors Anaplastic glioma Oligodendroglioma Ependymoma

Non-Glioma Medulloblastoma Schwannoma

Methylation Frequency Reference Among Studied Tumors 36/63 (57%) 25/46 (54.3%) 13/41 (31.7%) 37/53 (69.8%) 17/20 (85%) 41/44 (93.2%) 14/16 (88%) 5/5 (100%) 27/34 (79%) 19/24 (79.2%) 4/6 (66.7%) 1/10 (10%) 2/12 (16.7%) 60% 43/52 (83%) 17/44 (38.6%) 33/39 (85%) 12/15 (80%) 26/36 (72%) 54/140 (38%) 46/52 (88%) 19/53 (35.8%) 17/45 (38%) 13

20/40 (50% 33/41 (80%) 7/27 (26%) 2/7 (28%) 1/20 (5%) 1/26 (3%) 28/37 (76%) 9/44 (20%)

[174]1 [175]2 [269]3 [223]4 [177]5,B [272]D [170] [175] [176] [169]D [169]D [175] [175] [268]6,7,B [195]B,C [272]8,D [194]C [193] [192]910B [202] [214]11 [223] 4 [209]12 [211] [215]14,A [213]15,B [160]16 [213] 15,B [177]5,B [202]17 [210]A,C [271]B,C

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Table 2. Aberrantly methylated genes in CNS tumors (Continued) Gene (Location) CASP8 (2q33-34)

Tumor Type Ependymoma Medulloblastoma

p14ARF/ CDKN2A (9p21)

Medulloblastoma sPNET AT/RT Schwannoma Glioma Oligodendroglial tumors Astrocytoma Astrocytic tumors Oligodendroglioma Oligodendroglioma grade II Oligodendroglioma Anaplastic oligodendroglioma Oligodendroglioma grade II Ependymoma

p16INK4a/ CDKN2A (9p21)

Meningeoma Medulloblastoma Glioma Glioma Astrocytoma Astrocytic tumors Astrocytoma Glioblastoma Glioblastoma multiforme Oligodendroglial tumors Oligodendroglioma Anaplastic Oligodendroglioma Ependymal tumors Ependymoma Medulloblastoma

Methylation Frequency Reference Among Studied Tumors 1/27 (4%) 4/20 (20%) 90% 14/39 (36%) 81% 6/11 (55%) 8/24 (33%) 4/6 (67%) 5/44 (2.2%) 2/22 (9%) 14/34 (41%) 7/44 (0.16%) 23

6/29 (21%) 2/7 (29%) 18/41 (44%) 3/20 (15%) 10/20 (50%) 6/28 (21.4%) 10) 23/108 (21%) 1/2 (50%) 5/19 (26%) 2/44 (4.5%) 3/41 (7.3%) 10/42 (24%) 1/53 (1.9%) 13

3/30 (10%) 14/23 (60.9%) 5% 11/34 (32%) 9/41 (22%) 1/20 (5%) 26/123 (21%) 5/27 (18.5%) 4/ 23 (17.4%)

[160]16 [177]5,B [221]18,C [272]8,D [273]19 [168]20,C [169]47,A,C,D [169]47,A,C,D [271]B,C [150]21 [274]22,D [143]C [211] [275]24,B [143]C [213]15,B [275]24,B [149]25 [149]25 [152]B [143]C [143]C [272]B,D [269]3 [158] 26,D [223]4,B [211] [143]C [157]B [268]7,B [274] 22,D [213] 15,B [275] 24,B [152]27,B [160]16 [92]28,D

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Table 2. Aberrantly methylated genes in CNS tumors (Continued) Gene (Location) p16INK4a/ CDKN2A (9p21)

p15INK4b/ CDKN2B (9p21)

RB1 (13q14.2)

Tumor Type

Pituitary adenoma

Brain lymphoma Schwannoma Glioma Oligodendroglial tumors Oligodendroglioma Astrocytoma Glioblastoma multiforme Ependymal tumors Meningeoma Pituitary adenoma Oligodendroglioma Astrocytic tumors Primary glioblastoma Secondary glioblastoma Ependymoma Meningeoma Pituitary adenoma

p73 (1p36.3)

Schwannoma Various NS tumors Oligodendroglial tumors Anaplastic oligodendroglioma Astrocytoma Astrocytic tumors Glioblastoma Ependymoma

Schwannoma

Methylation Frequency Among Studied Tumors 2% 3/44 (6.8%) 30/42 (71.4%) 38/72 (52.8%) 20/24 (83.3%) 3/10 (30%) 5/44 (11.3%) 29

14/34 (41%) 1/7 (14%) 1/30 (3%) 2% 23/71 (32%) 1/19 (5%) 15/42 (35.7%) 1/41 (3%) 31

5/35 (14%) 9/21 (43%) 1/27 (4%) 1/7 (14%) 1/19 (5%) 8/30 (26%) 12/42 (28.6%) 2/44 (4.5%) 26/136 (19%) 17/44 (39%) 4/26 (15%) 25/53 (47.2%) 31 5/28 (18%) 9/27 (33%) 1/7 (14%) 1/20 (5%) 12/44 (27%)

Reference [221]18,C [272]B,D [276]B [277]B [278]B,C [279]B [271]B,C [142]30,C [274] 22,D [143]C [143]C [268]7,B [152]27,B [143]C [276]B [213]15,B [211] [280] [280] [160] 16 [213]125B [143]C [163] [276]B) [271]B,C [161]32,B [162]33,34 [164]D [223]4 [211] [164]D [160]16 [213]15 B [177]5 B [271]B,C

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Table 2. Aberrantly methylated genes in CNS tumors (Continued) Gene (Location) p73 (1p36.3)

WT1 (11p13) IRF7 (5q23-31) MT1A (16q13) OCT6 (1p34.1) CDH1/ECAD (16q22.1)

TIMP3 (22q12.1-13.2)

AR (Xq11.2-12) DBCCR1 (9q32-33)

Tumor Type Oligodendroglial tumors Anaplastic oligodendroglioma Astrocytoma Astrocytic tumors Glioblastoma Ependymoma

Schwannoma Astrocytoma Astrocytoma Astrocytoma Astrocytoma Astrocytoma Medulloblastoma Pituitary adenoma Glioma Oligodendroglioma Astrocytic tumors Diffuse astrocytoma Anaplastic astrocytoma Primary glioblastoma Secondary glioblastoma Ependymoma Medulloblastoma Schwannoma Brain tumors Astrocytoma Astrocytoma

Methylation Frequency Reference Among Studied Tumors 17/44 (39%) 4/26 (15%) 25/53 (47.2%) 31

5/28 (18%) 9/27 (33%) 1/7 (14%) 1/20 (5%) 12/44 (27%) 16/53 (30.2%) 3/27 (11%) females 11/26 (42%) males 16/53 (30.2%) 16/53 (30.2%) 17/53 (32%) 8% 3/23 (14%) 37 (50)% 29

10/41 (24%) 13

8/36 (22%) 2/10 (20%) 18/64 (28%) 20/28 (71%) 9/27 (33%) 2/7 (28%) 3% 8/44 (18%) 20/77 (26%) 22/27 (81.5%) females 0/26 males 1/53 (1.9%)

[162]33,34 [164]D [223]4 [211] [164]D [160]16 [213]15 B [177]5 B [271]B,C [223]4,B [223]4 B [223]4,B [223]4,B [223]4,B [221]18,C [92]D [222] [142]30,C [213]15,B [211] [246] [246] [246] [246] [160]16 [213]15B [221]18,C [271]B,C [245] [223]4B [223]4,B

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Table 2. Aberrantly methylated genes in CNS tumors (Continued) Gene (Location) MYOD1 (11p15) EPO (7q21) CDH13 (16q24.2) CCNA1 (13q12.3-q13) THBS1 (15q15)

GSTP1 (11q13qter) DAPK/DAPK1 (9q34.1)

SLC5A8 (12q23.1)

Tumor Type Glioma Astrocytoma Astrocytoma Astrocytoma Astrocytoma Glioma Astrocytic tumors Glioblastoma multiforme Ependymoma Schwannoma Oligodendroglioma Astrocytic tumors Ependymoma Glioma Oligodendroglioma Astrocytic tumors Ependymoma Schwannoma Glioma

ZNF342 (19q13.32) Oligodendroglioma N33/TUSC3 (8p22) ER/ESR1 (6q25.1) PTGS2 (1q25.2-25.3) CALCA (11p15.4)

29

1/53 (1.9%) 1/53 (1.9%) 3/53 (5.7%) 3/53 (5.7%) 29 13

14/42 (33%) 10/27 (37%) 16/44 (36%) 29/41 (70%) 23

2/7 (28%) 6/41 (14.6%) 27/41 (66%) 23

4/7 (57%) 2/44 (4.5%) 28/40 (70%) 16/22 (72.7%)

[142]30,C [223]4,B [223]4.B [223]4.B [223]4B [142]30C [211] [250]35 [160]16 [271]B.C [213]15.B [211]B [213]15.B [269]36 [213]15.B [211] [213]15B [271]B.C [94]37.38.D [266]39

Glioblastoma multiforme

61%

[268]7.40.B

Glioblastoma multiforme

59%

[268]7, 40.B

Glioma

29

[142]30.C

Glioma

29

[142]30.C

TMS1/ASC/ PYCARD Glioblastoma multiforme (16p12-11.2) PCDHGA11 (5q31) Astrocytoma MCJ/DNAJC15 (13q14.1)

Methylation Frequency Reference Among Studied Tumors

Ependymoma Medulloblastoma sPNET

10/23 (43%)

50/57 (87.7%) 2/20 (10%) 2/28 (7%) 3/10 (30%)

[186]D

[224]41.42.C [259]C [259]C [259]C

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Table 2. Aberrantly methylated genes in CNS tumors (Continued) Gene (Location) CTMP/THEM4 (1q21.3) EMP3 (19q13.3) BLU/ZMYND10 (3p21.3) SLIT2 (4p15.2) PTEN (10q23.3) NF2 (22q12.2) A

Tumor Type Glioblastoma

Methylation Frequency Ref. Among Studied Tumors 8/10 (80%) 43 [256]44.D

Glioma

16/41 (39%)

[235]45

Glioma

35/44 (80%)

[174]3

Glioma

37/63 (59%)

[230]46

27/77 (35%) 2/27 (7%) 14/23 (60.9%) 8/44 (18%)

[251] [160]16 [267] [271]B.C

Glioblastoma Ependymoma Schwannoma

hypermethylation not associated with reduction or loss of expression expression and/or reexpression not studied C CpG island not promoter associated or not further outlined D discrimination between partial and complete methylation 1 methylation increased with tumor grade: methylation detected in 4/10 grade II glioma, 8/15 grade III glioma, 24/38 grade IV glioma 2 glioma grade I-IV: 19 GBM, 5 anaplastic astrocytoma, anaplastic oligoastrocytoma, 5 anaplastic oligodendroglioma 3 histology not specified 4 astrocytoma grade I-IV: 14 pilocytic, 15 diffuse, 12 anaplastic, 12 GBM 5 studied ependymoma included all major clinical and histological subtypes, adult and pediatric ependymoma 6 HIC1 methylation in normal brain as well, but more extensive in tumor tissues 7 45 different tumors were studied and divided into 6 age groups, but results were not available for all genes in each tumor 8 background methylation in normal cerebellum, but methylation tumor specific 9 52 ependymoma: 21 WHO grade II, 26 grade III, 1 subependymoma, 3 grade I tumors and 1 ependymoma grade IV; methylation analysis of the 5´UTR and central region of HIC1 10 study of 4 different NotI restriction sites of the HIC1 gene situated in an EcoRI digestion fragment by Southern blotting; methylation also found in normal brain 11 study of 25 CpG sites within the promoter and hypermethylation defined as >50% of the sequenced CpG sites 13 hypermethylated in low-grade tumors in at least 45% of the cases (n=88: 24 diffuse astrocytoma, 21 anaplastic astrocytoma, 33 primary and 10 secondary glioblastoma) 14 93 tumor samples: 75 anaplastic astrocytoma and 18 tumors with an oligodendrolial component; methylation analysis of 40 tumors with a good yield of DNA 15 41 oligodendroglioma: 22 grade II oligodendroglioma, grade III anaplastic oligodendroglioma, 6 grade II-III mixed oligoastrocytoma) and 7 ependymoma B

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16

27 ependymoma: 22 WHO grade II and 5 grade III ependymoma; 14 adult and 13 pediatric tumors non glioma: 25 meningioma, 1 MB, whereas methylation was only detected in 1 MB 18 CpG island within 5´UTR that should reflect promoter methylation 19 The studied region is not located in the promoter region and actually has no features of a CpG island, therefore several experiments with cell lines have been undertaken to explore the relationship between methylation and gene silencing. In most cases methylation correlated with loss of gene function, however in some cases other inactivating mechanisms seem to matter. 20 CpG rich region of the 5´ flanking region studied; >75% methylation in the samples evaluated as methylated 21 study of different primary human tumors including glioma (histology not specified) 22 34 oligodendroglial tumors: 7 oligodendroglioma, 11 anaplastic oligodendroglioma, 8 oligoastrocytoma, 8 anaplastic oligoastrocytoma 23 hypermethylation in 15-50% of higher grade tumors and <10% of low-grade tumors (n=88: 24 diffuse astrocytoma, 21 anaplastic astrocytoma, 33 primary and 10 secondary glioblastoma) 24 29 oligodendroglioma (grade II) and 20 anaplastic oligodendroglioma (grade III) 25 hypermethylation or deletion of p14ARF 26 methylation detected reagardless of tumor grade 27 152 ependymal tumors: 15 subependymoma, 31 myxopapillary ependymoma, 68 WHO grade II and 38 WHO grade III ependymoma 28 weak methylation in normal cerebellum 29 measurable changes in methylation levels detected by MethyLight; as for TIMP3 and MYOD1 reduced levels of CpG island methylation have been detected 30 106 glioma: 46 astrocytoma of different grades, 7 oligoastrocytoma, 10 oligodendroglioma, 10 GBM 31 hypemethylation in 10-40% of all studied groups of astrocytic glioma (n=88: 24 diffuse astrocytoma, 21 anaplastic astrocytoma, 33 primary and 10 secondary glioblastoma) 32 136 tumors studied, including glioblastoma, anaplastic astrocytoma, ependymoma, mixed oligoastrocytoma, medulloblastoma, primary CNS lymphoma, and brain metastasis of solid tumors 33 investigation of methylation at the 5´region upstream and including the first exon of p73 34 44 oligodendroglial tumors: 19 oligodendroglioma, 14 anaplastic oligodendroglioma, 9 oligoastrocytoma, 2 anaplastic oligoastrocytoma 35 completely methylated, loss of expression and reactivation after demethylation 36 histology not specified 37 studied tumors include 17 WHO grade II astrocytoma, 10 grade II oliodendroglioma and 13 grade III oliodendroglioma 38 CpG island corresponding to the fragment 3D41 identified by RLGS analysis (NotI) 39 22 oligodendroglioma: 9 WHO grade II oligodendroglioma and 13 grade III anaplastic oligodendrolioma 40 methylation more frequent in tumors from patients >40 years of age and strong correlation between methylation of ER and N33 41 studied CpG island located in the first exon 42 57 astrocytoma: 34 grade II and III tumors, 23 GBM (grade IV) 43 mentioned cohort of tumor samples in which reduced CTMP mRNA levels have been detected 44 67 investigated CpG sites located between -525 and 233 of the CTMP gene 45 methylation detected in GBM and anaplastic astrocytoma 46 40 GBM and the remaining glioma samples consisted of all grades 47 promoter region not studied, but a 5’ CpG rich region that has been reported to correlate with CASP8 expression; sample evaluated as methylated if methylation level more than 50%; low levels of CASP8 methylation in control tissues (normal cerebellum and cerebrum) 17

In addition to the above described genes numerous genes have been reported to be methylated in brain tumors to a greater or lesser extent such as NF2 (neurofibromin 2), ZNF342 (zinc finger protein 342), WT1 (Wilms“ tumor 1), IRF7 (interferon regulatory factor

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7), MT1A (metallothionein 1A), OCT6/POU3F1 (Octamer-binding transcription factor 6) and AR (Androgen receptor), PTGS2 (prostaglandin-endoperoxide synthase 2) and CALCA (calcitonin-related polypeptide, alpha), ER (ESR1, estrogen receptor 1) and N33/TUSC3 (tumor suppressor cancidate 3), DAPK/DAPK1 (death-associated protein kinase 1), as well as GSTP1 (glutathione S-transferase P1) [142, 160, 211, 213, 223, 266-271]. Due to limited space and the multitude of methylation studies in various CNS tumors it is impossible to take all published data into account. A summary of a bulk of data is given in table 2.

Epigenetic Therapy In contrast to genetic alterations in cancer such as base pait mutations epigenetic changes may potentially be reversible. This feature might be used as the basis for cancer therapy to inhibit or reverse the process of epigenetic silencing. Epigenetic changes may occur early in malignant progression and have the potential to be detected even in precancerous tissues before tumor formation. Therefore strategies targeting the epigenome might also be the basis for cancer prevention [281]. Moreover the combinatorial use of conventional chemotherapeutic agents and epigenetic-based therapies may provide the opportunity to sensitize drug resistant tumors to established therapeutic approaches such as conventional chemotherapy or radiotherapeutic approaches [282]. Table 3. Representative DNA methyltransferase inhibitors DNMT Inhibitor 5-Azacytidine (Vidaza) 5-Aza-2´-deoxycytidine (Decitabine) 1-(beta-D-ribofuranosyl)1,2-di-hydro-pyrimidin-2one (Zebularine) Procainamide Procaine MG98

RG108

Comment • phase III approved for MDS (myelodysplastic syndromes) • phase II trials • cytidine analogue • stable in aqueous solution • oral application possible • 4-aminobenzoic acid derivative • agent to treat cardiac arrhythmias • 4-aminobenzoic acid derivate • anesthetic • DNMT1 antisense oligonucleotide • binds to DNMT1 and causes mRNA degradation • phase II trial • novel class of DNMT inhibitors

Reference [306] [294, 295, 307309] [310]

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Table 4. Representative histone deacetylase inhibitors HDAC Inhibitor TSA (Trichostatin A)

SAHA (Suberoylanilide hydroxamic acid)

Valproic acid

4-Phenylbutanoic acid

Butanoic acid (N-butyric acid) MS-275

N-acetyldinaline (CI-994) Depudecin Trapoxin A (TPX)

Desipeptide(FK228)

Apicidin

Comments • hydroxamic acid derivate • very potent, but reversible inhibior of HDAC • phase II trials, but high toxicity to patients • hydroxamic acid derivative • phase II trials • synthetic drugs, based on TSA structure • antitumor activity in solid and hematologic tumors has already been shown in phase I trial • short chain fatty acid • phase II trials • tested for the use in epilepsy and some cancers • short chain fatty acid • phase II trials • already tested for the use in epilepsy and some cancers • short chain fatty acid • clinical trials already started • benzamide • phase I trial • clinical activity in hematologic malignancies • benzamide • undergoing clinical trials • epoxide • natural product • epoxide • natural product, used as model for the design of novel drugs • irreversible inhibitor of HDAC • cyclic tetrapeptide • fungal metabolite • phase II trials • cyclic tetrapeptide • fungal metabolite

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[303, 304, 316-318]

[319]

[320, 321] [298, 322]

[296, 297, 323] [324] [325]

[326-328]

[329, 330]

Epigenetics, particularly DNA-methylation has the potential as a prognostic factor. Correlation between aberrant DNA-methylation and clinical parameters indicate that some genes are only methylated in certain tumor types and that some methylation patterns might be

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characteristic for certain risk groups [84, 283]. The main targets of epigenetic cancer therapy are DNA methyltransferases and histone deacetylases. Representative DNA methyltransferase inhibitors as well as histone deacetylase inhibitors are listed in table 3 and 4. 5-azacytidine, decitabine (5-aza2´-deoxycytidine) and zebularine [1-(beta-Dribofuranosyl)-1,2-dihydropyrimidin-2-one] are commonly used drugs targeting methylation [5]. 5-azacytidine and 5-aza2´-deoxycytidine are nucleoside analogues that operate through incorporation into the DNA and covalent binding of DNMT leading to their inactivation [4]. Zebularine is also incorporated into the DNA, but is stable in aqueous solution and is the first DNA demethylating agent that can be administered by the oral route [284]. As a drawback, zebularine demands high doses in the mouse model and therefore clinical studies in humans are difficult. A combination of zebularine with other demethylating agents seems to provide a promising means to lower its required dose for clinical approaches [285]. The disadvantage of nucleoside analogues is their instability in aqueous solution and their range of side effects, probably due to cytotoxic effects associated with the incorporation of these agents into the DNA. Other agents targeting DNA-methylation that are not incorporated into the DNA, are procainamide and procaine, that have originally been approved for the treatment of cardiac arrhythmias and as a local anesthetic, respectively [286, 287]. In addition to that a novel class of DNA methyltransferase inhibitors has been reported by Brueckner et al. This group has tested a new agent called RG108 that suits the catalytic DNMT1 domain and seems to act via blocking the active site of this enzyme [288]. Other approaches use analogues of the methyl donor SAM (S-adenosylmethionine) to inhibit cellular methyltransferases [289]. Novel approaches to target DNA-methylation include antisense constructs, RNA interference or ribozymes against DNA methyltransferases or other components of the DNA-methylation machinery [285]. Although the use of demethylating agents seems to be promising it has to be kept in mind that these agents might have severe side effects due to nonspecific action and could even promote the malignant transformation of cells [5]. The second group of anti-cancer drugs targeting the epigenome are histone deacetylase inhibitors (HDI). HDIs induce acetylation of histones, transcription factors and other proteins such as α-tubulin, HSP90 or β-catenin. In this context they lead to induction of cell differentiation, apoptosis as well as cell cycle arrest in G1 or G2/M phase [3]. One of the main mechanisms of action of HDAC inhibitors is the transcriptional reactivation of dormant tumor suppressor genes such as p21WAF1 [290]. Inhibition of HDAC activity in connection with chromosomal instability has to be kept in mind as it has been shown that inhibition of HDAC activity causes incorrect kinetochore localization of the mitotic checkpoint proteins and prolonged mitotic arrest leading to instable chromosomes [291]. HDIs have already been tested in in vitro and preclinical studies. It has been shown that they appear to be well tolerated. They have potential as anti cancer drugs, but non-specific side effects may not be disregarded [290, 291]. In some cases the combinatorial use of demethylating agents and HDAC inhibitors may be useful to alleviate gene repression because DNA-methylation and histone deacetylation cooperate in the process of gene silencing. It has been suggested that DNA-methylation dominates histone deacetylation in this context. It has thus been reported that the hypermethylated genes TIMP3, p15INK4b, p14ARF and p16INK4a could only be transcriptionally reactivated using TSA followed by 5-aza-2´-deoxycytidine treatment [81]. Furthermore the doses of demethylating agents might be reduced using combinatorial

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approaches with histone deacetylases, such as phenylbutyrate [292]. Although a number of substances have been clinically tested and 5-azacytidine has been appproved for treatment of myelodyplastic syndrome (MDS), there are several concerns regarding the use of epigenetic approaches for therapy of patients. Unspecific effects of epigenetic inhibitors that have been developed so far, especially DNMT inhibitors, may lead to non specific activation of genes and transposable elements in normal cells. Furthermore many of the tested agents have toxic, mutagenic or carcinogenic effects [293]. Several clinical studies exist for hematologic malignancies, but much work remains to be done regarding solid tumors, especially those of the CNS. Clinical trials of decitabine effects in solid tumors and combinations with conventional chemotherapeutic drugs have already been performed, but were not successful. Nevertheless improved dosing schedules as well as combinatorial approaches still need to be evaluated [294, 295]. To give some examples N-acetyldinaline, sodium phenylbutyrate, MS275 and depsipeptide have been studied in phase I clinical trials in solid tumors, excluding CNS tumors [296-300]. The effect of the DNMTase inhibitor fazarabine on solid tumors including high grade glioma has been studied in several phase II trials, but with poor activity, whereupon further investigation has been terminated [301, 302]. In the literature there are only few reports regarding clinical studies on demethylating agents and histone deactylase inhibitors used for the treatment of CNS tumors. Witt et al. used valproic acid to treat a child with glioblastoma multiforme after conventional treatment protocols had failed. Following valproic acid treatment, the clinical condition improved, but the tumor relapsed 16 months after initiation of treatment. Nevertheless valproic acid is tested in pediatric oncology trials [303]. A recent study evaluated the effects of valproic acid, as a non-enzyme inducing antiepileptic drug on survival and hematotoxicity, in patients with glioblastoma multiforme treated with standard chemotherapeutic agents [304]. A dose escalation study and pharmacologic study of phenylbutyrate orally administered to patients with recurrent malignant glioma has previously been published [305]. Thus the study of epigenetic mechanisms and the development of new epigenetics-based anti-cancer strategies remains an exciting challenge, as this field of research might provide a clearer understanding of human diseases including cancer and could open new therapeutic avenues.

Future Outlook In terms of understanding the basic mechanisms of epigenetic gene regulation, the influence of epigenetic changes on a global and a gene-specific level and the potential for clinical use of small molecule drugs targeting the epigenome questions clearly predominate over answers. Nevertheless a series of cues suggests the usefulness of epigenetic mechanisms in the management of patients with brain tumors. First of all as mentioned above gene-specific methylation events may represent usefull biomarkers for the prognostication of disease outcome and potentially response to therapy. In fact CSF is amenable to PCR-based analysis and might help in the early recognition of disease recurrence even before cells may be detected by microscopy. Furthermore as has been

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shown for high-grade glioma and response to temozolomide, certain methylation patterns may predict outcome and help in tailoring therapeutic strategies. In situ MSP may help detect subclones of methylated cells in tumors tissues and thus help to refine histologic diagnosis. Second HDI and DNMTase inhibitors hold great potential as therapeutic agents. HDI will certainly pass the blood-brain-barrier (BBB) due to their high lipophilicity. DNMTase inhibitors were based on their chemical structure not expected to pass the blood-brain barrier. However, as described by Chabot et al. 5-Aza-2´-deoxycytidine readily pass the BBB and during a continous infusion CSF levels reach up to 50% of those found in plasma [331]. Furthermore leukemic infiltrations of the meninges could be cleared by the use of this DNMTase inhibitor. Thus epigenetic therapy of leptomenigeal disease in CNS malignancies might be feasible. As agents in monotherapy epigenetically active substances will certainly not solve the problems of CNS cancer. Promising results have been achieved however with the addition of DNMTAse inhibitors and HDI to conventional chemotherapy. Some examples are anthracyclines, platinating agents and topoisomerase I competitors [332]. Most promising are combination therapies of HDI plus DNMTase inhibitors and conventional cytostatic drugs. Especially in the setting of pediatric brain tumors epigenetics hold great potential. These neoplasms may be seen as an aberration of development. Thus interfering with this aberration only during a critical stage of development may help to correct the aberrant developmental step. Examples for malignancies that might be influenced include such highrisk embryonal neoplasms as medulloblastoma and AT/RT. In conclusion the field of epigenetics in human brain tumors is only beginning to unravel. Much work remains to be done, but will clearly be an asset for the understanding of the pathomechanisms involved. Epigenetic therapy still needs to find its role in the clinics, however strategies such as continous low dose application of HDI (metronomic therapy) and combination strategies certainly warrant further attention.

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In: Trends in Brain Cancer Research Editor: Andrew V. Yang, pp. 253-280

ISBN 1-59454-972-9 ©2006 Nova Science Publishers, Inc.

Chapter VII

Neurotrophin Receptors and Heparanase: A Functional Axis in Human Medulloblastoma Invasion Dario Marchetti1,∗, Adam J. Kaiser1, Bryan E. Blust1, Robert E. Mrak2 and Neeta D. Sinnappah-Kang1 1

Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University-Baton Rouge, Baton Rouge, LA, USA. 2 Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR

Abstract Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Modern therapy has produced five-year survival rates as high as 70% for some MB patients, but this has come at the cost of significant long-term treatment-related morbidity. The cellular mechanisms involved in metastatic spread of medulloblastoma are largely unknown. Neurotrophins (NT) comprise a family of structurally and functionally related neurotrophic factors that are critical for central nervous system (CNS) development, and nerve growth factor (NGF) is the prototypic NT. NT act through two groups of structurally unrelated neurotrophin receptors (NTR): a family of receptor tyrosine kinases (Trks, mainly TrkA, TrkB, and TrkC) and a tumor necrosis factor receptor (TNFR)-like molecule called p75NTR. TrkC expression is a good prognostic indicator for MB. TrkC binds only to neurotrophin-3 (NT-3) whereas p75NTR binds to all NT family members. Importantly, little is known about the biological functions of p75NTR in primitive neuroectodermal tumors such as MB. In contrast, NTregulated heparanase (HPSE) is a unique ECM-degrading enzyme associated with tumor ∗

Correspondence concerning this article should be addressed to Dr. Dario Marchetti, Department of Comparative Biomedical Sciences, Room 2522, School of Veterinary Medicine, Louisiana State University-Baton Rouge, Baton Rouge, LA 70803, USA. Phone No: (225) 578-9897; Fax No: (225) 578-9895; E-mail: [email protected].

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Dario Marchetti, Adam J. Kaiser, Bryan E. Blust et al. angiogenesis and metastasis in a wide variety of cancers. However, the potential role of HPSE in MB and in MB invasive pathways has not been investigated. We have provided, for the first time, evidence of differential expression of HPSE in medulloblastoma, and we have shown a correlation between this expression and the invasive properties of three newly developed medulloblastoma cell lines. Equally important, we have demonstrated heparanase expression in 17 of 22 (77%) clinical medulloblastoma specimens analyzed by immunohistochemistry. This heparanase expression was found both in the cytoplasm and nucleus, with a particularly intense immunoreaction in the latter. Quantitative polymerase chain reaction revealed a negative correlation between expression of HPSE and expression of the NT-3 receptor TrkC, which is associated with a favorable clinical outcome in medulloblastoma. Activation of TrkC or TrkC/p75NTR by NT-3 was found to regulate HPSE activity and invasive properties of medulloblastoma. Taken together, our data provide initial evidence that HPSE functionality, in a context linked to TrkC and p75NTR activation, may play critical roles in medulloblastoma invasion and tumor progression.

Keywords: Neurotrophins, neurotrophin medulloblastoma, NT-3, cell invasion.

receptors,

heparanase,

TrkC,

p75NTR,

Introduction The pathogenesis of medulloblastoma (MB), the most common, malignant, and invasive brain tumor of the cerebellum in children, is still not well understood [1, 2]. Seventy percent of MB cases occur in individuals younger than 16 years of age [3]. Despite the use of craniospinal irradiation and intensive chemotherapy, MB metastatic disease remains the leading cause of treatment failure and the most significant clinical predictor of poor outcome [4, 5]. In recent years, progress has been made towards an improved understanding of the molecular genetic abnormalities that govern MB onset and/or progression. Several of these abnormalities appear to involve alterations in signaling systems that control normal cerebellar development. Scientists and physicians seeking to improve treatment outcomes for children with MB face two challenges. First, substantial mortality is still linked to both average- and high-risk MB patients. Second, children surviving after treatment suffer severe long-term side effects, i.e., cognitive impairments that affect their quality of life [4, 6]. An improved understanding of both the biology and the clinical relevance of these molecular genetic defects is essential to improve management of these tumors, thus reducing their disease- and treatment-related morbidity and mortality [5]. Through recent discoveries in cancer biology, it has become increasingly evident that normal development and tumorigenesis share many properties. Both processes involve alterations in cell proliferation, differentiation, invasion, and cell death. Altered expression of genes that are involved in normal developmental processes might therefore contribute to carcinogenesis. Key among these genes are those controlling signaling pathways involved in MB growth and spread. Elucidation of these pathways could lead to substantially improved clinical management of these neoplasms through more accurate prediction of disease risk and development of new targeted treatments.

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Neurotrophins (NT) are a family of neurotrophic factors that elicit cell-type-specific and highly diverse responses, such as differentiation, proliferation, survival, and apoptosis [2934]. Mature NT signal via two structurally unrelated neurotrophin receptors (NTR) comprised of (1) a family of receptor tyrosine kinases (Trks, mainly TrkA, TrkB, and TrkC), and (2) the TNFR-like molecule p75NTR, which binds to all NT members [29, 30]. Notably, TrkC is the putative receptor for NT-3 [35], a neurotrophin that is important in development of certain areas of the CNS. Available evidence suggests that NT/NTR function as critical determinants of metastatic tumor phenotype in the brain [15, 16, 27, 28]. Trk functions are generally trophic in nature, involving growth and survival, but they can be pro-apoptotic when over-expressed in medulloblastoma [38]. A homeostatic balance must be achieved to regulate signal transduction because the two NTR types (Trks and p75NTR) are able to independently transduce intracellular signals. Several groups have characterized interactions between p75NTR and TrkA, but the effect of p75NTR on TrkB and TrkC-mediated signaling remains poorly understood [38, 39]. All medulloblastomas appear to express TrkC, and high-levels of TrkC mRNA expression have been correlated with favorable clinical outcome, independent of age, gender or deletion of chromosome 17p (a marker for high-risk MB patients) [40-42]. Kim et al. [43] reported inhibition of medulloblastoma (DAOY cells) tumor growth through the induction of apoptosis when TrkC was activated by NT-3. In contrast, expression levels of p75NTR are not associated with either rate of disease progression or overall survival of medulloblastoma patients [44]. To date, the functional role of p75NTR in TrkC/NT-mediated signaling in medulloblastoma has not been investigated. Most of the molecular events associated with tumor growth, neovascularization, and metastasis are influenced by interactions between cancer cells and their extracellular matrix (ECM). Within the extracellular milieu, heparan sulfate proteoglycans (HSPG) are ubiquitous, being present on cell surfaces, embedded within the ECM, and present as soluble molecules [8, 15, 16]. Soluble HSPG are derived from molecules shed from the cell surface or from secreted proteoglycans. Heparan sulfate (HS) glycosaminoglycan chains can bind to and assemble ECM proteins. They can mediate both cell-cell and cell-ECM interactions [8, 15, 16]. Of equal importance, NT-regulated heparanase (HPSE) is an endo-β-D-glucuronidase that cleaves HS, a polysaccharide present on the cell surface, and the main constituent of ECM and of the vascular basal lamina [8, 15, 16]. Four groups independently reported in 1999 the successful isolation, cloning and expression of human HPSE, which represents the first and only example of purification/cloning of a mammalian HS-degradative enzyme [1821]. HPSE cDNA sequences from normal and metastatic cells are the same [18-21]. Additionally, by using anti-HPSE hammerhead ribozyme-, antisense- and small interfering RNA-mediated gene silencing, researchers have recently reported significantly reduced HPSE levels and tumorigenic properties in neoplastic cells transfected with these vectors, both in vitro and in vivo [22, 23]. HPSE is an important enzyme involved in invasive mechanisms associated with autoimmunity, inflammation, and key tumor metastatic events [15-18]. Since the cloning of the hpse gene and the development of specific probes, HPSE expression has been detected in metastatic cancers and in a variety of primary human tumors, whereas normal tissues do not

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express detectable levels of HPSE [17, 24-26]. Immunohistochemical studies have demonstrated that human HPSE is localized primarily in the perinuclear region, within lysosomes and late endosomes [9, 10], and is surface-linked and secreted [11]. Nuclear immunostaining of HPSE was also recently demonstrated in human gastric [12], esophagus [13], oral cavity and salivary gland primary tumors [14]. Notably, HPSE production and functionality are under NT regulation in brain-metastatic melanoma [16, 27, 28]. However, to date, there have been no reports linking HPSE to medulloblastoma. We have provided, for the first time, evidence of HPSE expression in medulloblastoma cell lines and clinical samples [72]. Of equal importance, we have also reported that high TrkC mRNA levels correlate with low HPSE levels, and vice versa, in MB cell lines. These findings, obtained in newly developed MB cell lines [45], led us to investigate the biological actions of activated TrkC on HPSE. On one hand, TrkC overexpression is associated with good survival, and this association has been linked to MB apoptosis in response to stimulation with NT-3 [43]. On the other hand, low expression of TrkC receptor is linked to poor survival of MB patients and to higher risk of metastasis [7, 43, 59, 60]. In addition to these findings, we have (1) examined whether low expression of TrkC alone in MB cell lines determines invasive properties when stimulated by NT-3, (2) studied the role of p75NTR in medulloblastoma when it is expressed at higher ratios compared to TrkC (p75NTR:TrkC), and (3) investigated the effects of NT stimulation on NF-kB activation in human MB cell lines.

Materials and Methods Cell Culture The three novel medulloblastoma cell lines (D556, D581 and D721) used in this study were a generous gift from Dr. Darrell Bigner (Director, Brain Tumor Program, Duke University Medical Center, Durham, NC). The comprehensive molecular cytogenetic investigation of chromosomal abnormalities for these cell lines can be found in Aldosari et al. [45]. Early-passage MB cells were grown in Richter’s Improved MEM (modified Earle’s Salts) medium (Gibco Invitrogen Corp., Grand Island, NY). Medium was supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT), and cells were incubated at 37°C in 5 7% CO2. All cell lines were grown in suspension. Cell viability was measured using Trypan blue exclusion. Briefly, a methodology involving alamarBlue (BioSource International, Camarillo, CA) was used to measure cell proliferation as per the manufacturer’s instructions. At the start of proliferation assays, a concentration of 1.0 x 106 cells/ml was used. One-hundred microliters of cells per cell line were then transferred into a 96-well plate in triplicates. Ten microliters of alamarBlue were subsequently added per well. Controls consisted of cell culture medium with dye (but without cells), and blank was medium without dye. Plates were incubated for 4 hrs (37°C, 5 - 7% CO2) before spectrophotometric readings were taken at 570 and 600 nm respectively.

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Calculations for the percentage of reduction of alamarBlue were performed according to the formula supplied by the manufacturer. To verify the extent of apoptosis (oligonucleosomal DNA fragmentation) during NT treatment, the Suicide-Track DNA Ladder Isolation Kit (CalBiochem, EMD Biosciences, San Diego, CA was used to recover fragmented and high-molecular weight genomic DNA from samples. D556 cells (1 x 106) were incubated for 24 hrs in serum-free medium (SFM) supplemented with bovine serum albumin (BSA; 0.1%) with different treatments of NT as indicated. Equal volumes of DNA from each treatment combination were resolved on a 1.5% agarose gel in tris-acetate-EDTA buffer (1X) and visualized with ethidium bromide. DNA markers and the positive control were supplied with the kit.

RNA Isolation and Two-Step Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Cells (105 – 107) were harvested and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Samples were treated with DNaseI. Total RNA (2 µg) from each cell line was used as templates to synthesize complementary DNA (cDNA) with random hexamers (Promega, Madison, WI). The same cDNA was later used for quantitation of HPSE gene expression. The following solutions were added to a final concentration of 1X M-MLV reverse transcriptase buffer: 2 µM dNTPs, 40 U rRNasin and 200 U M-MLV reverse transcriptase enzyme per tube. Tubes were then incubated for 60 min at 42°C, then for 5 min at 99°C, followed by a final incubation for 5 min at 4°C. Ten percent of volume of each synthesized cDNA was used as template in the following PCR cycle: 94°C for 3 min followed by 40 cycles at 94°C for 15 sec and 60°C for 1 min; and a final soak-cycle at 4°C (GeneAmp PCR System 7700, Applied Biosystems, CA). Each PCR reaction (25 µl) contained PCR Taq Buffer (1X), dNTPs (0.2 µM), primers (0.4 µM) and Taq polymerase enzyme (1U) (Promega). Controls consisted of identical conditions to the RT-PCR regimen, without the presence of the M-MLV reverse transcriptase enzyme (no RT reaction was performed) and were negative in all reactions. Three sets of primers were used during the PCR reaction: HPSE, TrkC and ribosomal 18S (as house-keeping gene). Forward and reverse primers for HPSE and 18S were from Murry et al [17] and Hashimoto et al [46] respectively. Primers for TrkC were: forward, 5’-GGA GAG ACA TCG TGC TGA AG-3’ and reverse, 5’-GAC AAT GTG CTC ATG CTG C-3’.

Quantitative PCR (Q-PCR) To analyze gene expression from cell lines, RNA (50 ng) was reverse-transcribed using the Sensiscript® kit (Qiagen, Valencia, CA) and random nanomers (US Biologicals, Swampscott, MA) according to the manufacturer’s recommendations. Q-PCR was performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). All samples were done in triplicates and each cell line was represented by three

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different passage numbers (within 20 - 25 passages each). The 18S rRNA assay reagents (Applied Biosystems) were used as an internal standard to normalize gene expression. The same HPSE primers used for RT-PCR were used in Q-PCR (excluding primers for 18S). TaqMan probe sequence for HPSE was from Murry et al [17]. Sequences (Integrated DNA Technologies, Coralville, IA) and probes for TrkA, TrkB, TrkC and p75NTR (Applied Biosystems) were as reported previously [47]. Data were analyzed via the SDS version 1.9 software (Applied Biosystems). The thermocycler parameters were as follows: 50°C for 2 min and 95°C for 10 min, followed by 60 cycles of 95°C for 15 sec and 60°C for 1 min.

Western Blot Analyses Cells were washed in phosphate-buffered saline (PBS) and lysed on ice with buffer containing Triton-X 100 (0.1%), sodium chloride (150 mM), Tris (20 mM, pH 7.4), a cocktail of protease inhibitors (Sigma, St. Louis, MO) and phenylmethylsulphonyl fluoride (100 mM) (Sigma) for 10 min. Protein concentration of the supernatant was determined by bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL). Equal quantities of lysates (300 µg at a concentration of 1 µg/ml) were incubated with 30 µg of pan-Trk antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 18 hrs. at 4°C. Immunocomplexes were captured by adding Protein-A Agarose (20 µl, Santa Cruz Biotechnology) and by gently rocking for 2 hrs at 4°C. The slurry was collected and washed three times with ice-cold PBS. It was then resuspended in Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) and boiled for 5 min. Equal volumes were separated on a 7.5% SDS-PAGE under reducing conditions and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). Membranes were then blocked (1% BSA, 0.05% Tween-20, in 1X TBS) before incubation with primary antibodies: TrkC (Santa Cruz) in 1X TBS containing BSA (0.01%) and Tween-20 (0.05%). The above-mentioned procedure was repeated for p75NTR detection using NGFR p75 (ME20.4) and NGFR p75 (H-92) antibodies (Santa Cruz Biotechnology) respectively. In experiments involving NT treatment, D556 cells (1.0 x 106 cells/well) were starved (6well format) in SFM for 2 hrs. Cells were then pre-treated in the presence or absence of NGF (1 µg/ml) for 30 min at 37°C and then treated with NT-3 (0 and 10 ng/ml) under identical conditions. After 15 minutes of incubation, cells were harvested as above. To detect phosphoNF-κB p65 (Ser536) (Cell Signaling Technology Inc., Beverly, MA) after NT treatments, total cell lysates from 106 cells were loaded per well on a 10% SDS-PAGE under reducing conditions. Equal loadings were evaluated using the antibody against the inactive form of NF-κB p50 (Santa Cruz Biotechnology). Specificity of NT-3 and TrkC autophosphorylation for NF-κB activation were confirmed using neutralizing antibodies to NT-3 (0.5 µg/ml) (R&D Systems, Minneapolis, MN) and the tyrosine kinase inhibitor K252a (0.5 µg/ml) (CalBiochem), respectively. Secondly, an inhibitory ligand to NF-κB (UpState, Lake Placid, NY) was used as a negative control to inhibit the translocation of transcriptionally active NFκB to the nucleus. Bound antibodies were detected with an anti-rabbit/horseradish peroxidase conjugate (Upstate, Lake Placid, NY). MagicMark XP Western Protein Standard (Invitrogen,

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Carlsbad, CA) was used as the molecular marker on the blots. The Visualizer Western Blot kit (Upstate) and CL-XPosure films (Pierce Biotechnology) were used for detection.

Immunohistochemistry (IHC) Serial sections from human medulloblastoma tissue samples were used in this study. Immunoreactions were performed on the automated immunostainer (Dako Corp., Carpinteria, CA). The protocol was as previously described (17) with minor modifications. Briefly, paraffin-embedded sections were deparaffinized and rehydrated. Endogenous peroxidase activity was quenched with H2O2 (3.0%). Pretreatment with proteinase K (Dako Corp.) was performed on all slides. Slides were blocked with horse serum (30 min) and incubated with the primary antibody at 25°C. The mouse monoclonal antibody directed against HPSE (generous gift from Dr. Motowo Nakajima, Johnson and Johnson, Toyko, Japan) was diluted 1:100. Following rinsing, slides were incubated with HRP labeled polymer (avidin- and biotin-free) conjugated goat anti-mouse IgG (EnVision, Dako Corp.). Peroxidase activity was determined using the NovaRED substrate kit (Vector Laboratories, Inc., Burlingame, CA), and cell nuclei were conterstained with Mayer’s hematoxylin. No immunoreaction was seen in the absence of primary antibody (negative control).

HPSE Activity Assays The Takara Elisa kit (Takara Mirus Bio, Madison, WI) was used in this assay for the quantitation of HPSE enzymatic activity. Cells (0.5 - 1.0 x 108) were harvested per cell line and lysed using the extraction buffer supplied with the kit. The assay was then carried out according to the manufacturer’s protocol. The kit is based on the principle that HS loses its ability to bind to basic fibroblast growth factor (bFGF) when digested by a HS-degrading enzyme. Biotinylated HS is used as a substrate for the enzyme. The undegraded substrate bound to bFGF is then detected with avidin-peroxidase, and the absorbance is measured at 450 nm. All samples were done in duplicates, and readings were taken in triplicates. For NT treatment, D556 cells (8 x 106 cells/well) were starved (6-well format) in SFM for 2 hrs. Cells were then treated in the presence or absence of nerve growth factor (NGF) (1 µg/ml) for 30 min at 37°C and treated with NT-3 (0, 0.1, 1.0, and 10 ng/ml) under identical conditions. After 12 hrs of incubation, cells were harvested as above using the Takara extraction buffer. Specificity of NT-3 effects and TrkC autophosphorylation for HPSE in vitro activity was confirmed using neutralizing antibodies to NT-3 (0.5 µg/ml) (R&D Systems, Minneapolis, MN, USA) and the tyrosine kinase inhibitor K252a (0.5 µg/ml) (CalBiochem), respectively. Readings, per treatment, were taken in triplicates. These experiments were performed twice independently. In Vitro Invasion Assays In vitro invasion assays were performed using the modified Boyden’s chamber and 12well plates [48]. Invasiveness of MB cell lines was measured through Matrigel™-coated

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Transwell filter inserts (Costar, Corning Incorporated, Corning, NY). Transwell inserts with 12-µm pores were coated with Matrigel™, and 300 µl of cell suspension (4.0 x 105 cells/ml) in serum-free medium (with 0.1% BSA) was added in triplicate wells. N-formyl-L-methionylL-leucyl-L-phenylalanine was added (5 µM) to the lower wells as a chemoattractant in serum-containing medium. After 36 hrs of incubation at 37°C, cells remaining on the upper surface of the membrane were removed with a cotton swab, and filters were fixed and stained using the Hema-3 staining kit (Fischer Scientific International, Fairlawn, NJ). The total number of invaded cells per well was counted using an inverted microscope. In order to study the in vitro invasive behavior of D556 cells, dose-dependent effects of NT-3 (0, 1.0, 10, and 50 ng/ml), in the presence or absence of NGF (1 µg/ml), were carried out for 24 hrs. NGF was added 30 min prior to adding NT-3, both directly into the upper chambers. The protocol used was as stated above. Total cells per well were then counted. All invasion assays were performed at least two times independently.

Statistical Analyses Immunoreactions of each human medulloblastoma tumor section was assigned scores ranging from 0 to 3+ as follows: negative (0), weakly positive (1+), moderately positive (2+), and positive (3+). For IHC determinations, positive cases were those that reacted with an intensity score of ≥ 1+, the baseline mark for immunopositivity. Sections were analyzed by pathologists blinded to treatment groups. The binomial test was used for statistical analysis [49]. All other data were analyzed by the general linear model (GLM) procedure of Statistical Analysis System software version 9.0 [50]. Meaningful treatment comparisons were made via single-degree of freedom contrasts.

Results HPSE/NTR mRNA Expression in Medulloblastoma Cell Lines We analyzed the expression levels of HPSE and NTR in three medulloblastoma cell lines considered in this study. First, D556 cell lines showed the highest proliferation rate followed by D581 and D721 cell lines (Figure 1). The differences between proliferation slopes generated by these cell lines were highly significant (p < 0.0001). Second, we found HPSE mRNA to be expressed in all three MB cell lines (Figures 2 and 3) with D721 having significantly higher levels of HPSE transcripts than did D556 or D581 cell lines (Figure 3). TrkA was not detected in any of the cell lines studied and TrkB was only detected in D581 cell lines (Figure 4A). TrkC mRNA was detected only in D581 and D721, with a ratio of 1711:1 (Figure 4B). Third, we observed p75NTR presence in all MB cell lines studied, and the ratio of D556p75NTR: D581p75NTR: D721p75NTR was 4: 5: 1 at the mRNA level (Figure 4C). Within the D581 cell line, the ratio of TrkB: TrkC: p75NTR was 1: 150: 3 (Figure 4A). For D721, the ratio of TrkC: p75NTR was 1: 12 (Figure 4D). In summary, quantitative real-time

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PCR of NTR expression revealed that D556 and D721 cell lines are p75NTR - dominant systems whereas the D581 cell line is a TrkC - dominant system.

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TrkC and p75NTR Protein Expression in MB Cell Lines Western blot analyses showed that TrkC protein was present in all three MB lines with the gp142 kDa TrkC (mature form) being more highly expressed in D581 than in D556 or D721 cell lines (Figure 5A). Although mRNA was not detected by RT-PCR, low levels of TrkC protein in D556 cell lines indicated expression of the trkC gene. We conclude that the mRNA ratio of TrkC: p75NTR was much lower for D556 than for D721 cell lines (see also above). p75NTR expression levels in medulloblastoma cell lines are shown in Figure 5B, where a protein at 68 kDa was detected for p75NTR. p75NTR is usually detected at a size of approximately 75 kDa, and this difference probably reflects post-translational modifications of this molecule. p75NTR mRNA and protein levels in the three cell lines were not completely concordant, raising the possibility that there can be various factors that regulate p75NTR protein stability.

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Figure 5. Western blot analysis of NT-3 receptors TrkC and p75NTR. Cell lysates (300 µg) were immunoprecipitated with pan-Trk antibody and subjected to immunoblotting with anti-TrkC antibody. Arrow indicates the 142 kDa mature form of glycosylated TrkC. Cell lysates (500 µg) were immunoprecipitated with anti-p75NTR mouse monoclonal antibody (ME20.4) and subjected to immunoblotting with anti-p75NTR rabbit polyclonal antibody (see “Materials and Methods” for details). Arrows indicates the 68 kDa mature form of p75NTR and a non-specific 62kDa protein respectively. The MagicMark Western standard marker was used to estimate the molecular weight of proteins of interest.

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HPSE Activity and the Invasive Properties of Medulloblastoma Cell Lines Next, we measured HPSE activity using a Takara Elisa kit. D556 and D721 cell lines showed significantly higher HPSE activity than did D581 cell lines (Figure 6A). HPSE activities of MB cell lines were only significantly different at the 10% confidence level (p = 0.0868, R2 = 80%, CV = 22%).

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We then determined the invasion capacities of D556 and D721 MB cell lines by seeding cells onto Matrigel-coated filters in Transwell units. Cells in the top chamber must degrade the Matrigel to penetrate the membrane filter and adhere to the underside of the filter. Such degradation of the ECM is an important factor in tumor-cell invasion and metastasis, and is mediated by a variety of degradative enzymes including proteases and glycosidases such as HPSE. D556 and D721 cell lines showed significantly higher invasive capabilities than did the D581 cell line (Figure 6B). ANOVA analysis revealed that invasive properties of the cell lines were significantly different (p = 0.0078, R2 = 80%, CV = 40%). Although D721 cell lines had highest levels of HPSE mRNA transcripts (Figure 3), their HPSE activity and invasive properties were not significantly higher when compared to D556 cell lines. This phenomenon could be attributed to the fact that D556 cells have a significantly higher proliferation rate, i.e., D556 are more aggressive in growth compared to D721 cells (p < 0.0001, Figure 1). Another possibility is that the activity assays are reflective of HPSE enzyme extracted only from cytoplasm and cell surface (excluding possible nuclear HPSE) [12-14] whereas at the mRNA level, the results reflect the relative copy numbers of the gene expressed by each cell line. We cannot exclude the possibility that expression of other proteases (e.g., matrix metalloproteinases [MMPs]) [51] contribute to degradation of ECM in these invasion assays. We used the D556 cell line in all subsequent experiments because it showed high levels of HPSE mRNA (Figure 3) and high invasive behavior (Figure 6A). This cell line also had the lowest levels of TrkC mRNA and protein (Figure 5A). The ratio of p75NTR: TrkC was greater than one for D721 cell lines.

HPSE Expression in Human Medulloblastoma Clinical Samples To translate our in vitro HPSE findings to in vivo settings, we performed Immunohistochemistry (IHC) to detect the presence of HPSE in clinical medulloblastoma samples. 77% of MB samples (17 out of 22 analyzed) analyzed by IHC expressed ≥ 1+ level of HPSE content [72]. A representative clinical sample with HPSE immunopositivity is shown in Figure 7B. This patient sample showed an intense, nuclear granular reaction of HPSE with only a weak cytoplasmic staining.

NT Effects on DNA Fragmentation To verify that the results from NT-treated invasion and HPSE assays were not adversely affected by apoptosis (as previously reported [43]), we carried out DNA fragmentation assays. Fragmentation patterns (Figure 8) showed apoptotic ladders of very low intensities in all lanes [52, 53], but no differences were seen in patterns between control samples and samples incubated for 24 hrs with NT (Figure 8). This rules out apoptosis as a confounding factor, enabling us to conclude that our results reflect true differences in NT-modulating invasion and HPSE activity.

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Figure 7. HPSE immunopositivity in MB clinical samples. A representative example of a human MB tumor sample immunopositive for HPSE is shown. A. Hematoxylin and eosin staining. B. Adjacent section showing HPSE immunopositivity. HPSE positivity was observed in the ECM surrounding MB cells using a specific monoclonal antibody to human HPSE and a peroxidase procedure. In addition, an intense, nuclear HPSE reaction was observed (arrows) with only a weak cytoplasmic reaction. HPSE immunopositivity was scored 3+. C. Negative control showing no reaction in the absence of primary HPSE antibody. Only nuclei were counterstained in blue using Mayer’s hematoxylin. Images were taken at an original objective magnification of 40X. Bars represent 20 µm.

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Effects of TrkC or TrkC/p75NTR NT-3-Mediated Activation on HPSE Activity We analyzed NT-3 effects on HPSE activity as shown in Figure 9A. ANOVA revealed that the differences in HPSE activity were highly significant for this assay (R2 = 89%, CV = 2%). All statistically significant treatment contrasts have been tabulated in Table 1A. The most important finding is that HPSE activity in D556 cell lines was significantly decreased in the presence of NGF, compared to no NGF treatment, in the absence of NT-3 (p = 0.0094, Figure 9A and Table 1A). This finding indicates that NGF-ligated p75NTR significantly downregulates HPSE activity. Additionally, after p75NTR was ligated in the presence of an excess of NGF, with NT-3 thus binding only to TrkC, the decreased HPSE activity in D556 cells was highly significantly at a NT-3 concentration of 10 ng/ml (p < 0.0001) (Figure 9A and Table 1A) compared to cells without NGF pretreatment at the same NT-3 concentration.

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NT -3 [ng/ml] Figure 9. Dose-dependent effects of NT on HPSE activity and on the invasive behavior of D556 cells. Cell were incubated in the presence or absence of NGF (1 µg/ml), followed by presence or absence of NT-3 (0, 0.1, 1.0, and 10 ng/ml respectively). At the end of incubation (12 hrs), cells were harvested and processed as described in “Materials and Methods”. All results in the bar graphs are expressed as mean ± SD (n = 2 – 3). In the absence of NT-3 (0 ng/ml), NGF pre-treatment significantly decreased HPSE activity of D556 cells (* p = 0.0094). In the presence of NT-3 (10 ng/ml), NGF pre-treatment significantly decreased HPSE activity (** p < 0.0001) (see also Table 1A). D556 cells were incubated in the presence or absence of NGF (1 µg/ml) and then incubated with varying NT-3 concentrations (0, 1.0, 10, and 50 ng/ml respectively) for 24 hrs using a modified Boyden’s chamber. Non-invasive cells were removed and invasive cells were fixed and stained (see “Materials and Methods” for details). In the absence of NT-3 (0 ng/ml), NGF pre-treatment significantly (* p = 0.0047) decreased invasion by D556 cells. In the presence of NT-3 (1.0 and 10 ng/ml), NGF pre-treatment significantly decreased invasion [p = 0.0003 (**) and p = 0.0371 (***) respectively] at the two concentrations of NT-3 (see also Table 1B).

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A comparison of all cells pre-treated with NGF with all cells without NGF pre-treatment, using the general linear method (GLM) procedure, showed a significant effect on HPSE activity of D556 cell lines (p = 0.0001, Table 1A). This was independent of the dosedependent response to NT-3 and whether or not p75NTR was ligated. By using specific inhibitors, we verified that the effects from NT treatment were not due to non-specific stimulations (data not shown). These results indicate a possible role of activated TrkC/p75NTR in affecting HPSE functionality that can be cell-type-specific. Table 1. Effects of NT treatments on D556 cell invasion and HPSE activity determined via single degree of freedom contrasts A. HPSE activity assay (1/450nm) Treatment contrast All +NGF vs. all –NGF -NGF(0ng NT-3) vs. all +NGF -NGF(0ng NT-3) vs. all –NGF -NGF(0ng NT-3) vs. –NGF(10ng NT-3) -NGF(0ng NT-3) vs. +NGF(0ng NT-3) +NGF(10ng NT-3) vs. –NGF(10ng NT-3)

p value 0.0001 0.0024 0.3810† 0.0272 0.0094 < 0.0001

Treatment contrast with p value marked with † and all other treatment contrasts not listed above were found not to be significant (see Figure 9A).

B. Invasion Assay (number of invaded cells/membrane). Treatment contrast All +NGF vs. all –NGF -NGF(0ng NT-3) vs. all +NGF -NGF(0ng NT-3) vs. all -NGF -NGF(0ng NT-3) vs. -NGF(1ng NT-3) -NGF(0ng NT-3) vs. -NGF(50ng NT-3) -NGF(0ng NT-3) vs. +NGF(0ng NT-3) -NGF(0ng NT-3) vs. +NGF(1ng NT-3) -NGF(0ng NT-3) vs. +NGF(50ng NT-3) +NGF(0ng NT-3) vs. -NGF(1ng NT-3) +NGF(1ng NT-3) vs. -NGF(1ng NT-3) +NGF(10ng NT-3) vs. -NGF(10ng NT-3) +NGF(10ng NT-3) vs. +NGF(50ng NT-3) +NGF(50ng NT-3) vs. -NGF(1ng NT-3)

p value 0.0002 0.0034 0.9391† 0.0502 0.0168 0.0047 0.0058 0.0028 0.0003 0.0003 0.0371 0.0355 0.0002

Treatment contrast with p value marked with † and all other treatment contrasts not listed above were found not to be significant (see also Figure 9B).

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Effects of TrkC- or TrkC/p75NTR- NT-3-Mediated Activation on MB Cell Invasiveness We examined dose-dependent effects of NT-3 (at 0, 1.0, 10, and 50 ng/ml) on the invasiveness of D556 cell lines (p75NTR-dominant system), in the presence or absence of NGF (1 µg/ml) pre-treatment (Figure 9B). NGF is known to bind to p75NTR but not to TrkC (or to TrkB) [29-31]. Vesa et al [39] used NGFs p75NTR-blocking capacity in a study of BDNF effects on a TrkB/p75NTR-expressing cell line, and found that NGF at high concentrations had p75NTR-blocking effects similar to those obtained using a rabbit polyclonal anti-p75NTR antibody raised against the extracellular domain of rat p75NTR (REX) [54]. Another group reported that ligation of p75NTR with BDNF or with REX had no effect on NT-3 binding to TrkA [55]. For our studies, all statistically significant treatment contrasts are shown in Table 1A. Invasiveness significantly decreased (p = 0.047) when cells were treated with only NGF and without NT-3. Since TrkC does not bind to NGF, this response to NGF is mediated by p75NTR. Blocking p75NTR through ligation with an excess of NGF (leaving NT-3 free to bind only to TrkC) resulted in significantly decreased invasive behavior of D556 cells at NT-3 concentrations of 1.0 and 10 ng/ml (p = 0.0008 and 0.0371 respectively) (Figure 9B and Table 1B) compared to cells without NGF pre-treatment at those same NT-3 concentrations. In the absence of p75NTR ligation and blockage with NGF (no NGF treatment), NT-3 probably acts through both p75NTR and TrkC receptors. These differences in invasive properties are highly significant (R2 = 92%, CV = 22%). In summary, a collective comparison of all cells pre-treated with NGF against cells without NGF pre-treatment using the GLM procedure showed a significant effect of NT-3 on the invasive behavior of medulloblastoma cells (p = 0.0002, Table 1B). This effect was independent of the dose-dependent response to NT-3 and whether or not p75NTR is ligated. These results indicate a possible functional role of TrkC/p75NTR in the invasive behavior of a p75NTR-dominant medulloblastoma cell line.

NT-Stimulation on NF-κB Activation Andela et al (2000) provided evidence for the involvement of the transcription factor NFκB as a major regulator of genes important in tumor metastasis.They also reported an upregulation of anti-metastasis genes when NF-κB signaling was blocked. One pro-metastasis gene that could be down-regulated using a dominant negative inhibitor of NF-κB was hpse. Accordingly, we examined the effects of NT-stimulation on NF-κB activation in D556 cell lines. We chose NT-3 concentrations of 0 and 10 ng/ml to correspond with the observed significant effects on HPSE activity and cell invasion at these concentrations. MB cells pretreated with NGF showed significantly lower levels of phospho-NF-κB than did cells without NGF pre-treatment (Figure 10). After NGF pre-treatment, NT-3 (10 ng/ml) stimulation via TrkC and p75NTR increased phosphorylation of NF-κB. Conversely, NGF pre-treatment (1 µg/ml), in the absence of NT-3, resulted in decreased NF-κB phosphorylation. In the presence of an excess of NGF, phosphorylation was further reduced by addition of NT-3, which under these circumstances stimulates cells only via TrkC because p75NTR is blocked by

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an excess of NGF [39] (Figure 10). Thiese results indicate a role for the TrkC/p75NTR axis in the regulation of NF-κB in human medulloblastoma. + NGF (1 µg/ml)

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Discussion Residual tumor and metastatic disease are two clinical prognostic factors linked with poor outcome in medulloblastoma [56]. Even with recent gains in survival of MB patients, secondary tumor growth is observed in up to 40% of cases, resulting in poor prognosis [57]. The cellular mechanisms underlying metastatic behavior in medulloblastoma remain obscure. Matrix metalloproteinases (MMPs) and HPSE have been linked to metastatic behavior in many tumor types. However, Özen et al [51] investigated four MMPs and their inhibitors in medulloblastoma and found that MMPs expression did not predict prognosis independent of clinical parameters. Our work on NTR and HPSE expression in medulloblastoma cell lines [70] provides, for the first time, four main observations: 1) HPSE is expressed in medulloblastoma cell lines and clinical samples; 2) HPSE content negatively correlates with TrkC at the mRNA level; 3) TrkC or TrkC/p75NTR activation affects cell invasive properties and HPSE activity in vitro; and 4), a TrkC-dominant medulloblastoma cell line (D581) is less invasive and has lower HPSE activity than do p75NTR-dominant cell lines (D556 and D721).

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The critical roles of HPSE in cancer metastasis, angiogenesis, and inflammation have been well-documented [8, 14-16]. In order to verify that HPSE expression in medulloblastoma was not restricted to in vitro cell cultures, we investigated human MB clinical samples. HPSE immunopositivity in 77% of MB samples analyzed [72] confirmed the presence of HPSE expression in primary MB and its potential relevance in this neoplasm. During development, TrkC is expressed by most mature granular cells of the cerebellum [58]. The potential functionality of Trks in medulloblastoma was first suggested by a clear correlation between TrkC mRNA expression and patient survival [44]. This effect appears to be the result of TrkC activation and transduction of differentiation and pro-apoptotic NT-3 signals [43] leading to a decreased tumor growth. In addition to TrkC, some MB tumors also express other members of the Trk family, i.e., TrkA and TrkB, as well as NT other than NT3, i.e., NGF and BDNF [59]. Anatomical co-localization of NT/NTR has been demonstrated and suggests that autocrine/paracrine loops exist in the case of BDNF/TrkB and NT-3/TrkC [59, 60]. The MB cell lines used in this study express both TrkC and p75NTR, at both the mRNA and protein levels, but do not express detectable levels of TrkA [61]. Only D581 cells express detectable levels of TrkB, and only at the mRNA level. All cell lines used expressed the nt-3 gene at the mRNA level (data not shown). Grotzer et al [42] found high TrkC mRNA expression in a majority of primitive neuroectodermal tumors (PNETs) examined, particularly in those PNETs showing neuronal differentiation. This latter observation, however, was not statistically significant due to the relatively small size of the subgroup available for analysis. Xenograft models of prostatic [62, 63] and pancreatic cancers [64], and of neuroblastoma [65], suggest that Trk inhibition can both increase the rate of apoptosis and decrease metastatic spread. Finally, dose-dependent movement of tumor cells in response to BDNF and NT-3 have been demonstrated in vitro [64]. In the presence of Trk, p75NTR acts as an accessory receptor modifying Trk function [38, 66]. The structural basis for p75NTR and TrkC interactions in medulloblastoma has not been determined. Segal et al [44] measured the relative expression of NT/NTR in medulloblastoma tumor samples from 12 patients. They concluded that patients with high indices of tumor TrkC have a significantly longer interval before disease progression than those with low indices. They also found that levels of TrkB expression are not linked to differences in disease progression. In their study, the level of p75NTR expression was not significantly associated either with the rate of disease progression or with overall survival. Conversely, the activation of p75NTR (and TrkC) does appear to be important in melanoma cell invasiveness through the upregulation of secreted HPSE and subsequent ECM degradation [28, 48, 67-69]. Our data reveal a similar function for p75NTR in medulloblastoma. We found that when p75NTR was first ligated and blocked with an excess of NGF, NT-3 - stimulated TrkC significantly decreased invasive properties, HPSE activity, and NF-κB phosphorylation in a p75NTR - dominant system. When p75NTR was unbound (no NGF treatment), and NT-3 was able to activate both p75NTR and TrkC, the in vitro invasive properties of our MB cell lines significantly increased at low NT-3 concentrations, (i.e., at 1.0 and 10 ng/ml respectively). Because NGF acts as a ligand for p75NTR, our results do not exclude the possibility that bound p75NTR was unable to alter TrkC ability to respond to NT-3.

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More importantly, in the absence of NGF p75NTR is free to bind NT-3. Under these conditions we show an increase in invasive properties at low concentrations of NT-3 (i.e., 1.0 and 10 ng/ml respectively) and NF-κB phosphorylation. These results strongly suggest that events occurring between p75NTR and TrkC, either directly or indirectly, are critical for inhibiting NT-3 signals via TrkC and thus promoting tumor invasion. Mischel et al [55] used a Xenopus oocyte microinjection assay and found that ligated p75NTR inhibited the ability of TrkA to respond to NT-3 by altering the conformation of the TrkA-binding site. Whether this molecular phenomenon occurs in medulloblastoma is not known and is worth investigating. To our knowledge, the relationship we describe between HPSE and TrkC/p75NTR has not been previously documented in medulloblastoma. A direct comparison between NT treatment effects on both the HPSE activity and invasive properties of MB D556 cells was not possible due to differences in assay environments. However, these separate assays do establish a link between HPSE functionality and dose-dependent NT-3 activation of TrkC. In addition, p75NTR appears to play a role in regulating both HPSE activity and the invasive behavior of medulloblastoma cell lines. Regulation of NF-κB phosphorylation by NT stimulation via TrkC/p75NTR also indicates possible roles of these receptors in the expression of prometastasis and anti-metastasis genes. Such signaling pathways leading to the up-regulation of the metastatic potential of medulloblastoma cells could be targeted for therapeutic interventions. Complications of radiation treatment include growth dysfunction and cognitive impairments that substantially decrease quality of life for many long-term medulloblastoma survivors. In PNETs with high TrkC mRNA expression and with no evidence of leptomeningeal tumor dissemination, therapy with reduced craniospinal radiation retains efficacy but reduces toxicity and, therefore, improves quality of life for survivors [42]. In conclusion, our data support the idea that HPSE-1, in a context linked to TrkC and p75NTR, plays a critical role in MB tumor progression. Preliminary studies on HPSE expression in medulloblastoma clinical specimens [70] highlight cytoplasmic and nuclear localizations of HPSE in tumor cells as an important determinant to consider in future studies of this cancer type.

Acknowledgements We thank Drs. Darrell Bigner (Duke University Medical Center, Durham, NC, USA) for providing MB cell lines used in these studies, and Dr. Motowo Nakajima (Johnson and Johnson, Tokyo, Japan) for providing the mouse monoclonal antibody to HPSE. We also thank Dr. Manjit S. Kang, Professor of Quantitative Genetics (LSU-AgCtr), for help with SAS analyses and for reviewing the manuscript, Dr. Daniel B. Paulsen, Professor of Pathology (LSU-SVM) to review clinical samples, Julie Millard and Sherry Ring (LSUSVM) for their help in performing I.H.C. analyses, and Dr. Andrea Greiter-Wilke (Hoffmann-LaRoche, Basel, Switzerland) for designing the RT-PCR primers to TrkC. Finally, we express our gratitude to Jason Blust for his editorial help. This work was supported by grants from the National Institutes of Health (NIH) (CA086832 and CA103955)

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to DM. Research described in this article was also supported in part by a grant from Phillip Morris USA Inc. and Phillip Morris International (to DM).

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In: Trends in Brain Cancer Research Editor: Andrew V. Yang, pp. 281-301

ISBN 1-59454-972-9 ©2006 Nova Science Publishers, Inc.

Chapter VIII

Psychiatric Manifestations of Brain Tumors Subramoniam Madhusoodanan1,∗, Abhishek Sinha2, Despina Moise3 and Sidhartha Sinha4 1

St. John’s Episcopal Hospital, NY and State University of New York Health Science Center, Brooklyn, NY 2 Royal Free University College of Medicine, London, UK 3 New Zealand Department of Medical Services, Wangenui, 4 Department of Surgery, Addenbrooke’s Hospital, Hills Road, Cambridge, UK

Abstract Psychiatric manifestations, even though uncommon with brain tumors may be the presenting symptomatology in some cases. If diagnosed early and treated satisfactorily, there may be complete resolution of the presenting symptoms. Various authors have attempted to categorize psychiatric symptoms based on the location of the tumor. Neuro imaging should be considered in patients with new onset psychosis, recurrence of previously well controlled mental symptoms or occurrence of new mental symptoms and in patients who remain refractory to psychiatric treatment. Our review of the published cases over the past 54 years indicate that neither tumor location nor type is correlated with any particular psychiatric symptoms. Mood symptoms have been noted in a significant number of cases and could be a harbinger to an evolving tumor of the brain.

Keywords: Psychiatric, Manifestations, Brain, Tumors.

∗

Correspondence concerning this article should be addressed to Dr. Subramoniam Madhusoodanan, M.D. Department of Psychiatry, St. John’s Episcopal Hospital, 27 Beach 19th Street, Far Rockaway, NY 11691. Tel# 718-869-7375; Fax# 718-869-8532; E-mail address: [email protected].

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Introduction Brain tumors presenting primarily with psychiatric symptoms are a rare occurrence. They represent an organic cause of mental disturbance that, if diagnosed at an early stage, can often be treated with complete resolution of the presenting symptoms. The presentation can vary greatly. The human brain consists of 20 billion neurons spread over an area of 2 square meters. The current view is that it does not consist of discrete higher centers segregated from one another in specific lobes of the brain, but is made up of a large number of interconnected networks spread throughout a number of different areas. Hence lesions in the brain can produce a myriad of symptoms depending on the functions of various networks underlying the damaged area. Thought, affect, mood and perception are the entities that, once disrupted, can dominate the psychiatric picture. Their neural origins remain elusive. It is intriguing that so complex an entity is rarely disrupted by the physical trauma caused by neoplastic lesions, yet appears readily susceptible to psychological trauma [1]. In the majority of cases brain tumors present with specific neurological signs due to mass effect of the tumor compressing critical structures within the brain, such as the cerebral vasculature and the underlying ground tissue. It is only in rare instances that tumors present primarily with psychiatric symptoms; this is reflected in the paucity of research in this area.

Higher and Lower Centers Brain tumors can occur in any part of the central nervous system, from the lobes of the neocortex to more primitive centers such as the pituitary and the thalamus. Cortical tumors are thought to exert their effect through the disruption of neural networks that underlie them, either directly through physical compression of the brain tissue and its vascular supply or more indirectly through alterations in the ionic constituents that are involved in neuroregulation and disruption of neurotransmitter levels, implicated in psychiatric disease. Tumors of lower centers, especially tumors of the pituitary, have a different patho physiological mechanism responsible for psychiatric symptoms. Aberrant hormone production may produce electrolyte disturbances which may contribute to the presenting psychopathology [2].

Case Reports Psychiatric manifestations of brain tumors are infrequent and there are only few studies reported in the literature. The bulk of the research data comes from isolated case reports. The

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case histories outlined below show that almost any kind of psychiatric manifestation can be the presenting symptom of a brain tumor. This ranges from simple neurotic symptoms, such as anxiety and depression [3], to frank psychotic symptoms, such as auditory hallucinations [4]. In certain cases the presenting symptomatology may be related to an evolving entity, with symptoms becoming more severe or new ones occurring over time. Sometimes this change in symptomatology proves to be the first clue to the presence of a lesion responsible for the change in affect. Ghadirian et al [5] describe a single patient who progressed through a number of psychiatric disorders before a secondary cause was determined. Their patient presented initially with symptoms of anxiety. Over a two month period she began to complain of depressive symptoms and finally she started experiencing visual hallucinations. The relationship between tumor location and the manner of presentation has been an area of debate. Some authors have correlated tumor location and psychiatric symptoms, while others have seen no such concordance. The largest case study was done is by Filley and Kleinschmidt- DeMasters [6]. They studied 8 patients, retrospectively with frontal and temperolimbic brain tumors, who presented with psychiatric illness and attempted to correlate tumor location with neurobehavioral symptoms. Patient 1 presented with apathy, social withdrawal and poor selfcare which were refractory to anti-depressant medications. A Computed Tomography (CT) scan showed the presence of an eight centimeter bifrontal mass that was histologically diagnosed as a benign meningioma. Patient 2 presented with apathy, irritability, anomia and a right hemiparesis. CT scanning revealed a 4cm lesion involving the left frontal lobe and the genu of the corpus callosum. Patient 3 presented with severe depression and extensive weight loss. A Magnetic Resonance Imaging (MRI) revealed a 3cm mass in the left frontal lobe that was reported to be a squamous cell carcinoma. Patients 4 and 5 presented with frank psychotic symptoms. Visual and auditory hallucinations were documented. CT scanning revealed tumor masses encroaching upon the left (patient 4) and right (patient 5) temporal lobes. Patient 6 presented with new onset of disorganized thinking and flight of ideas and other signs of mania. A 3cm butterfly lesion (glioblastoma multiforme) was found to penetrate both temporal lobes. Patient 7 presented with increased tremulousness, paraesthesia and diaphoresis. CT scanning revealed a pituitary tumor encroaching upon the right medial temporal lobe. The final patient presented with apathy, amnesia and poor affect. An MRI scan showed a large mass extending into the thalami and fornical columns. In their work they found that lesions located in the frontal lobes of the brain tend to produce psychiatric symptoms such as personality changes and depression, whereas those in the temperolimbic area tend to produce psychotic symptoms, such as hallucinations. A study by Avery [7] of seven patients with frontal lobe meningiomas, who presented with symptoms ranging from depression to mania, seems to support this concept. Ko and Lok [8] reported on four patients who presented with psychiatric symptomatology that were secondary to brain tumors. One patient presented with depressive symptoms, which included depressed mood, emotional lability and personality changes. He also had significant amnesic symptoms, especially for recent events. CT scanning revealed multiple metastatic lesions in the right fronto-parietal region. Chest radiography localized the

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primary tumor site to the left upper lung zone. Following surgery and chemotherapy, psychiatric symptoms improved. The second patient, a 61 year old man, presented with a six week history of difficulty with verbal expression and short term memory problems. No focal neurological signs were present. A CT scan revealed numerous metastatic lesions in the left frontal and parietal lobes. A chest radiograph revealed the primary tumor in the left lower lung zone. Surgical intervention was not advised. Their third patient, a 53 year old housewife presented with a one year history of paranoid ideations. In addition she too experienced short term memory problems. She was described as being irritable and quarrelsome, but had no other psychiatric symptoms. Abdominal examination revealed a ballotable right kidney. CT scanning confirmed the presence of a space occupying lesion in the right kidney. CT scans also demonstrated a metastatic lesion in the left parieto-occipital region, with significant shift of nearby midline structures. Due to the advanced nature of the disease, surgical resection was not advised and the patient died six months later. Their fourth patient presented with deteriorating memory, change in behavior and visual agnosia. In addition there was evidence of self neglect and poor personal hygiene. On examination she had some aphasia and agnosia and was not orientated to time and place. A CT scan showed a large, left sided parietal lobe tumor that extended to the temporal lobe with significant midline shift of neighboring structures. Operative removal was recommended, but she decided against it. She was discharged but readmitted in a comatose state three weeks later, from which she never recovered. A case report by Madhusoodanan et al [9] described a 79 year old woman who presented to the emergency room complaining of depression and not wanting to be discharged. Psychiatric evaluation of the patient indicated a possible major depressive episode with feelings of anger and agitation. No psychotic symptoms were noted. A non-contrast CT of the head showed a 5cm left parietal tumor mass with significant vasogenic edema. Operative treatment was undertaken and a significant portion of the tumor removed, this was followed by chemotherapy and radiotherapy. Following the procedure there was a temporary remission of her depressive symptoms. Binder [10] studied three patients who presented to psychiatric hospitals with altered behavior and/or thinking. Patient A was a 52 year old woman admitted to hospital for alterations in her behavioral state in the preceding week. She had been found to be wandering in the streets looking confused and exhibiting poor self-care and personal hygiene. At the time of presentation neurological signs were absent or minimal. Over the next 24 hours her condition deteriorated. She became more confused and started to develop neurological signs including hemiparaesis, increased reflexes and right facial weakness. Emergency angiography revealed a large vascular mass in the left thalamus. Despite aggressive medical treatment she continued to deteriorate and subsequently died. An autopsy revealed a glioblastome multiforme in the left thalamus compressing the lateral ventricle. The second patient in his case series was a 40 year old woman who was admitted for a sudden change in behavior. She had pressure of speech and delusional thoughts that she could speak to the dead. On examination no neurological signs were present. She was diagnosed to have a manic episode and was discharged with appropriate medications. However she was readmitted following deterioration in her mental status- with severe paranoid delusional thoughts. On her second admission a CT scan was done which revealed a hyperdense mass in

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the left lateral ventricle. Surgery was recommended and despite initial reservations, the tumor was resected. Following surgery all psychiatric symptoms resolved. The third patient in his case series was a 32 year old man who had a long history of chronic schizophrenia. He had needed many hospitalizations during this time due to rage attacks, when he would become hyperactive and violent with inappropriate affect. He was admitted to the hospital following a severe headache lasting for a month. A CT scan showed a large bilateral occipital meniginoma. Surgical removal led to a very positive long-term outcome, with a 6 year follow up showing a complete absence of rage attacks. Binder however noted that the mode of psychiatric presentation was a poor indicator of tumor location and that many other factors may influence clinical presentation. Sokolski and Denson [11] described a 51 year old female who had a long psychiatric history with manic episodes. Since the age of 21 she had had at least 9 manic episodes. She was referred by her treating psychiatrist for a double blind placebo controlled study of topiramate in bipolar disorder due to breakthrough mania following 8 months of euthymia on 1500mg of valporate. Initially she showed improvement with regard to sleep, mood and agitation. However after 2 months it was noted that she appeared to have moderate hyperactivity, talkativeness and agitation. In addition it was noted that she also had mild nausea and dizzy spells. She also started having daily derealization episodes which were preceded by a distinct aura in which she felt cold and experienced a burning acid-like smell. A CT scan revealed a 2.5cm mass in the right medial temporal lobe, displacing the right lateral ventricle and right hippocampus. Surgical resection followed by radiation therapy was provided. However the tumor, (histologically a grade IV invasive astrocytoma) following a 2 year remission period associated with improved psychiatric symptoms, returned with a fatal progressive course that was unresponsive to anti-neoplastics and radiation therapy. Kohler [12] described a 35 year old woman with a medical history of a left frontal neurocytoma that had undergone two incomplete resections and radiation treatment. She presented to the neuropsychiatry unit following a long psychiatric history, of dysphoria, apathy and hopelessness not responding to antidepressant medications. In the unit she was described as being reclusive, tearful and had abnormally slow motor and verbal responses. MRI showed a large, complex mass in the left lateral ventricle with left frontal encephalomalacia. Due to the nature of the tumor and its inability to be surgically excised and non-response to pharmacological treatment it was decided to try Electro Convulsive Therapy (ECT) to alleviate psychiatric symptoms. This proved to be very effective, with the patient appearing bright and cheerful with improved motor and verbal responses. Uribe VM [13] described a case of a 41 year old man who presented with a 3 month history of forgetfulness, poor appetite, loss of sexual desire, insomnia, nightmares, episodes of rage and problems maintaining interpersonal relationships. On examination he showed a low stream of thought, obsessive ruminations about the loss of his wife. There was impairment of both short term memory and abstract thinking. In two days time he began to complain of severe headache and became disorientated to time and place. He also developed neurological symptoms for the first time, which included gait unsteadiness and a hemiparesis. CT imaging was suggestive of a glioblastome multiforme in the left tempero-parietal region. The tumor was surgically removed. Unfortunately, long-term follow up was not reported in

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this case. Uribe links affective disorders to right hemispheric dysfunction and schizophrenia like psychosis to left hemispheric dysfunction. Blackman and Wheler [14] described a 12 year old boy with excessive anxiety and school phobia which were found to be secondary to a fourth ventricular tumor. During his initial hospitalization he was found to be extremely anxious and tearful. A diagnosis of overanxious disorder of childhood, school phobia and associated learning difficulties was made and he was treated with desipramine hydrochloride and psychotherapy. Despite treatment his symptoms recurred and he was readmitted to the hospital three months later. At this time, in addition to his behavioral symptoms he was noted to have increased reflexes on the left side, left intention tremor, and mild ataxia. A CT scan showed a midline mass in the fourth ventricle, which was densely calcified with significant obstructive hydrocephalus of the third and lateral ventricles. The patient underwent neurosurgical excision and shunt insertion. Even though the post-operative course was difficult the child eventually recovered with improvement in psychiatric symptomatology but with mild residual neurological deficits. There are three case reports in which patients presented with amnesic syndromes secondary to the presence of an astrocytoma. Gillespie et al [15] described a case of a 33 year old woman who presented with memory loss and headaches. She also admitted to having several complex partial seizures. CT scanning showed an area of low attenuation around the left temporal area. Over the next year she began to develop daily episodes in which she would cry, look frightened and only be partially responsive, despite medical treatment. At this time she was admitted and an MRI was done which showed an extensive non-enhancing lesion of the left temporal lobe. Despite operative treatment, the tumor was not removed in its entirety and she continued to experience anterograde amnesia and occasional seizures. Shimuachi et al [16] reported in 1989 a case of amnesia secondary to a bilateral hippocampal glioblastoma. The patient underwent a right temporal lobectomy, but the outcome was not described in the paper. Umemura et al [17] described a patient with amnesia which was secondary to a left sided temporal lobe astrocytoma, that had spread to the opposite side through the brain stem. The patient underwent a left-sided craniotomy, radiotherapy and interferon-beta therapy. Following this treatment regime, significant improvement was noted in his memory symptoms. Wilcox and Naranjo [18] described a case of a 38 year old man who initially presented with a 10 month history of headaches. He was subsequently referred to a psychiatrist for ‘bizarre behavior’ and paranoid ideations. Endocrine work up showed deranged hormonal profiles and CT imaging showed a bulky pituitary gland, with a bulge on its superior aspect. The patient was managed with hormonal replacement therapy and steroids. He responded satisfactorily to the regime and his psychotic symptoms improved. There are other reports in which pituitary tumors presented with psychiatric disturbances. Wilcox [19] described the case of a middle aged woman who presented with a 6 month history of depressed mood and fatigue, and later complaining of panic attacks. She described having panic attacks, palpitations, feelings of dread and respiratory discomfort. She was admitted with a very severe panic attack. A dexamethasone suppression test was negative and a CT scan revealed a microadenoma of the left anterior pituitary lobe. The tumor was removed surgically and the patient’s psychiatric symptoms improved dramatically. Rueda –Lara et al [20] also reported

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on two cases in which pituitary masses presented with psychiatric symptoms. In their cases psychotic symptoms dominated the clinical presentation. The first patient had florid delusions (in which she thought she was pregnant with the child of Jesus Christ) and auditory and visual hallucinations. Their second patient had passive suicidal ideation and paranoid delusions in which he believed someone was trying to harm him. Both patients had clinical signs that pointed to neuro-endocrine pathology. The latter had signs Cushing syndrome and the former acromegalic signs. MRI showed a bulky pituitary in the first patient and a giant sellar and supra sellar pituitary adenoma in the second patient. Peduncular hallucination is an uncommon psychiatric symptom and there are two case reports where the patient with a brain tumor presented with this symptom. Miyazawa et al [21] described a case of peduncular hallucinosis due to a pineal meninginoma. In their report they describe a 53 year old woman who presented with sudden onset of headaches and visual hallucinations. The hallucination occurred several times a day and lasted for several minutes. CT scanning revealed a mass in the pineal gland. The patient required two operations to remove the tumor. After the second operation the patient experienced visual hallucinations to a much smaller degree (once a month post-operatively, as opposed to several times a day preoperatively). Long-term follow up showed that she enjoyed a normal life with no neurological deficits. Maiuri et al [22] described two cases in which large posterior meniginomas led to peduncular hallucinations. The first case involved a 69 year old woman with a two month history of progressive ataxia and visual hallucinations. MRI showed a right tentorial meniginoma with infratentorial extension and significant mass effect on the pons and the midbrain. The histological diagnosis was fibroblastic meningioma. Following surgical removal, the patient had no further hallucinatory episodes. The second patient developed peduncular hallucination post-operatively after removal of a meniginoma. MRI revealed edema of cerebellar parenchyma and midbrain. The patient was treated with dexamethasone and carbamazepine with a good response There are four case reports of patients with ventricular colloid cysts presenting as psychiatric symptoms. Lajara-Nanson [23] described the case of a 48 year old man with no prior psychiatric history presenting with personality changes and mood disturbances. His wife reported that he was making inappropriate sexual comments towards her and their 12 year old daughter. In addition he had paranoid ideation, claiming that people were plotting against him. CT scanning revealed a mass in the third ventricle. MRI showed a hyperintense ovoid mass consistent with a colloid cyst. He underwent surgical resection of the mass but despite operative management he continued to have paranoid ideations. However his sexual inappropriateness did improve. Lobosky [24] et al reported a series of three patients with ventricular cysts who all presented with changes in personality, memory disturbances and emotional lability. All three patients underwent surgical resection and their’ psychiatric symptomatology subsequently improved. Jones AM [25], in 1993 also reported a similar case in which a patient with mental retardation presented with changes in personality, aggressive behavior and emotional lability. Imaging showed the presence of a ventricular cyst which was removed surgically leading to amelioration of the psychiatric symptoms. Finally Upadhyaya et al26 described a middle-aged housewife who was admitted following suicidal attempt. She was severely depressed and she gradually developed delusional thoughts. A CT scan showed

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a ventricular cyst of the third ventricle. Following surgical removal she remained symptom free, except for one episode of persecutory delusions 6 weeks after the surgery. In certain instances pre-existing psychiatric conditions confound the clinical presentation of the brain tumor. Carson et al [27] described a nine year old boy with a choroid plexus papilloma of the third ventricle. This patient had a three year history of behavioral problems including temper tandrums and Attention Deficit Hyperactivity Disorder (ADHD). He gave a history of hearing voices which commanded him to go to the gun shop and kill others and himself. He also became aggressive and violent at school. While institutionalized, his psychosis worsened. He was described as running around in circles constantly and smearing feces on the walls. He was treated with haloperidol with good result. While in the institution an MRI showed a third ventricular tumor on the right side with mild to moderate hydrocephalus. The patient underwent surgical resection and recovered completely. He had no psychotic episodes or evidence of any mood disorder since the surgery. He continued to be medicated for ADHD. Anorexia nervosa has been reported as a presenting feature of a number cases of brain tumors. However it is difficult to ascertain the diagnostic validity of anorexia nervosa, where all diagnostic criteria (especially fear/dread of obesity) have been met. There are ‘atypical’ presentations in which not all criteria have been met and may represent the non-specific signs and symptoms occurring with any neoplasm (such as loss of appetite, weight loss and cachexia). Lin et al [28] described a case of anorexia nervosa in a 19 year old man. MRI revealed a large mass with extensive infiltration occupying areas including the surapsellar area, hypothalamic region, third ventricle, pineal region, lateral ventricle and corpus callosum. Before any further studies could be conducted the patient died due to central herniation. Anorexia nervosa, secondary to a neoplasm, has been reported by various authors. Although there seems to be a preponderance for females to develop the condition, studies have shown that a third of the cases of anorexia related to masses in the brain do occur in males [29]. Tumors of the hypothalamus show a propensity for causing this type of psychiatric disturbance, although other tumor types have been associated with it [30-36]. Changes in behavior and personality may be first noticed in the work place and there are three case reports where psychiatric changes were first noticed by the employer of the patient. Carroll and Neal [37] described two cases in which altered behavior and poor work performance were the first psychiatric signs of a brain neoplasm. Following work place concerns over their change in behavior, investigations were performed which showed the presence of a craniopharyinginoma in one patient and a prolactinoma in the other patient. Jamieson and Wells [38] reported a single patient, who had no prior history of psychiatric illness. His employer was concerned about his excessive working hours. The patient presented with a manic episode characterized by euphoria, pressured speech and frequent changes in the tone and volume of his voice. He also admitted to poor sleeping pattern. Surgery and radiotherapy failed to eradicate the tumor, but his mania responded well to lithium treatment. Khaun et al [39] described a single case in which a patient initially presented with depression and poor work performance. He did not respond to antidepressant medication. CT scanning showed a vascularised tumor of the right thalamus. Surgical removal was not advised but long-term follow up was not discussed in this report.

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Nagaratnam et al [40] described an interesting case of a patient with venous angioma presenting and paranoid delusions. In their report a 78 year old woman, with no prior psychiatric history, was found wandering in the streets complaining that the “tax man was out to get her.” There were no other neurologic symptoms. She also showed mild short term memory problems. CT scanning showed a series of abnormal vessels in the anterior frontal lobe, running along the left lateral ventricle suggestive of a venous angioma. Management was not discussed in this case report. Fisher and Harper [41] described a 65 year old man who presented with depression. Initial scanning and blood tests were all normal. It was only on repeat scanning that a neoplastic lesion was found. Unfortunately the patient passed away and histological study on autopsy revealed a Central Nervous System lymphoma. They suggested that a negative scan by itself cannot exclude the presence of a neoplastic lesion but a continued evaluation and repeat scanning may be needed in atypical cases with psychiatric symptoms. Bluestein and Seeman [42] reported on three patients who presented with psychiatric disorders which were later found to be secondary to cerebral tumors. The first patient presented with mild euphoria and rambling, disconnected speech. The second patient was a 40 year old man who was seen by his psychiatrist and diagnosed with depression and somatization and obsessive compulsive personality. The last patient was a 16 year old girl who presented with thought disorders and auditory hallucinations. Unfortunately in their case report, management and outcome were not discussed. They suggested that speech disorder should be carefully reviewed in any psychiatric patient. Burch et al [43] caution the labeling of symptoms in patients “hysterical” because it could cloud long term observation. Their patient had a three and half year history of hysterical symptoms (attributed to the breakdown of her marriage) and a plethora of neurological symptoms including (ataxia, vertigo and nystagmus) which were dismissed as conversion disorders stemming from her hysteria. She finally died in 1971 and an autopsy revealed an extensive glioblastoma multiforme involving the pons and medulla oblongata. Moise et al [44] in a recent case report described a 29 year old woman who was treated for Post Traumatic Stress Disorder (PTSD) and Borderline Personality traits who later developed depressive symptoms and memory loss. Brain imaging showed the presence of a left thalamic tumor which was later confirmed as glioblastoma multiforme. She received surgical treatment and radiation therapy and continued antidepressant treatment with partial improvement in her psychiatric symptoms. Brain tumors can also present with more unusual symptoms. Burns and Swerdlow [45] described the case of a man who exhibited paedophilia and impulsive sexual behavior in spite of preserved moral knowledge. A detailed neurological examination found constructional apraxia and agraphia. Neuro-imaging revealed a right orbitofrontal tumor and the symptoms resolved following the resection of the tumor. Durst and Rosca-Rebaudengo [46] report on a unique case of Koro secondary to a tumor of the corpus callosum. Their patient was treated satisfactorily with ECT and remained symptom free despite continued growth of the tumor as evidenced by serial CT scanning.

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Discussion Some authors have suggested that there may be an association between tumor location and the psychiatric presentation. In general it was suggested that brain tumors affecting the frontal lobe were associated with psychiatric symptoms such as abulia, personality change and depression. Patients with temperolimbic tumors were noted to have auditory and visual hallucinations, mania, panic attacks and amnesia [6]. This assumption could be supported by underlying physiological mechanisms.

The Frontal Lobe and Neurospsychiatric Illness The frontal lobes make up approximately one third of the total cortical area and mediate a variety of wide ranging functions; from social interaction to more primitive oculomotor functions [47]. Anatomically it is thought that the lobes consist of five parallel circuits, linking regions of the frontal cortex to the striatum, globus pallidus/ substantia nigra and thalamus. Each of these five circuits comprises a discrete, closed loop that is capable of functioning independently. However while each loop is capable of independent functions, within each loop lie ‘open elements’ that are capable of being modulated by external inputs from neighboring circuits [48]. Physiological studies and extrapolation from patients suffering from lesions involving the frontal lobe support the idea that these five neural paths are capable of mediating motor and oculomotor functions, as well as executive functions such as motivation and socially responsible behavior. More specifically, the dorso-lateral prefrontal- subcortical circuit mediates the organization of information to facilitate a response; the anterior cingulated subcortical circuit is required for motivated behavior; and the lateral orbitofrontal circuit allows the integration of limbic and emotional information into contextually appropriate behavioral responses [49-51]. Hence disruption of the frontal lobe and its circuits tend to produce neurotic symptoms (disorders of action, apathy and lability), rather than symptoms due to inappropriate perception or of stimulus integration.

Temporal-Limbic Connections Hallucinations arise from improper processing of exogenous stimuli. While the nuances of visual and auditory perception, and how they are coded for and networked in the neocortex, are yet to be explored, physiologic studies have shown that the temporal lobe plays an important role in sensory perception. Electrophysiological studies have shown that the primary auditory cortex lies on the superior surface of the temporal lobe, in the gyrus of Heschl [52, 53]. In addition certain parts of the visual pathway also lie deeply invested in the temporal cortex [54, 55]. The inferior temporal gyrus in particular seems to be of paramount importance in perceiving and recognizing visual stimuli. Destruction of these visual representation centers results in agnostic deficits. Neurobiological studies on animal models have suggested that a variety of neural circuits play an important role in declarative memory

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and its access. These circuits are thought to lie in the parahippocampal region, the hippocampus and associative areas in the parietal and temporal cortices [56, 57]. Hence physiologic theory, as it stands, can in part explain why disruption of the temporal region and the hippocampal/ parahippocampal regions may lead to amnesia-like symptoms and hallucinatory episodes.

The Hypothalamus The human hypothalamus plays a central role in human regulation, with almost every component of the neuraxis being subjected to some degree of control by this component of the central nervous system. Numerous studies have suggested that the hypothalamus can play a significant role in caloric intake and changes in weight. The ventrolateral hypothalamus seems to contain a feeding center, while the ventromedial aspect contains a satiety center [58, 59]. Given this, it is not surprising to see an association between anorexic disease and tumors of the hypothalamus. However studies also indicate that tumors affecting the limbic system also have a tendency to cause anorexic symptoms [29].

Pathophysiological Complications Even though a casual relationship between tumor location and type the psychiatric presentations seems interesting, the majority of brain tumors present with neurological signs, suggesting other factors may be responsible for the clinical presentation. Furthermore the bulk of the case reports do not support physiologic theory. The authors of a number of case reports suggest that the anatomical location of the brain tumor is often a poor guide to the psychiatric symptomatology. This poor correlation is probably due to the fact that a variety of factors, only one of which is the anatomical location of the tumor, may contribute to the presenting psychiatric features in a patient with a cerebral tumor.

Tumor Construct and Growth Rate Tumor type also plays a role in symptomatology. Studies by a number of authors have suggested that slow growing, often benign, tumors tend to be associated with a smaller incidence of psychiatric symptoms than more rapidly growing, malignant tumors [60, 61]. The increased incidence of mental symptoms in the latter is unknown although its has been suggested that slow growing tumors offer the nervous system a chance for adaptive compensatory changes, but not with more rapidly growing tumours [62, 63]. In addition it has been suggested that high grade tumors have a greater incidence of causing raised intracranial pressure, which can contribute to mental symptoms. The fact that malignant tumors are capable of invading and compromising brain tissue to a much greater extent than low grade tumors also explains why they tend to produce more mental symptoms (and neurological

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symptoms for that matter). It has been noted that metastatic tumors in the brain were associated with an even greater incidence of mental symptoms than any primary tumor. This is likely to be due to metastatic tumors being scattered throughout the brain substance and the additive effects of the constitutional symptoms associated with the primary tumor itself (lethargy, apathy, tiredness etc).

Secondary Effects Brain tumors can lead to destruction and compression of the neural ground tissue. In addition to simple physical compression tumors are, capable of invading neural vasculature and causing an ischaemic necrosis often beyond the physical boundary of the tumor mass. Furthermore cerebral edema, a pathological response associated with many intracranial pathologies, also occurs as a result of brain tumors. The effect of this increased amount of cerebrospinal fluid is often further exacerbated by the physical obstruction of cerebrospinal fluid outflow paths that sometimes occur with tumors of the brain [64]. Since the brain is devoid of a lymphatic system any excessive fluid accumulation cannot be removed by drainage through this pathway. Instead cerebral fluid drains much more slowly via the cerebrospinal pathways. Hence any tumor that compromises vascular permeability and produces additional cerebral edema and obstructs this cerebrospinal drainage pathway can lead to increased intracranial pressure, adding to the mass effect of the tumor itself. Raised intracranial pressure can lead to fluctuations in levels of consciousness, difficulty in thinking, emotional dullness and apathy [65]. Via these secondary effects it is possible for a brain tumor to affect parts of the brain beyond its physical boundaries. These secondary effects produce a pathological extension beyond the simple location of the brain tumor.

Individual Variations Certain individuals are predisposed to certain types of psychiatric conditions. These predispositions arise from genetic, social, economic and concurrent medical factors. In these individuals the presence of the tumor may simply serve as a catalyst or a critical event that ultimately leads to the manifestation of symptoms. In patients with a past psychiatric history, the clinical picture can be further complicated by on going psychiatric management. Effective management in these cases may result in symptom amelioration, but over time as the tumor mass enlarges and invades more brain tissue, this can lead to additional symptoms or recurrence of old ones. Hence in these patients there is often very poor correlation between presenting mental symptoms and tumor location. Initial symptoms which were probably more directly related to the tumor location, may be considered functional and treated or controlled with psychotropic medications.

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Relevance Brain tumors presenting primarily with psychiatric symptoms remain a rare medical entity. However if diagnosed at an early stage, can be treated satisfactorily often with complete resolution of the presenting psychiatric symptoms. The evidence shows that surgical resection remains the best choice, in terms of offering a resolution of the psychiatric symptoms associated with brain tumors; but even in non-operative cases (or when surgery is palliative), chemotherapy, radiotherapy and electroconvulsive therapy have been shown to be helpful. While psychiatric symptoms secondary to a brain tumor are a rare occurrence, certain pitfalls could be avoided in the assessment of patients. A complete medical history and examination is of paramount importance; both to detect relevant symptomatology from an extra-cranial primary source and also to detect any subtle neurological signs. In patients with a prior psychiatric history, new onset psychiatric symptomatology or sudden recurrence of psychiatric symptoms that were previously well controlled, warrant concern. Neuro-imaging may be considered if other factors such as medication non-adherence and psychosocial issues are ruled out. Patients who are refractory to conventional medical treatment should also warrant suspicion. Our analysis of the 66 cases (table 1 and 2) for the past 54 years, indicate that neither tumor location nor type is correlated with any particular type of psychiatric symptoms. However mood symptoms (depression/mania/apathy) have been noted in 42% of the cases (n=28). Out of the 8 cases with anorexia nervosa/ anorexia, five cases were associated with tumors of the hypothalamic region. As we had discussed earlier, the use of the term “anorexia nervosa” in these case reports, does not appear to be supported by valid diagnostic criteria. It is possible that some cases of anorexia might have been labeled as anorexia nervosa. It could still be conceptualized that anorexia nervosa/ anorexia may be a presenting symptom of a hypothalamic tumor. Table 1. Psychiatric symptoms, tumor location and tumor type in reported cases Reference Dyck Griffith

Psychiatric symptoms

Tumor location

Auditory hallucinations Sylvian fissure Depression Olfactory area

Ghadirian et al Depression and anxiety followed by visual hallucinations Filley, Psychotic symptoms Kleinschmidt- (perceptual DeMasters disturbances) Filley, New-onset manic Kleinschmidt- symptoms DeMasters Filley, Apathy, social Kleinschmidt- withdrawal, poor selfDeMasters care

Right temporal lobe

Tumor type Lipoma Esthesioneuroblastoma Meningioma

# of cases 1 1

Remarks

1

Temporal

Low grade oligoastrocytoma

2

Bitemporal

Glioblastoma multiforme

1

Bifrontal

Benign meningioma

1

Other case due to oligodendroglioma

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Subramoniam Madhusoodanan, Abhishek Sinha, Despina Moise et al. Table 1. (Continued)

Reference

Psychiatric symptoms

Tumor location

Filley, KleinschmidtDeMasters Filley, KleinschmidtDeMasters Filley, KleinschmidtDeMasters Filley, KleinschmidtDeMasters Avery

Apathy, irritability, anomia, right hemiparesis Severe depression, extensive weight loss

Left frontal and genu of corpus callosum Left frontal

Tremuloussness, paresthesia, diaphoresis

Avery

Tumor type Immunoblastic lymphoma

# of cases 1

Remarks

Squamous cell carcinoma

1

Pituitary and left medial temporal Thalami and fornical columns Olfactory groove

Gonadotrophic cell pituitary adenoma

1

Gonadotrophic cell pituitary adenoma

1

Meningioma

1

Euphoria, drowsiness and apathy

Tuberculum sellae

Meningioma

1

Avery

Depression, apathy

Right cribriform Meningioma plate

2

Avery

Cribriform plate

Meningioma

1

Avery

Apathy, change in work behaviour Apathy, tiredness

Right Meningioma sphenoidal ridge

1

Avery

Euphoria

Olfactory groove Multiple metastatic right fronto-parietal lesions Multiple metastatic left fronto-parietal lesions Left parietooccipital metastatic lesion Left parietal extending to temporal lobe with midline shift

Meningioma

1

Origin in right lung

1

Origin in right lung

1

Origin in right kidney

1

No surgical intervention due to advanced stage

Unknown- surgery refused- no autopsy report given

1

Resolution of depressive symptoms after surgery, chemotherapy and radiation therapy

Left parietal

High grade glial neoplasm with sporadic cells

1

Left thalamic

Glioblastoma multiforme

1

Resolution of depressive symptoms after surgery, chemotherapy and radiation therapy Patient died in spite of aggressive medical treatment

Apathy, amnesia, poor affect Mania, euphoria

Ko and Lok

Depressive symptoms, emotional lability, amnesia for recent events Ko and Lok Expressive aphasia, short-term memory difficulties, no focal neurologic signs Ko and Lok Paranoid ideation, irritability, short-term memory problems Ko and Lok Deteriorating memory and disorientation to time and space, behavioral changes, visual agnosia, aphasia, self neglect Madhusoodanan Recent depressive et al symptoms, anger and agitation Binder

Behavioral changes, confusion with neurological signs developing after 24 hours

Some residual psychiatric disturbance following resection Some residual psychiatric disturbance following resection Post-op.manic and euphoric episode before resolution of symptoms (1case); improvement after surgery (2nd case) Patient did well following removal Patient died before surgery could be carried out

Improvement in psychiatric symptoms after surgery and chemotherapy No surgical intervention

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Table 1. (Continued) Reference Binder

Binder

Blustein and Seeman Blustein and Seeman Blustein and Seeman Khuan et al Sokolski and Denson

Kohler

Uribe VM

Carson et al

Blackman and Wheler

Gillespie et al, Unemura et al, Shimuachi et al Nagaratnam et al Fisher and Harper

Sudden behavioral changes followed by paranoid delusions; no focal neurological signs New-onset rage attacks on background of chronic schizophrenia Thought disorder, auditory hallucinations Depression

Right lateral ventricle

meningioma

# of cases 1

Bilateral occipital

meningioma

1

Left parietooccipital Right temporal

1

Disconnected speech, mild euphoria Depression, poor work performance Breakthrough manic symptoms with mild nausea and dizzy spells, daily derealisation episodes with olfactory auras Depressive symptoms refractory to antidepressants, following surgical resection of left frontal neurocytoma Depressive symptoms with rage episodes, forgetfullness, disturbance in short-term memory and abstract thinking, later -onset headaches, disorientation,gait unsteadiness, hemiparesis Pediatric psychosisAggression, violence, hallucinations Pediatric case of excessive anxiety and school phobia with lateronset neurological symptoms Amnesia

Left, posterior frontal Right Thalamus

Porencepahlic cyst Grade I astrocytoma glioma

Psychiatric symptoms

Tumor location

Tumor type

Remarks Complete resolution of symptoms after surgical intervention Disappearance of rage attacks after surgical removal

1 1 1

Right medial temporal, displacing right ventricle and right hippocampus

Grade IV invasive astrocytoma

1

Improvement of psychiatric symptoms with surgical resection

Left lateral ventricle, left frontal encephalomalacia

Neurocytoma

1

Good response to ECT

Left temporoparietal

Glioblastoma multiforme

1

Third ventricle

Choroid plexus papilloma

1

Symptoms improved after surgical removal

Choroid plexus papilloma

1

Improvement of symptoms after surgical resection and shunt insertion

Astrocytoma

3

Improvement with treatment

Paranoid delusions

Fourth ventricle with obstructive hydrocephalus of ventricles III and IV Left temporal (two cases), bilateral hippocampal (one case) Left frontal lobe

Venous angioma

1

Depression

Limbic system

CNS lymphoma

1

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Reference

Tumor location

Headaches and psychotic symptoms

Pituitary (one case), pineal (one case)

One caseunknown; pineal meningioma

Depression, headache Moise and Madhusoodanan Memory loss

Right thalamus

Glublastoma multiforme

Maiuri et al

Posterior masses Meningioma

2

Pituitary

Hormone producing adenoma unknown

2

Arachnoid cyst

1

Teratoma Craniopharyngioma Pinealoma Ectopic pinealoma Pineal teratoma

1 1 1 1 1

Pinealoma Hormone producing adenoma Metastatic tumours from unknown primary source

1 1

Glioblastoma multiforme

1

Colloid cyst

1

Miyazawa et al

Hallucinations

Rueda-Lara et al Delusions, hallucinations Lin et al Anorexia nervosa

Wolanczyk et al Anorexia nervosa, delusions, catatonia Berek et al Anorexia nervosa Climo Anorexia nervosa Swann I Anorexia nervosa Daly et al Anorexia nervosa Weller and Anorexia nervosa Weller Nicholson et al Anorexia Nervosa Wilcox Panic attacks

1

Hypothalamic region, third ventricle, pineal region, lateral ventricle, corpus callosum Right parietal lobe Third ventricle Hypothalamus Hypothalamus Hypothalamus Hypothalamus Pineal gland Pituitary

Jamieson and Wells

Mania

Right occipital, temporal and parietal lobes

Burch et al

Hysteria

Upadhyaya AK and Sud PD Burns and Swerdlow Durst R, RoscaRebaudengo Lajara-Nanson, Lobosky, Jones AM

Depression and delusional ideation Paedophilia

Medulla oblongata and pons Third Ventricle

Koro Personality changes and emotional lability

Tumor type

# of cases 2

Psychiatric symptoms

Right orbitofrontal Corpus callosum Ventricular

Remarks Improvement with hormone/steroid treatment and surgical removal, respectively Partial improvement of symptoms with surgical treatment and antidepressants Improvement with treatment

1

1

Died before 46th birthday despite surgical and medical management

1 Lipoma or dermoid tumour 3 unknown, 2 ventricular cysts

1 5

Biopsy not done due to position of tumour mass Improvement with surgery

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Table 2. Tumor locations and types of symptoms Tumor location

Symptoms

Frontal

-Apathy, social withdrawal, poor selfcare, depression -Paranoid delusions -Mild euphoria -New-onset paraphilia -Depression, emotional lability, amnesia -Expressive aphasia, memory difficulties -Mood changes -Amnesia -Perceptual disturbances -Anorexia and psychotic symptoms -Depression -Psychotic symptoms -Memory disturbances -Apathy, amnesia, affective changes -Behavioral changes -Depression Rage attacks Anorexia nervosa Mood changes- mania or depression -Personality changes, emotional lability -Depression -Anorexia nervosa -Psychosis -Anxiety Apathy, depression, euphoria or tiredness -Psychosis -Panic attacks -Tremulouasness, diaphoresis,paresthesia -Anorexia nervosa -Headaches,psychotic symptoms -Koro -Amnesia -Depression -Psychotic symptoms -Hysteria -Psychotic symptoms -Mania

Fronto-parietal

Temporal

Parietal, parieto-occipital, parieto-temporal

Thalamic

Occipital Hypothalamic Olfactory Ventricular

Cribriform plate, sphenoidal, tuberculum sellae Pituitary

Pineal gland

Corpus callosum Hippocampal Limbic Posterior masses Medulla and pons Sylvian fissure Widespread metastases (occipital, temporal, parietal)

# of cases 3 1 1 1 1 1 4 2 2 1 2 2 1 1 1 2 1 5 3 6 2 1 2 1 5 2 1 1 1 1 1 1 1 2 1 1 1

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Conclusion Brain tumors can in rare instances present with psychiatric symptomatology. Mood symptoms have been noted in a significant number of cases. There is often a poor correlation between tumor site and the presenting psychiatric symptoms. This poor correlation may be due to the secondary effects of the tumor, the individual health and psycho-social issues of the patient and the exact type of the tumor itself. Early detection and treatment can result in complete resolution of symptoms and a greater quality of life. Neuroimaging should be considered with new onset psychosis, recurrence of previously well controlled mental symptoms or occurrence of new mental symptoms and in patients who remain refractory to psychiatric treatment.

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[10] [11]

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[14] Blackman M, Wheler GH. A case of mistaken identity: a fourth ventricular tumor presenting as school phobia in a 12 year old boy Can J Psychiatry. 1987 Oct;32(7):584-7. [15] Gillespie JS, Craig JJ, McKinstry CS. Bilateral astrocytoma involving the limbic system precipitating disabling amnesia and seizures. Seizure. 2000 Jun;9(4):301-3. [16] Shimauchi M, Wakisaka S, Kinoshita K. Amnesia due to bilateral hippocampal glioblastoma. MRI finding. Neuroradiology. 1989;31(5):430-2. [17] Umemura A, Yamada K, Masago A, Tanigawa M, Nakaaki S, Hamanaka T. Pure amnesia caused by bilateral temporal lobe astrocytoma--case report. Neurol Med Chir (Tokyo). 1997 Jul;37(7):556-9. [18] Wilcox JA, Naranjo J. Psychiatric manifestations of pituitary tumors. Psychosomatics. 1997 Jul-Aug;38(4):396-7. [19] Wilcox JA. Pituitary microadenoma presenting as panic attacks. Br J Psychiatry. 1991 Mar;158:426-7. [20] Rueda-Lara MA, Buchert S, Skotzko C, Clemow LP. Psychiatric symptoms masking pituitary adenoma in Spanish speaking immigrants. Gen Hosp Psychiatry. 2003 SepOct;25(5):367-71. [21] Miyazawa T, Fukui S, Otani N, Tsuzuki N, Katoh H, Ishihara S, Nawashiro H, Wada K, Shima K. Peduncular hallucinosis due to a pineal meningioma. Case report. J Neurosurg. 2001 Sep;95(3):500-2. [22] Maiuri F, Iaconetta G, Sardo L, Buonamassa S. Peduncular hallucinations associated with large posterior fossa meningiomas. Clin Neurol Neurosurg. 2002 Jan;104(1):41-3. [23] Lajara-Nanson WA. Neuropsychiatric manifestations of a third ventricular colloid cyst. W V Med J. 2000 Jul-Aug;96(4):512-3. [24] Lobosky JM, Vangilder JC, Damasio AR. Behavioral manifestations of third ventricular colloid cysts. J Neurol Neurosurg Psychiatry. 1984 Oct;47(10):1075-80. [25] Jones AM. Psychiatric presentation of a third ventricular colloid cyst in a mentally handicapped woman. Br J Psychiatry. 1993 Nov;163:677-8. [26] Upadhyaya AK, Sud PD. Psychiatric presentation of third ventricular colloid cyst. A case report. Br J Psychiatry. 1988 Apr;152:567-9. [27] Carson BS, Weingart JD, Guarnieri M, Fisher PG Third ventricular choroid plexus papilloma with psychosis. Case report. J Neurosurg. 199.7 Jul;87(1):103-5. [28] Lin L, Liao SC, Lee YJ, Tseng MC, Lee MB Brain tumor presenting as anorexia nervosa in a 19-year-old man. J Formos Med Assoc. 2003 Oct;102(10):737-40. [29] Chipkevitch E. Brain tumors and anorexia nervosa syndrome. Brain Dev. 1994 MayJun;16(3):175-9, discussion 180-2 [30] Weller RA, Weller EB. Anorexia nervosa in a patient with an infiltrating tumor of the hypothalamus. Am J Psychiatry. 1982 Jun;139(6):824-5 [31] Daly JJ, Narbarro JDN, Powell T. A case of anorexia. Br Med J 1973: 2:156-61 [32] Swann I. Anorexia nervosa--a difficult diagnosis in boys. Illustrated by three cases. Practitioner. 1977 Mar;218(1305):424-7. [33] Climo LH. Anorexia nervosa associated with hypothalamic tumor: the search for clinical-pathological correlations. Psychiatr J Univ Ott. 1982 Mar;7(1):20-5.

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[34] Berek K, Aichner F, Schmutzhard E, Kofler M, Langmayr J, Gerstenbrand F. Intracranial germ cell tumor mimicking anorexia nervosa. Klin Wochenschr. 1991 Jul 22;69(10):440-2. [35] Wolanczyk T, Komender J, Brzozowska A. Catatonic syndrome preceded by symptoms of anorexia nervosa in a 14-year-old boy with arachnoid cyst. Eur Child Adolesc Psychiatry. 1997 Sep;6(3):166-9. [36] Nicholson M, Keitel H, Williams J, Millican F, Lourie RS, Lopresti M, Stevens H, Guin GH. Pinealoma with associated hypernatremia and symptoms of anorexia nervosa. Clin Proc Child Hosp Dist Columbia. 1957 Jul;13(7):133-45. [37] Carroll N, Neal LA Diencephalic tumors presenting as behavioral problems in the workplace. Occup Med (Lond). 1997 Jan;47(1):52-4. [38] Jamieson RC, Wells CE. Manic psychosis in a patient with multiple metastatic brain tumors. J Clin Psychiatry. 1979 Jun;40(6):280-3. [39] Khuan TC, Dass D, Majeed H. Psychiatric presentation of thalamic tumor - a case report. Med J Malaysia. 1979 Sep;34(1):38-41. [40] Nagaratnam N, Ghougassian DE, Wong K, Walker S. Psychiatric presentation of a venous angioma of the frontal lobe. Br J Clin Pract. 1990 Jan;44(1):34-5. [41] Fisher R, Harper C Depressive illness as a presentation of primary lymphoma of the central nervous system. Aust N Z J Psychiatry. 1983 Mar;17(1):84-90. [42] Blustein J, Seeman MV. Brain tumors presenting as functional psychiatric disturbances. Can Psychiatr Assoc J. 1972;17(2):Suppl 2:SS59-63 [43] Burch EA Jr, Hutchison CF, Still CN. Hysterical symptoms masking brain stem glioma. J Clin Psychiatry. 1978 Jan;39(1):75-8. [44] Moise D, Madhusoodanan S. Psychiatric symptoms associated with brain tumors – A clinical enigma. CNS Spectrums. In Press. [45] Burns JM, Swerdlow RH. Right orbitofrontal tumor with pedophilia symptom and constructional apraxia sign. Arch Neurol. 2003 Mar;60(3):437-40. [46] Durst R, Rosca-Rebaudengo P. Koro secondary to a tumor of the corpus callosum. Br J Psychiatry. 1988 Aug;153:251-4. [47] Alexander GE, DeLong MR, Strick PL. Parallel organization of functionally segregated circuits linking basal ganglia and cortex. Annu Rev Neurosci. 1986;9:357-81. [48] Mega MS, Cummings JL. Frontal-subcortical circuits and neuropsychiatric disorders. J Neuropsychiatry Clin Neurosci. 1994 Fall;6(4):358-70. [49] Cummings JL. Frontal-subcortical circuits and human behavior. Arch Neurol. 1993 Aug;50(8):873-80. [50] Logue V, Durward M, Pratt RT, Piercy M, Nixon WL. The quality of survival after rupture of an anterior cerebral aneurysm Br J Psychiatry. 1968 Feb;114(507):137-60. [51] Barris RW and Schuman HR. Bilateral anterior cingulate gyrus lesions; syndrome of the anterior cingulate gyri. Neurology. 1953 Jan;3(1):44-52. [52] Imig TJ, Reale RA. Patterns of cortico-cortical connections related to tonotopic maps in cat auditory cortex. J Comp Neurol. 1980 Jul 15;192(2):293-332. [53] Celesia GG, Puletti F. Auditory cortical areas of man. Neurology. 1969 Mar;19(3):21120.

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[54] Albright TD, Desimone R, Gross CG. Columnar organization of directionally selective cells in visual area MT of the macaque. J Neurophysiol. 1984 Jan;51(1):16-31. [55] Tanaka K, Saito H, Fukada Y, Moriya M. Coding visual images of objects in the inferotemporal cortex of the macaque monkey. J Neurophysiol. 1991 Jul;66(1):170-89. [56] McDonald RJ, White NM A triple dissociation of memory systems: hippocampus, amygdala, and dorsal striatum. Behav Neurosci. 1993 Feb;107(1):3-22. [57] Eichenbaum HB et al. Learning and Memory: Systems Analysis. Fundamental Neuroscience. Acadaemic Press 1999. Chapter 56; pgs1455-1486 [58] Duggan JP, Booth DA. Obesity, overeating, and rapid gastric emptying in rats with ventromedial hypothalamic lesions. Science. 1986 Feb 7;231(4738):609-11. [59] Stellar E. The physiology of motivation. Psychol Rev. 1954 Jan;61(1):5-22. [60] Keschner M et al. Mental symptoms associated with brain tumor: A study of 530 verified cases. Am Med Assoc 1938; 110:7114-718. [61] Busch E. Psychical symptoms in neurological disease. Acta Psychiatr et Neurol Scand 1940; 15:257-90. [62] Seitz RJ, Huang Y, Knorr U, Tellmann L, Herzog H, Freund HJ. Large-scale plasticity of the human motor cortex. Neuroreport. 1995 Mar 27;6(5):742-4. [63] Duffau H. Lessons from brain mapping in surgery for low-grade glioma: insights into associations between tumor and brain plasticity. Lancet Neurol. 2005 Aug;4(8):476-86. [64] Ellison D et al. Neuropathology. London Mosby, 1998. [65] Kleihues P et al. Pathology & Genetics of Tumors of the Nervous System. Lyon IARC Press 2000.

In: Trends in Brain Cancer Research Editor: Andrew V. Yang, pp. 303-321

ISBN 1-59454-972-9 ©2006 Nova Science Publishers, Inc.

Chapter IX

The Waterjet Instrument in Neurosurgery: A Detailed Account of its Clinical Potential after More than 150 Procedures Joachim Oertel1,∗, Jürgen Piek2, Henry W.S. Schroeder3 and Michael R. Gaab1 1

Department of Neurosurgery, Hannover Nordstadt Hospital, Germany, 2 Department of Neurosurgery, University of Rostock, Germany, 3 Ernst Moritz Arndt University Greifswald, Germany.

Abstract The waterjet instrument is currently under clinical evaluation in neurosurgical procedures, and precise tissue dissection with vessel preservation has been demonstrated experimentally. The present study focuses on the general application technique of the device and on the distinct clinical situations in which the device possesses peculiar advantages compared with conventional techniques based on the experience of more than 150 procedures. The waterjet instrument has been applied in more than 150 intracranial procedures including gliomas (°1-4), metastases, meningiomas, acoustic neurinomas, epidermoids cysts, and epilepsy surgery. The instrument was used in combination with conventional methods for tissue dissection and tissue aspiration. All cases were prospectively followed up to 2 years. Intraoperatively, the waterjet was easy to handle. While it was applied in a similar fashion as the ultrasonic aspirator in most tumours, the instrument possessed peculiar ∗

Correspondence concerning this article should be addressed to Dr. Joachim Oertel, M.D., Ph.D. Department of Neurosurgery, Hannover Nordstadt Hospital, Klinikum Hannover, Haltenhoffstrasse 41, 30167 Hannover, Germany. Phone / Fax: +49-511-970-1245 / -1606; Email: [email protected].

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Joachim Oertel, Jürgen Piek, Henry W.S. Schroeder et al. advantages in the dissection of tumours from the intact adjacent brain parenchyma and in the separation of brain tissue from the arachnoid membranes. In the first, the parenchyma was precisely dissected and preserved vessels could be coagulated at a wide distance to the surrounding brain. With this technique a significant reduction of surgical blood loss was observed, and the tissue dissection was minimally traumatic. In the latter, the arachnoid membranes were easily preserved while the brain tissue was precisely cut. Our results indicate (i) that the waterjet enables tissue dissection and subsequent vessel coagulation without damage to the remaining brain tissue, and (ii) that it might be well suited for special indications such as subpial dissections. In all, it appears to be more suited for tissue dissection than the CUSA under certain conditions particularly if minimally traumatic surgery with minimal blood loss is of major importance.

Introduction The reduction of intraoperative blood loss and parenchymal trauma is a major goal in surgical procedures. Each well established technique, such as laser surgery, thermal coagulation, and ultrasonic aspiration has its own disadvantages i.e. thermal damage to the surrounding tissue [35]. Several research projects have focussed on the development of an instrument that combines highly precise parenchymal dissection with preservation of surrounding tissue and a reduction of blood loss. Since the early 80ies, the waterjet dissection technique is under continuous investigation as a new promising new technique in surgical procedures. This technique is based on the principle that water is pushed through an 80- to 120-µm-diameter nozzle under various pressures. Industrially, this technique is suited for cutting of metal, wood, stone and plastic [38]. Actually, the first report of the waterjet technique in the medical literature was an anecdotal trauma case caused by a high-pressure industrial jet [14]. In the early 1982, Papachristou and Barters reported on the use of the waterjet in liver surgery [25] and stated that the jet washed the intrahepatic matrix away, leaving the ducts and blood vessels undamaged leading to a reduction of blood loss when this procedure was applied in 45 lobectomies in dogs and in four liver resections in humans [25]. Subsequently, other studies confirmed that the use of the waterjet dissector enables to reduced parenchymal trauma and blood loss in liver surgery, compared with blunt dissection or ultrasonic aspiration [1, 31]. At present, this procedure is widely used in liver resections and reports have been frequent [1, 2, 5, 6, 8, 27, 30, 31, 32, 41]. Also, possible indications of the waterjet dissector in other surgical disciplines such as kidney [7, 26], bone [3], vascular [12], and craniomaxillofacial surgery [10] as well as dermatology [36] and ophthalmology [4, 13] are currently under investigation. To become a useful neurosurgical tool, the waterjet instrument should improve parenchymal dissection under vessel preservation allowing a reduction of intraoperative blood loss and parenchymal trauma. The first experimental results of application of the waterjet technique in neurosurgical experiments were published by Terzis and coworkers [39] in 1989. They reported precise dissection of the brain parenchyma accompanied by preservation of vessels larger than 20 µm in cadaveric porcine brains. No clinical studies by this group followed. The study group of the authors of this chapter works with a newly

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developed waterjet dissector that has been approved for clinical applications in Europe and the United States since 1996. Several experimental cadaver studies in the pig and in vivo studies in the rabbit have shown that the waterjet technique allows the accurate dissection of brain parenchyma with preservation of blood vessels [17, 19, 22, 29]. In the first clinical applications of the new waterjet device, resection of various lesions, such as gliomas [24, 28], metastatic brain lesions [18, 28], meningiomas [23, 28] and normal brain in epilepsy surgery [20, 28] was performed without complications. However, the ideal application of the device, its indications and contraindications in the brain remain under investigation. Particularly, the correct application of the device in brain surgery remains under debate. In the presented series of currently more than 150 intracranial waterjet procedures, the indication and application of the waterjet instrument has changed. Thus, the authors present here their current way of application of the device based on the experience gained in more than 150 intracranial procedures.

Clinical Material and Methods Patient Population One-hundred-and-fifty-four patients (79 male and 75 female patients; mean age 52 years, range 12–81 years) underwent surgery with the aid of the waterjet between August 1997 and August 2005. Eighty-five patients suffered from gliomas of WHO grades 1-4 [11]. In 21 patients who underwent surgery for epilepsy, the waterjet dissection technique was used. There were 18 procedures performed for solitary metastases, 13 for meningiomas, six for epidermoid cysts, three for hemangioblastomas, two for acoustic neurinomas, and six for various other lesions. The WHO classification of brain tumours was used in all cases [11].

Description of the Instrument Between August 1997 and August 1999, the first generation of waterjet instruments (Müritz 1000; Andreas Pein Medizintechnik, Schwerin, Germany) was used in 21 cases. Since September 1999, its successor, the Helix Hydro-Jet (Figure 1a; Erbe Elektromedizin, Tübingen, Germany) has been used in all subsequent cases. The waterjet is generated via a medium converter with electronically controlled hydraulics. Waterjet pressures ranging from 1 to 150 bars are generated. The instrument is connected to a pencil-like handpiece consisting of a narrow nozzle that is 100 or 120 µm in diameter, and a surrounding suction tube (Figure 1b and 1c). Through this hand-piece, sterile 0.9% isotonic saline is emitted. The pressure and suction is manually preset using the control unit. During surgery, the waterjet application and pressure can be adjusted within the preset range by a foot pedal. The waterjet system has been approved by the regulatory authorities in Europe and the United States for surgical use in humans.

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Figure 1. a. Helix Hydro-Jet instrument (Erbe Elektromedizin, Tübingen, Germany). b. Equipment for waterjet dissection with handpiece, saline cartridge and suction bag. c. 120µm handpiece with the emitted waterjet (arrow).

Surgical Procedure and Follow Up The waterjet instrument was used for lesion resection in combination with conventional neurosurgical procedures. Directly after each procedure, the intensity and quality of the waterjet dissection, the instrument’s usefulness, handling aspects, and ability to preserve blood vessels were noted. Also, pressures and adjustments used as well as complications were encountered. Intraoperative blood loss and oedema formation were monitored. A neuronavigation system was used for intraoperative guidance in most cases. In epilepsy surgery, electrocorticography was used for localization of epileptic foci. Follow-up review included clinical examination and postoperative MR studies after various time intervals.

Comparison of Waterjet Dissection with Ultrasonic Aspiration To compare the dissection and aspiration qualities of the waterjet device with an ultrasonic aspirator, both techniques were applied in parallel in 15 glioma cases. Advantages and disadvantages of each technique were noted. Results of this comparison (first 12 cases)

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have been published elsewhere [24]. However, since the authors consider this comparison to be of major importance to understand the potential of waterjet dissection in the brain, the results of all 15 cases are again presented.

Result General Handling Aspects Intraoperatively, the waterjet device is easy to use. The handling is similar to an ultrasonic aspirator handpiece. However, since the handpiece is smaller and lighter than most ultrasonic aspirators, it is even rather easier to use than an ultrasonic aspirator handpiece. The handpiece allows perfect guidance of the waterjet in a straight direction. In all our procedures, the device was used with a nonfragmented stream and permanent suction. In general, the device can be used in two different ways. On the one hand, aspiration of tissue is possible similar to the application of an ultrasonic aspirator. On the other hand, separation of lesions and / or tumours from the adjacent intact brain parenchyma can also be performed with the waterjet.

Figure 2 a,b. Dissection of the brain parenchyma in right temporal glioblastoma by continuous movements of the handpiece. The arrow indicates a preserved vessel.

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Figure 2 c,d. Pre- and 3 months postoperative MRI of the right temporal glioblastoma showing complete tumour removal.

The nozzle tip was held in direct contact with the tissue but with no application of any mechanical force. The cutting depth was controlled with the aid of the operating room microscope, and the waterjet pressure was adjusted so that a dissection depth of 5 to 10 mm resulted. Tumour debulking and resection, as well as parenchymal resection in epilepsy surgery were performed using this procedure. Additionally, in firm tumours we used the instrument to dissect the lesion from the brain and to develop a plane between them. To form this plane, the beam was directed exactly at the brain–tumour border, with the nozzle tip at the tissue or at maximum in a distance of 2 to 3 mm. The jet was reflected to the brain– tumour border and dissected the lesion from the brain. In these cases, the waterjet pressure was set lower than the pressure required for dissection of the tumour. Blood vessels were spared at pressures below 25 bars in all cases investigated (Figure 2a,b). Subsequently, the vessels were coagulated with bipolar diathermia and cut with microscissors. No complications due to the application of the device were found. Overall, pressures between 3

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and 45 bars were used. In all pathologies a variable dissection of the lesions and a good separation of the lesion from the brain were observed. The results are summarized in the tables 1-4. Table 1. Application of the waterjet in gliomas Aetiology

Number / location

Resection

Waterjet Pressure Complications

Astrocytoma °I Astrocytoma °II

1 (cerebellar) 12 (frontal 5, parietal 2, temporal 5) 2 (frontal) 4 (frontal 3, occipital 1) 15 (frontal 7, temporal 6, occipital 1, parietal 1) 1 (frontal) 50 (temporal 16, parietal 8, frontal 17, occipital 8, cerebellar 1)

Total 1 Total 11, subtotal 1

0 0

Total 2 Total 4

Oligoastrocytoma °II Oligodendroglioma °II Astrocytoma °III

Oligodendroglioma °III Glioblastoma

12 6-10

Usefulness Tumour Aspiration High High

Usefulness Tumour Separation High High

0 0

3-10 8

High High

High High

Total 14, subtotal (intended) 1

0

5-13

Moderate

High

Total 1 Total 41, subtotal (intended) 7, subtotal (unintended) 1, biopsy 1

0 0

10 3-17

High Moderate – poor

High High

Gliomas Eighty-five patients (51 male and 34 female; mean age 59 years, range 12-81 years) were operated on for glioma. Part of these data (51 patients) has been published earlier [24]. The histological diagnosis according to the WHO organization [11] was as follows: one Grade I astrocytoma, 12 Grade II astrocytomas, two Grade II oligo-astrocytomas, four Grade II oligodendrogliomas, 15 Grade III anaplastic astrocytomas, one Grade III oligodendroglioma, and 50 Grade IV glioblastomas. Eighty-three tumours were located supratentorial and two in the cerebellum. Of the supratentorial localization, most tumours were found in the frontal and temporal lobes. The waterjet was intensively used, and pressures ranging from 3 to 17 bars (mostly 5 to 10 bars) were set. For tumour separation from the brain slightly lower pressures than for tumour aspiration were used. In all procedures, the tumour could be separated from the brain with the waterjet. Tumour aspiration was possible in all but one case of a very firm glioblastoma but tended to be more time consuming in high grade tumours. However, in direct comparison with the ultrasonic aspirator, the aspirator works much faster than the waterjet in tumour tissue aspiration. With the waterjet technique, almost no bleeding was observed during the resection of even well-vascularized glioblastomas. No specific complications were observed. Please refer to figures 2a-d for intraoperative pictures and preand postoperative MR images as well as to table 1 for a detailed presentation of the waterjet results in gliomas.

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Metastases Eight male and ten female patients underwent surgery for solitary brain metastasis. On ten of those patients a report was presented in EJSO in 2003 [18]. The mean age scored 54 years with a range of 21 to 70 years. Lesions were located in the frontal lobe 10, parietal lobe 2, temporal lobe 2, and in the cerebellum 6. The waterjet was intensively used and pressures from 4 to 12 bars, mostly 8 bars, were applied. The waterjet was easy to use in all metastases procedures. In all cases, the device was applied for tumour aspiration as well as tumour separation from the healthy brain. Interestingly, very variable results were observed depending on the characteristics of the metastases in both procedures. In metastases firmer than the surrounding intact brain parenchyma, separation was achieved by formation of a plane between the metastasis and the brain. In metastases softer than the brain parenchyma, also the jet was directed slightly towards the tumour, and also a plane at the brain tumour border was formed. However, separation of the lesion from the brain was then rather achieved by tumour aspiration. With those techniques, all metastases could be accurately separated from the brain. In all cases, vessels at the brain-tumour border were preserved with the applied pressures. Tumour aspiration could also be obtained in all metastases. However, very firm metastases required much higher pressures up to 25 bars. With these pressures, blood vessels were often harmed. No complications due to the waterjet dissector occurred. Please refer to figures 3a-d for intraoperative pictures and pre- and postoperative MR images as well as to table 2 for a detailed presentation of the waterjet results in metastases.

Figure 3 a,b. Separation of a left precentral metastasis from the brain by continuous movements of the handpiece.

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Figure 3 c,d. Pre- (T1 with gadolinium) and 24-hours postoperative MRI (T2) of the left precentral solitary metastasis of a lung cancer patient showing complete tumour removal.

Table 2. Application of the waterjet in metastases

Number Resection

Waterjet complications

Lung cancer

4

Total 4

0

Breast cancer Rhadomyosarcoma Hypernephroma Malignant Melanoma Unknown Larynx

3 1 2 1 2 1

Total 4 Total 1 Total 2 Total 1 Total 2 Total 1

0 0 0 0 0 0

Aetiology

Tumour firmness 3 firm, 1 soft 3 soft 1 soft 2 firm 1 soft 2 firm 1 firm

Waterjet pressure (bars) 10-20

Usefulness Tumour Aspiration Poor

Usefulness Tumour Separation High

4-10 5-8 12-25 4-6 8-20 12

High High Poor High Poor Poor

High High High High High High

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Meningiomas The waterjet technique was applied in 13 meningioma patients (three males, ten females, mean age 61.2 years, range 46-70 years). The data of eleven patients have been reported earlier [23]. Eight patients suffered from tumours of the convexity, two each from meningiomas of the sphenoid plane and sphenoid wing, and one each from tumours of the olfactory groove and the frontal falx. An MRI was performed prior to surgery in all cases. Neuronavigation was applied for determining the craniotomy localization in six cases. No complications clearly due to the waterjet application were observed. Because meningiomas are mostly much firmer than the surrounding brain, higher pressures had to be used for tumour dissection. Optimum dissection was found at pressures higher than 20 bars. At these pressures and particularly at pressures higher than 25 bars, blood vessels were also dissected, and in eight of 13 cases a dense network of tumour trabeculae remained. Thus, bleeding increased without achieving satisfactory tissue aspiration. In one case, these high pressures actually led to the perforation of the tumour capsule at the opposite side. In five cases of selected rather soft meningiomas, tumour aspiration was possible under vessel preservation at 6-15 bars. Separation of the meningiomas from the surrounding brain was possible in all cases. Please refer to figures 4a-d for intraoperative pictures and pre- and postoperative MR images as well as to table 3 for a detailed presentation of the waterjet results in meningiomas.

Figure 4 a,b. Separation of a right frontal meningioma from the brain by continuous movements of the handpiece.

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Figure 4 c,d. Pre- and 2-months postoperative MRI (T1 with gadolinium) of the meningioma showing complete tumour removal.

Table 3. Application of the waterjet in meningiomas

Localization

Waterjet complications

Tumour firmness

Waterjet pressure (bars)

Usefulness Tumour Aspiration

Usefulness Tumour Separation

0 0

2 soft 2 firm

8-15 10-35

Moderate Poor

High High

0

1 soft

15-20

Moderate

High

Total 6

0

12-25

Tumour perforation with 25 bars

Poor moderate Poor

High

Total 1, Subtotal 1

4 firm, 2 soft 2 firm

Number

Resection

Sphenoid plane Sphenoid wing

2 2

Olfactory groove Convexity

1

Total 2 Total 1, Subtotal 1 Total 1

6

Falx

2

10-25

High

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Epilepsy Twenty patients underwent surgery for temporal lobe epilepsy and one patient for frontal lesionectomy. On a part of these patients has been reported earlier [20]. Nine males and 12 females accounted to this group; the mean age was 34 years with a range of 13 up to 70 years. In the patients with temporal lobe epilepsy, an anterior- or two-thirds temporal lobectomy tailored by electrocorticography plus an amygdalohippocampectomy were performed. Temporal lobectomy was usually performed using only the waterjet. At pressures of 4 – 10 bars, vessels were preserved, whereas the parenchyma was easily dissected. Particularly for subpial dissection, the waterjet appeared well suited. It allowed precise subpial dissection with preservation of the arachnoid membrane covering the oculomotor nerve or the cerebral peduncle. No complications due to the waterjet dissector were observed. Please refer to figures 5a-d for intraoperative pictures and pre- and postoperative MR images as well as to table 4 for a detailed presentation of the waterjet results in epilepsy surgery.

Figure 5 a,b. Separation of the left temporal lobe by continuous movements of the handpiece. T: temporal lobe; arrow indicates the separation plane.

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Figure 5 c,d. Pre- (flair sequence) and 3-months postoperative MRI (T2 sequence) of the temporal lobe epilepsy patient.

Table 4. Application of the waterjet in epilepsy surgery and various other aetiologies

Aetiology

Number

Resection

Waterjet complications

Tumour firmness

Waterjet pressure (bars)

Usefulness Usefulness Tumour Tumour Aspiration Separation

21

Temporalobectomy 21, Frontal lesionectomy 1

0

Parenchym a

4-10

Poor

High

6

Total 6

0

Very soft

4-8

High

High

3 2

Total 3 Total 2

0 0

Rather firm 4-10 Rather soft 5-10

Moderate Moderate

High ???

2

Total 2

0

Moderate

High

High

Epilepsy

Epidermoid / Dermoid cyst Haemangioblastoma Acoustic neurinoma Primitive neuroectodermal tumours

10-12

Other Aetiologies The various other indications for waterjet dissection were six dermoid cysts, three haemangioblastomas, two acoustic neurinomas, two primitive neuroectodermal tumours, one cavernous haemangioma, and three biopsies. In the dermoid cysts, the waterjet is well suited for aspiration of the cyst contents as well as it is well suited for separation of the lesion capsule from the brain parenchyma. In the haemangioblastoma cases, the waterjet was primarily applied for tumour dissection from the brain. This was obtained very reliable and accurate. In the acoustic neurinomas, the waterjet was applied for tumour aspiration only. This was performed without any problems in both cases. However, at present, no results are

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present for separation of the tumour from the cranial nerves. In the pediatric brain tumours, the waterjet was used for tumour aspiration as well as tumour separation. This was possible in both cases. A reliable parenchyma dissection and tissue aspiration was noted in the other aetiologies. In these various cases, pessures of 8 to 25 bars were applied, and no complications due to the waterjet were noted.

Complications Only one complication clearly related to the application of the waterjet was noted in over 150 cases. This consisted of a perforation of the tumour capsule at the opposite side in a falx meningioma. However, no neurological deficits resulted from this accident. Retrospectively, this perforation was clearly related to the high pressure applied of up to 25 bars. At present, the authors of this chapter only use pressures of up to 15 bars in their daily routine. Under these conditions, it appears to be very unlikely that a deep perforation of a tumour capsule could occur again. In the 154 procedures, one transient oculomotor palsy, one transient hemiparesis and two epidural wound infections occurred in epilepsy surgery cases. Out of all patients, two brain abscess formations (one metastasis, one meningioma) were noted. In one malignant melanoma, a subcutaneous satellite metastases occurred after an intracranial waterjet operation within three months after surgery.

Conclusion In general surgery, especially liver and kidney surgery, the waterjet instrument has been established as an additional valuable surgical tool since the first report of its application in 1982 [25]. In comparison with conventional techniques, a reduction in blood loss and operation time has been shown [1, 31]. In contrast to other new techniques such as laser ablation [37], no thermal damage to the surrounding parenchymal structures occurs. In neurosurgery, with its still high perioperative risk particularly for tumour patients [15], the waterjet device could represent an addition to the neurosurgical armamentarium. The waterjet device could allow a reduction in intraoperative blood loss and postoperative oedema formation due to precise tissue dissection, vessel preservation, and minimum trauma to the adjacent brain parenchyma. However, the clinical experience in neurosurgery is very limited. The goal of the research group of the authors of this chapter is to evaluate the advantages and disadvantages of the application of the waterjet device in intracranial procedures. Up to date, various cadaver experiments [17, 21, 29] and experimental in-vivo studies [19, 22] have been performed. Those studies demonstrated that the brain parenchyma can be dissected very precisely with the waterjet instrument. Second, there is a very good correlation of the applied waterjet pressure and the resulting dissection depth. Third, even smallest vessels can be preserved under pressures which allow a precise and accurate dissection of brain parenchyma. This results in-vivo in a reduction of blood loss compared with an ultrasonic aspirator [19]. In clinical neurosurgical cases, the waterjet device has been applied in more than 150 procedures so far [18, 20, 23, 24, 28]. It has been shown that the instrument

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can safely be applied in gliomas [24], meningiomas [23], metastases [18], epilepsy surgery [20] and various other pathologies. In comparison with conventional techniques such as ultrasonic aspiration, the operative blood loss can be reduced [20, 24]. However, the waterjet instrument remains under debate in the neurosurgical community. Particularly the correct intraoperative application has been described as difficult and hindering. Here, the authors of this chapter present a detailed account of the waterjet technique with special reference to the intraoperative application technique and the results with this technique in distinct surgical pathologies.

General Handling Aspects Intraoperatively, the waterjet device has been shown to be easy to use like an ultrasonic aspirator. With the hand piece, the beam can be perfectly guided. The authors use a nonfragmented stream and permanent suction in all their procedures since with a fragmented stream or pedal steered suction severe bubble formation can occur. Based on their experience, the device has been used for tumour aspiration as in a debulking procedure and for tumour separation from the brain. With direct contact between the nozzle tip and the target tissue, bubble formation ceased to be a problem, and the cutting depth can be easy controlled by the microscope. Also, various intracranial pathologies can be accurately resected as well as aspirated with this technique. While tumour aspiration with the waterjet resembles the aspiration with an ultrasonic aspirator, precise dissection of a lesion from the adjacent intact brain parenchyma under preservation of blood vessels and without application of any thermal force represents a quality unique to the waterjet technique. Thus, further research with this technique is required to evaluate its potential in minimally invasive neurosurgery.

Gliomas and Metastases The prognosis of gliomas has been under continuous evaluation since the 60ies and 70ies [15]. At present, study data point to a role of the extent of resection for the prognosis of these patients. In solitary metastases, the role of surgery is still under debate. However, the authors of this chapter rather prefer to operate on solitary metastases than to start directly with radiotherapy. With the waterjet, gliomas as well as metastases have been the most frequent pathologies treated. There, the new technique has been shown to allow a precise resection of the tumours from the brain under preservation of vessels resulting in a reduction of blood loss. There is no significant tumour tissue aspiration in rather firm metastases. In gliomas, tumour aspiration is possible but can be much faster achieved with the ultrasonic aspirator. Thus, for tumour aspiration such as debulking of the tumour mass, the waterjet appears not to be well suited – at least in the opinion of the authors of this chapter.

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Epilepsy Surgery In epilepsy surgery, temporal lobe resection is a complex procedure harbouring a significant risk for complications [21]. Preservation of the pial-arachnoid layer to avoid - or at least reduce - the risk for injury of the oculomotor nerve and / or the pedunculus cerebri appears to be of major importance. There, it is the impression of the authors of this chapter that the waterjet instrument might be very valuable to preserve this layer leading to a reduction of the surgical risk. However, at present, study data are missing to support this observation [20]. Thus, the device seems to be very valuable for selected epilepsy surgery cases although further research in the field of epilepsy surgery is urgently needed.

Meningioma Surgery For a decision about the potential of the waterjet technique in meningiomas, the patient data set from 13 procedures is rather limited. However, based on this data, the authors consider the waterjet in general for not useful in meningioma surgery despite there was a satisfactory tumour separation from the surrounding brain in all cases. But, this tumour separation is also easily achieved with conventional techniques using cottonoids and has even been reported earlier with the use of a simple syringe [40]. Thus, highly precise separation of the meningioma from the surrounding brain with a 120µm waterjet is rarely required. For tumour debulking, the typical meningioma tumour matrix with dense trabeculae possesses a resistance to the waterjet to high for satisfactory tissue aspiration. In most cases, waterjet pressures would be required which resulted in dissection of tumour vessels. These pressures might even possess a risk of tumour capsule perforation and injury of the adjacent brain parenchyma as reported [23]. Thus, at present, the authors apply the waterjet exclusively in soft highly vascularized meningiomas when vessel preservation is achievable and seems to be of major importance. In fact, this indication is rare and the waterjet was applied by the authors in only two procedures for meningiomas within the last three years.

Complications The authors could pretend particular risks of the waterjet technique in neurosurgery for metastatic spreading and an increased infection rate because of the intense rinsing during surgery. Additionally, there could be an increased risk for damage to surrounding brain structures with waterjet high pressures. In our 154 procedures only one case of a subcutaneous metastatic spread was observed. This patient suffered from a malignant melanoma in the end stage of her disease. Thus, there seems to be no data pointing to an increased risk for metastatic spreading with the waterjet. Two patients suffered from brain abscess formation after cranial surgery (1.3%). Thus, compared to reported infection rates, there is no increased risk for brain abscess formation with application of the waterjet. Also, therer is no significant rate of other waterjet related complications. Thus the device can be considered safe. In all our procedures, only one complication clearly related to the waterjet

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technique was observed. In this case, a high pressure for dissection of the rather firm meningioma was used, and the tumour was perforated at the opposite base. Since this occurred at the frontal base, no neural structures were lesioned by the high pressure jet (25 bars). However, the authors concluded that firm tumours which required pressures higher than used for vessel preservation (20 – 25 bars) are not suited for waterjet aspiration. In all, the waterjet technique enables a precise brain tumour dissection and separation of the tumour from the surrounding intact brain parenchyma with preservation of even very small blood vessels in most cases. The technique appears to possess best qualities for glioma and epilepsy surgery cases as well as selected others. It also is well suited for special indications such as subpial dissections. In comparison with conventional techniques, it allows a reduction of surgical blood loss. Thus, it appears to be most valuable if minimally traumatic surgery with minimal blood loss is of major importance. Currently, studies are ongoing investigating whether or not the waterjet instruments enables a higher radicality in brain tumour surgery. As already under observation in preclinical studies, there may exist further indications for this instrument in endoscopic neurosurgery [9] or intracerebral hematoma evacuation [15]. Particularly in endoscopic applications with its potentially devastating risk of damage to blood vessels [33], the waterjet technique might play an important role in the future. Also, the authors could imagine an advantage of the waterjet in skull base surgery when preservation of cranial nerves and important vessels is a major issue [34]. However, further studies will have to show that the waterjet allows dissection of skull base processes such as meningiomas or neurinomas under preservation of cranial nerves and vessels.

Acknowledgements/Disclosure Most parts of the studies presented were supported by two grants of the Else-KrönerFresenius-Stiftung, Bad Homburg, Germany. The authors state that they have no financial interest in the waterjet device described. All animal experiments reported were performed under Principles of Laboratory Animal Care (NIH publication no.86–23, revised 1985).

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Joachim Oertel, Jürgen Piek, Henry W.S. Schroeder et al. Horie T: [Liver resection by water jet.] Nippon Geka Gakkai Zasshi 90:82–92, 1989 (Jpn). Hubert J, Mourey E, Suty JM, et al: Water-jet dissection in renal surgery: experimental study of a new device in the pig. Urol Res 24:355–359, 1996. Izumi R, Yabushita K, Shimizu K, et al: Hepatic resection using a water jet dissector. Surg Today 23:31–35, 1993. Jakob S, Kehler U, Reusche E, et al: Endoskopischer Einsatz des Water Jet Dissektors im Hirnkammersystem—ein experimentelle Studie. Zentralbl Neurochir 61:14–21, 2000. Kaduk WM, Stengel B, Pohl A, et al: Hydro-jet cutting: a method for selective surgical dissection of nerve tissue. An experimental study on the sciatic nerve of rats. J Craniomaxillofac Surg 27: 327–330, 1999. Kleihues P, Berger PC, Scheithauer BW: The new WHO classification of brain tumours. Brain Pathol 3:255–268, 1993. Kobayashi M, Sawada S, Tanigawa N, et al: Water jet angioplasty — an experimental study. Acta Radiol 36:453–456, 1995. Lipshitz I, Bass R, Loewenstein A: Cutting the cornea with a waterjet keratome. J Refract Surg 12:184–186, 1996. Matzker J: Aussergewohnliche Kehlkopfverletzung. Laryngol Rhinol Otol 58:68–69, 1979. Mukai H, Yamashita J, Kitamura A, et al: Stereotactic Aqua-Stream and Aspirator in the treatment of intracerebral hematoma. An experimental study. Stereotact Funct Neurosurg 57: 221–227, 1991. Oertel J, von Butlar E, Schroeder HWS, Gaab MR: Prognosis of gliomas in the 70ies and today. Neurosurgical Focus 18(4):E12, 2005. Oertel J, Gaab MR, Knapp A, et al: Water jet dissection in neurosurgery: experimental results in the porcine cadaveric brain. Neurosurgery 52:153–159, 2003. Oertel J, Gaab MR, Piek J: Waterjet resection of brain metastases — first clinical results with 10 patients. Eur J Surg Oncol 29: 407–414, 2003. Oertel J, Gaab MR, Pillich D-T, et al: Comparison of waterjet dissection and ultrasonic aspiration: an in vivo study in the rabbit brain. J Neurosurg 100: 498-504, 2004. Oertel J, Gaab MR, Runge U, et al: Waterjet dissection versus ultrasonic aspiration in epilepsy surgery. Neurosurgery 56:ONS-142-ONS-146, 2005. Oertel J, Gaab MR, Runge U, et al: Neuronavigation and complication rate in epilepsy surgery. Neurosurg Rev 27: 214-217, 2004. Oertel J, Gaab MR, Schiller T, et al: Towards waterjet dissection in neurosurgery: experimental in-vivo results with two different nozzle types. Acta Neurochir (Wien) 146: 713-720, 2004. Oertel J, Gaab MR, Warzok R, et al: Waterjet dissection in the brain: review of the experimental and clinical data with special reference to meningioma surgery. Neurosurg Rev 26: 168–174, 2003. Oertel J, Wagner W, Gaab MR, et al: Waterjet dissection of gliomas - experience with 51 procedures. Minim Invas Neurosurg 47: 154-159, 2004 .

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[25] Papachristou DN, Barters R: Resection of the liver with a water jet. Br J Surg 69:93– 94, 1982. [26] Penchev RD, Losanoff JE, Kjossev KT: Reconstructive renal surgery using a water jet. J Urol 162:772–774, 1999. [27] Persson BG, Jeppsson B, Tranberg KG, et al: Transection of the liver with a water jet. Surg Gynecol Obstet 168:267–268, 1989. [28] Piek J, Oertel J, Gaab MR: Waterjet dissection in neurosurgical procedures: clinical results in 35 patients. J Neurosurg 96: 690–696, 2002. [29] Piek J, Wille C, Warzok R, et al: Waterjet dissection of the brain: experimental and first clinical results. Technical note. J Neurosurg 89:861–864, 1998. [30] Rau HG, Meyer G, Cohnert TU, et al: Laparoscopic liver resection with the water-jet dissector. Surg Endosc 9:1009–1012, 1995. [31] Rau HG, Schardey HM, Buttler E, et al: A comparison of different techniques for liver resection: blunt dissection, ultrasonic aspirator and jet-cutter. Eur J Surg Oncol 21:183–187, 1995. [32] Rau HG, Schauer R, Pickelmann S, et al: Dissektionstechniken in der Leberchirurgie. Chirurg 72:105–112, 2001. [33] Schroeder HWS, Oertel J, Gaab MR: Incidence of complications in neuroendoscopic surgery. Child’s Nerv Syst 20: 878-883, 2004. [34] Schroeder HWS, Oertel J, Gaab MR: Endoscope-assisted microsurgical resection of epidermoids in the cerebellopontine angle. J Neurosurg 101: 227-232, 2004 . [35] Schurr MO, Wehrmann M, Kunert W, et al: Histologic effects of different technologies for dissection in endoscopic surgery: ND:YAG laser; high frequency and water-jet. Endosc Surg Allied Technol 2:195–201, 1994. [36] Siegert R, Danter J, Jurk V, et al: Dermal microvasculature and tissue selective thinning techniques (ultrasound and water-jet) of short-time expanded skin in dogs. Eur Arch Otorhinolaryngol 255:325–330, 1998. [37] Suhm N, Gotz MH, Fischer JP, et al: Ablation of neural tissue by short-pulsed lasers— a technical report. Acta Neurochir 138: 346–349, 1996. [38] Summers DA: Waterjetting Technology. London: E & FN Spon, 1995. [39] Terzis AJ, Nowak G, Rentzsch O, et al: A new system for cutting brain tissue preserving vessels: water jet cutting. Br J Neurosurg 3:361–366, 1989. [40] Toth S, Vajda J, Pasztor E, et al: Separation of the tumor and brain surface by “water jet” in cases of meningiomas. J Neurooncol 5:117–124, 1987. [41] Une Y, Uchino J, Shimamura T, et al: Water jet scalpel for liver resection in hepatocellular carcinoma with or without cirrhosis. Int Surg 81:45–48, 1996.

Index A access, 7, 133, 157, 178, 197, 291 accumulation, 10, 12, 18, 21, 66, 105, 123, 124, 195, 214, 292 accuracy, 43, 51 acetone, 110 achievement, 95, 96 acid, 14, 18, 27, 80, 135, 152, 182, 184, 197, 212, 217, 225, 226, 228, 249, 250, 258, 285 activation, ix, xii, 11, 14, 18, 49, 59, 82, 83, 108, 120, 124, 128, 131, 134, 137, 138, 140, 153, 154, 155, 160, 162, 164, 165, 197, 199, 203, 216, 228, 231, 248, 254, 256, 258, 268, 269, 271, 272, 273, 274 active site, 164, 165, 225, 227 acute lymphoblastic leukemia, x, 167, 175, 188, 189 acute promyelocytic leukemia, 251 adaptation, viii, 2, 14, 165 adenoma, 215, 247, 287, 299 ADHD, 288 adhesion, 13, 14, 15, 16, 18, 80, 134, 210, 212, 215 adhesions, 217 adolescents, 189 ADP, 197, 199, 231, 232 adulthood, x, 193, 200 adults, x, xi, 4, 5, 20, 45, 53, 58, 60, 63, 67, 68, 69, 71, 73, 78, 82, 83, 84, 86, 122, 146, 179, 181, 182, 184, 189, 191, 194, 213 adverse event, 39 affect, 2, 4, 5, 15, 16, 120, 121, 180, 254, 282, 283, 285, 292, 294 agar, 18 age, ix, x, 2, 3, 4, 5, 7, 9, 11, 12, 19, 23, 26, 31, 32, 33, 35, 37, 39, 49, 76, 90, 92, 120, 121, 122, 124,

132, 136, 168, 176, 181, 182, 193, 195, 203, 214, 223, 224, 232, 254, 255, 285, 305, 309, 310, 312, 314 ageing, 203, 233 agent, 18, 44, 67, 69, 136, 172, 175, 178, 179, 225, 227, 242, 248, 249, 250, 251 age-related, 132 aggressive behavior, 287 aggressive therapy, 168 aggressiveness, 29, 31, 129, 213 aging, 168, 202 agnosia, 284 AIDS, 169, 170, 171, 173, 185, 186, 189 alcohol, 93, 94, 98 alkaloids, 19 allele, 200 alopecia, 35 alternative, viii, 2, 31, 32, 60, 109, 126, 129, 133, 135, 158, 160, 164, 165, 173, 231, 241 alternatives, 195 alters, 162 amino acids, 213 amnesia, 283, 286, 290, 291, 294, 297, 299 amygdala, 301 anabolism, 21 anatomy, 13 androgen, 279 anemia, 93 aneuploid, 20 anger, 284, 294 angiogenesis, ix, xi, 10, 13, 14, 17, 18, 21, 48, 50, 51, 56, 58, 62, 63, 73, 79, 83, 84, 87, 101, 104, 119, 125, 126, 131, 137, 138, 141, 147, 148, 150,

324

Index

151, 158, 164, 165, 202, 210, 212, 216, 245, 254, 273, 275, 276 angiography, 284 angioma, 55, 289, 295, 300 angioplasty, 320 angiotensin converting enzyme, 147 angiotensin II, 157, 158, 159, 160, 162, 164 angulation, 20 animal modeling, 131 animals, 16, 17, 29, 124, 131, 135, 216 anorexia, 288, 293, 299, 300 anorexia nervosa, 288, 299, 300 ANOVA, 266, 268, 269 antagonism, 156, 157, 162 antibody, 9, 110, 140, 208, 209, 258, 259, 264, 267, 271 anticancer drug, 18, 19, 52, 59, 60, 64, 72, 87 antidepressant, 285, 288, 289 antigen, 9, 61, 65, 66, 98, 110, 111, 115, 134, 135, 137, 138, 140, 143, 169 antigenicity, 134 antigen-presenting cell, 134, 135, 137 antitumor, 226, 250 anxiety, 283, 286, 293, 295 apathy, 285, 290, 292 APC, 240 aphasia, 284 apoptosis, 10, 11, 14, 15, 16, 18, 31, 59, 77, 78, 85, 87, 104, 132, 150, 151, 153, 154, 155, 156, 161, 163, 202, 210, 211, 213, 215, 227, 240, 245, 251, 255, 256, 257, 266, 273, 278, 279 appetite, 285 apraxia, 289, 300 arabinoside, 184, 185, 191 arginine, 197 arrest, 24, 36, 60, 70, 132, 210, 212, 227, 239, 240, 245 arterial hypertension, 35 artery, vii aspiration, 96, 304, 306, 307, 309, 310, 312, 315, 317, 320 assessment, viii, ix, 2, 3, 30, 45, 51, 58, 63, 69, 90, 91, 100, 114, 124, 293 association, 11, 38, 39, 47, 49, 122, 126, 180, 212, 213, 229, 230, 240, 241, 256, 277, 290, 291 assumptions, 21 asthenia, 39 astrocytoma, ix, 9, 11, 12, 13, 19, 47, 51, 57, 62, 67, 68, 69, 71, 72, 76, 78, 83, 85, 96, 101, 106, 109, 112, 113, 114, 119, 121, 122, 123, 124, 128, 130,

135, 137, 138, 139, 143, 165, 194, 204, 210, 215, 217, 223, 224, 298, 299 astrogliosis, 146 asymptomatic, 7 ataxia, 174, 286, 287, 289 atherosclerosis, 174 ATP, 197, 217 attachment, 18, 133, 198, 199 attacks, 285, 286, 295, 296, 297, 298 attention, xi, 30, 76, 105, 170, 185, 194, 229 Attention Deficit Hyperactivity Disorder, 288 attitudes, 42 auditory cortex, 290 autoimmunity, 135, 143, 255 autopsy, 169, 180, 284, 289, 294 axon, 215, 244

B BAC, 109 basal ganglia, 171, 300 basal lamina, 255 base pair, xi, 194, 268 BBB, 229 behavior, 3, 5, 19, 23, 28, 42, 121, 127, 130, 133, 260, 266, 269, 271, 272, 274, 279, 284, 286, 288, 290 behavioral change, 294, 295 behavioral problems, 288, 300 benign, 4, 42, 80, 85, 140, 194, 212, 283 bias, 33 binding, 9, 11, 19, 105, 126, 128, 138, 147, 151, 153, 154, 156, 160, 197, 199, 201, 203, 210, 213, 216, 217, 225, 227, 230, 233, 236, 239, 240, 245, 268, 269, 271, 274 bioavailability, 250 biological activity, 159, 165, 278 biological processes, 100 biological systems, 277 biomarkers, viii, 90, 104, 128, 129, 130, 229, 248 biopsy, 10, 23, 26, 28, 31, 33, 59, 67, 68, 69, 78, 171, 309 biotin, 108, 111 bipolar disorder, 285 bladder, 105, 276 , 309, 312 blocks, 197, 200, 217 blood, ix, xii, 8, 10, 18, 28, 37, 45, 56, 67, 69, 80, 92, 113, 114, 120, 132, 133, 140, 145, 147, 150,

Index 161, 162, 163, 169, 172, 176, 229, 289, 304, 306, 310, 312, 316, 317, 319 blood flow, 10 blood vessels, 120, 132, 162, 304, 305, 306, 310, 312, 317, 319 blood-brain barrier, 8, 18, 69, 80, 113, 114, 120, 133, 150, 161, 163, 172, 229 body, vii, 126, 133, 182, 207 body fluid, 207 bone marrow, 127, 135, 140, 171, 178, 180, 181, 182, 188 boys, 299 brachytherapy, 84 brain, vii, viii, ix, xi, xii, 1, 2, 3, 13, 15, 19, 22, 26, 27, 28, 29, 30, 31, 34, 36, 37, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 79, 80, 81, 82, 83, 85, 86, 87, 89, 90, 91, 98, 107, 108, 109, 112, 113, 114, 119, 120, 121, 122, 123, 127, 128, 131, 133, 136, 137, 140, 141, 142, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 158, 159, 161, 162, 163, 168, 169, 170, 171, 172, 173, 174, 185, 186, 187, 194, 195, 202, 204, 210, 211, 213, 214, 215, 216, 217, 223, 224, 225, 229, 237, 240, 242, 245, 246, 247, 253, 254, 255, 256, 276, 277, 279, 281, 282, 283, 286, 287, 288, 290, 291, 292, 293, 298, 300, 301, 304, 305, 307, 308, 309, 310, 312, 315, 316, 317, 318, 319, 320, 321 brain abscess, 316, 318 brain activity, 46 brain functioning, 3, 36 brain growth, 52 brain stem, 68, 300 brain tumor, vii, ix, xi, 27, 31, 45, 48, 50, 58, 59, 63, 68, 69, 83, 87, 90, 108, 122, 127, 137, 145, 146, 150, 161, 194, 195, 213, 214, 240, 253, 254, 287, 291, 292, 293 brainstem, 4, 12, 69 brainstem glioma, 12 branching, 215 Brazil, 89, 90, 246 breakdown, 289 breast cancer, 90, 115, 216, 241, 243, 244, 245 breast carcinoma, 107, 236 breeding, 131 burning, 285

325

C cachexia, 288 cadaver, 305, 316 , 217, 225 calcium, 104, 152, 161, 215 caloric intake, 291 cancer, vii, viii, 17, 18, 19, 51, 52, 56, 57, 58, 59, 61, 64, 66, 70, 75, 79, 80, 81, 85, 86, 87, 89, 90, 91, 92, 93, 94, 98, 99, 104, 107, 108, 109, 116, 128, 129, 130, 131, 135, 139, 140, 142, 143, 146, 148, 149, 150, 152, 153, 154, 155, 156, 157, 160, 161, 162, 163, 165, 177, 188, 189, 190, 194, 195, 201, 202, 203, 204, 205, 206, 208, 209, 210, 211, 213, 214, 215, 216, 217, 225, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 241, 245, 246, 247, 248, 249, 254, 255, 273, 274, 275, 276, 278, 311 cancer cells, vii, 98, 99, 149, 153, 154, 157, 201, 202, 203, 208, 209, 210, 211, 214, 233, 245, 248, 249, 255 cancer progression, 56, 107, 109, 148, 149, 157 candidates, 104, 134, 162 capillary, 134, 237 capsule, 312, 315, 316, 318 carbon, 63, 200 carcinogen, 90, 108 carcinogenesis, 57, 125, 244, 254 carcinoma, 164, 169, 215, 244, 245, 250, 294 cardiovascular disease, 147, 154 cardiovascular function, 147 carrier, 217 case study, 283 cast, 231 catalyst, 292 categorization, 101 cauda equina, 53 causal relationship, 131 CD8+, 135 cDNA, 15, 61, 69, 87, 100, 106, 116, 127, 135, 136, 139, 255, 257 cell, ix, xi, 3, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 28, 46, 47, 48, 49, 50, 51, 52, 58, 60, 61, 62, 65, 66, 68, 70, 71, 72, 75, 76, 79, 82, 83, 84, 85, 86, 98, 100, 101, 104, 106, 109, 110, 111, 116, 117, 119, 120, 121, 122, 123, 124, 125, 126, 127, 130, 132, 133, 134, 135, 136, 137, 139, 140, 141, 143, 144, 145, 146, 147, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 160, 163, 168, 169, 173, 174, 175, 177, 178, 179, 180, 181, 184, 186, 187, 189, 195, 202, 206, 208, 209, 210, 211, 212, 213,

326

Index

215, 217, 224, 227, 238, 239, 240, 241, 242, 244, 245, 247, 250, 251, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 268, 269, 270, 271, 272, 273, 274, 276, 277, 278, 279, 280, 294 cell body, 133 cell culture, 16, 18, 131, 150, 256, 273 cell cycle, 9, 11, 12, 15, 16, 18, 20, 47, 60, 62, 104, 123, 124, 126, 132, 195, 210, 212, 217, 227, 238, 239, 245 cell death, 10, 132, 153, 154, 211, 240, 254 cell line, xi, 10, 16, 17, 18, 21, 48, 49, 51, 52, 68, 72, 79, 82, 84, 86, 106, 130, 131, 134, 140, 141, 144, 149, 160, 179, 210, 211, 212, 213, 215, 217, 224, 240, 242, 247, 254, 256, 257, 259, 260, 261, 262, 263, 264, 265, 266, 268, 269, 270, 271, 272, 273, 274, 278 cell surface, 15, 16, 154, 155, 169, 255, 266 cellular automaton, 63 central nervous system, vii, viii, x, xi, 3, 5, 14, 16, 29, 54, 55, 57, 58, 59, 63, 67, 71, 84, 89, 93, 106, 120, 121, 122, 132, 133, 134, 139, 140, 141, 146, 147, 165, 167, 168, 170, 172, 180, 181, 183, 185, 186, 187, 188, 189, 190, 191, 193, 194, 195, 214, 215, 253, 275, 279, 282, 291, 300 centromere, 109, 230 cerebellar development, 254 cerebellum, 4, 224, 254, 273, 310 cerebral aneurysm, 300 cerebral hemisphere, 47, 67, 69, 73, 77, 86, 122 cerebrospinal fluid, 72, 175, 188, 251, 292 cerebrovascular disease, 174 cerebrum, 79, 121, 122, 224 cervical cancer, 250 cervix, 248 channels, 61, 80 chemoprevention, 247 chemotaxis, 21 chemotherapy, x, 17, 24, 25, 26, 35, 37, 38, 39, 40, 42, 43, 44, 46, 48, 49, 63, 67, 68, 69, 72, 79, 80, 81, 82, 87, 96, 135, 136, 167, 171, 172, 173, 174, 175, 176, 178, 179, 182, 184, 185, 186, 187, 188, 189, 195, 214, 217, 225, 229, 242, 249, 254, 284, 293, 294 childhood, x, xi, 47, 183, 188, 189, 193, 194, 211, 239, 240, 253, 286 children, x, xi, 5, 51, 73, 78, 79, 121, 122, 174, 175, 176, 177, 178, 179, 184, 188, 189, 193, 194, 213, 217, 254, 278, 319 chloroform, 100 choroid, 288, 299

chromatography, 236 chromosome, 10, 11, 12, 38, 46, 55, 59, 60, 61, 63, 73, 79, 84, 108, 109, 123, 124, 125, 137, 139, 177, 195, 200, 210, 211, 213, 214, 215, 216, 217, 255 chronic myelogenous, 249 cirrhosis, 321 classes, ix, 9, 32, 120, 129, 215 classification, ix, 3, 4, 8, 13, 16, 24, 28, 49, 51, 55, 56, 61, 64, 70, 71, 81, 84, 90, 91, 101, 103, 104, 109, 114, 115, 116, 119, 121, 122, 126, 128, 130, 138, 139, 140, 181, 237, 305, 320 cleavage, 199, 204 clinical diagnosis, 140 clinical examination, 306 clinical presentation, 179, 285, 287, 288, 291 clinical trials, x, 26, 28, 39, 72, 78, 135, 136, 157, 167, 177, 183, 184, 226, 228 cloning, 165, 206, 234, 255, 276 clustering, 16, 101, 104, 128, 129, 207 clusters, 69, 128, 244 , vi, xi, 19, 67, 72, 73, 81, 137, 147, 151, 155, 163, 168, 169, 173, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 193, 194, 195, 217, 218, 219, 220, 221, 222, 223, 224, 225, 228, 229, 253, 255, 295, 300 CO2, 256 coagulation, xii, 304 coding, 101, 106, 203, 213 codon, 11 cognition, 35 cognitive deficit, 30 cognitive dysfunction, 30 cognitive function, viii, 2, 30, 36, 48, 64, 84 cognitive impairment, 254, 274 cohort, 64, 73, 173, 224 collaboration, 42, 92 collagen, 133 colon, 159, 163, 215, 217, 233, 246, 250 colon cancer, 163, 215, 217 colonization, 66 colorectal cancer, 105, 114, 215, 235, 237, 244 common findings, 124 common rule, 20, 25 communication, 176 community, 42, 317 compensation, viii, 2, 3, 29, 30, 36, 53, 54 competition, 21, 22 complementary DNA, 257 complex partial seizure, 286

Index complexity, 130, 184 complications, vii, 190, 191, 305, 306, 308, 309, 310, 311, 312, 313, 314, 315, 316, 318, 321 components, ix, 9, 10, 14, 16, 32, 47, 90, 98, 133, 145, 146, 148, 152, 153, 157, 195, 225, 227 composition, 19, 57, 133, 134 compounds, 18, 27, 76, 237 comprehension, 36, 57 computed tomography, 47, 69, 78, 84, 86, 170, 171 concentration, 25, 43, 120, 154, 155, 256, 257, 258, 268, 269 conception, 29 concordance, 51, 63, 283 condensation, 239 conditioning, 22 conduct, 28 conduction, 120 confidence, 22, 176, 177, 265 confidence interval, 22, 176, 177 configuration, 8, 14, 33, 52 conflict, 232 confusion, 294 connectivity, viii, 2, 3, 36, 54 consciousness, 94, 292 conservation, 22 consolidation, 173, 182 contamination, 96, 105, 176 context, xi, 6, 24, 25, 34, 44, 123, 147, 148, 149, 154, 155, 157, 176, 178, 180, 201, 203, 208, 210, 212, 227, 254, 274 contralateral hemisphere, 60, 94 control, 7, 11, 15, 18, 35, 42, 53, 54, 56, 57, 61, 68, 75, 92, 93, 94, 98, 100, 104, 106, 115, 120, 126, 135, 147, 149, 150, 154, 155, 156, 164, 199, 204, 210, 224, 230, 232, 238, 239, 254, 257, 258, 259, 261, 262, 266, 267, 291, 305 control group, 75 convergence, 277 conversion, 190, 213, 235, 289 conversion disorder, 289 cooling, 108 cornea, 320 corpus callosum, 2, 36, 54, 94, 171 correlation, xi, 9, 11, 23, 53, 55, 56, 60, 65, 76, 84, 85, 92, 101, 125, 132, 180, 210, 211, 214, 224, 254, 273, 277, 291, 292, 298, 316 cortex, 36, 47, 49, 52, 53, 54, 55, 59, 63, 66, 71, 75, 78, 79, 159, 290, 300, 301 corticosteroids, 171, 173 cotton, 260

327

coupling, 160 covering, 314 cranial nerve, 175, 177, 180, 316, 319 craniotomy, 52, 69, 70, 77, 81, 86, 286, 312 CSF, 20, 32, 135, 157, 170, 172, 175, 176, 177, 180, 188, 194, 229 CT scan, 46, 170, 284, 285, 286, 287 cues, 229 culture, ix, 16, 124, 131, 145, 153, 155, 256 cycles, 38, 105, 181, 257, 258 cyclophosphamide, 174 cyst, 159, 171, 287, 295, 296, 299, 300, 315 cytoarchitecture, 121 cytochrome, 19 cytogenetics, 108, 109, 123 cytokines, 11, 13, 14, 15, 50, 135 cytology, 95, 96, 170 cytometry, 9, 51, 84 cytoplasm, xi, 112, 113, 122, 212, 254, 266 cytosine, 182, 184, 185, 191, 199, 203, 209, 213, 232, 233, 237 cytostatic drugs, 229 cytotoxicity, 19, 144

D damage, xii, 29, 61, 80, 162, 174, 304, 316, 318, 319 data analysis, 105, 117, 129 data set, 94, 130, 318 database, 52, 86, 94, 168 death, vii, ix, 1, 7, 10, 21, 58, 62, 90, 145, 147, 152, 182, 211, 216, 217, 225, 240 decision making, vii, 1, 28, 31, 44, 57 decisions, 31 declarative memory, 291 defects, 146, 147, 152, 177, 213, 242 defense, 101, 134 deficit, vii, viii, 1, 2, 3, 23, 29, 30, 31, 44, 46, 56, 156 definition, ix, 3, 5, 8, 28, 51, 52, 90, 91, 114, 175 deformation, 14 degenerate, 242 degradation, 11, 198, 210, 212, 225, 238, 250, 266, 278, 279 delivery, 44, 186, 187 delusions, 287, 288, 289, 295, 296, 297 demand, 28, 184 dementia, 174 dendritic cell, 134, 135, 140, 141, 143, 144

328

Index

density, 10, 15, 22, 25, 27, 28, 45, 67, 126, 128, 132, 143, 150, 199, 206, 237 deoxyribonucleic acid, 51 deposits, 180 depression, 73, 283, 284, 288, 289, 290, 293, 294, 297, 298 depressive symptoms, 283, 289, 294 deregulation, 217 derivatives, 158 dermatology, 304 desorption, 130 destruction, 4, 292 detection, 3, 7, 31, 36, 45, 56, 78, 105, 107, 108, 110, 111, 115, 116, 127, 130, 143, 170, 204, 206, 207, 208, 209, 210, 236, 237, 238, 258, 259, 298 determinism, 6 developing brain, 14 deviation, 22, 70 dexamethasone suppression test, 286 diagnostic criteria, 288 diagnostic markers, 142 diet, 136 differential diagnosis, 59, 168 differentiation, 15, 16, 46, 61, 67, 70, 76, 120, 121, 123, 127, 146, 152, 202, 215, 227, 250, 254, 255, 273, 276, 279 diffusion, 18, 21, 22, 24, 28, 55, 57, 65, 80, 86 diffusion-weighted imaging, 65 digestion, 100, 107, 108, 111, 204, 223 diploid, 21 discomfort, 286 discrimination, 28, 76, 223 disease progression, 38, 255, 273 disorder, 286, 289, 295 displacement, 86 dissociation, 57, 200, 301 distribution, 4, 21, 27, 43, 55, 82, 92, 121, 132, 146, 152, 162 diversity, 5, 7, 9, 25 division, 127 DNA, viii, xi, 9, 10, 11, 12, 15, 19, 48, 51, 52, 62, 65, 70, 71, 76, 77, 79, 80, 81, 83, 90, 92, 97, 100, 101, 104, 105, 106, 107, 108, 109, 114, 115, 116, 117, 125, 131, 134, 138, 139, 142, 150, 161, 164, 194, 195, 196, 197, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 212, 213, 214, 223, 225, 227, 228, 229, 230, 232, 233, 234, 235, 236, 237, 238, 241, 242, 243, 244, 248, 249, 257, 258, 266, 268 DNA breakage, 197

DNA ploidy, 48, 77, 83 DNA repair, 138, 199, 202, 210, 213, 242, 243 DNA sequencing, 106 dogs, 251, 304, 321 domain, 126, 154, 212, 213, 217, 225, 227, 230, 233, 236, 239, 240, 241, 246, 271, 278 dominance, 83 dosing, 228 double helix, 230 down-regulation, 15, 139, 241 drainage, 292 Drosophila, 232, 233, 244 drug action, 18 drug delivery, 25, 81, 178 drug efflux, 18 drug resistance, 18, 19, 25, 44, 49, 69, 72, 75, 80, 104, 202, 217 drug therapy, 104 drugs, viii, 2, 14, 16, 17, 19, 25, 66, 90, 142, 147, 152, 155, 157, 214, 225, 226, 227, 228, 248 dry ice, 98 DSC, 56 dura mater, 180, 190 duration, 34, 39, 40, 42, 44, 80, 158, 184 DWI, 28, 43 dysphoria, 285 dysplasia, 20

E ECM, xi, 216, 253, 255, 266, 267, 273 ECM degradation, 273 ecology, 18, 21, 232 edema, 7, 14, 24, 122, 147, 150, 171, 173, 284, 287, 292 electrolyte, 147, 282 electrophoresis, 100, 129, 268 embryogenesis, 16, 200 emergence, 21 emission, 45, 47, 48, 52, 53, 56, 58, 62, 69, 73, 75, 78, 84, 100 emitters, 26 emotional information, 290 encephalopathy, 35 encoding, 129, 143, 212, 239 endocrine, 147, 287 endothelial cells, 13, 14, 66, 71, 74, 112, 113, 120, 132, 134, 150, 163 endothelium, 112, 113, 174 environment, 17, 21, 40, 42, 104, 120, 127, 133

Index environmental change, 125 enzymatic activity, 259, 276 enzyme-linked immunosorbent assay, 116 enzymes, 9, 19, 67, 76, 106, 107, 108, 147, 148, 156, 197, 200, 209, 230, 266 ependymoma, 212, 213, 214, 216, 223 epidemiology, 3, 56, 81 epigenetics, 195, 228, 229, 232, 248 epilepsy, xii, 6, 7, 23, 30, 32, 34, 35, 37, 39, 40, 42, 44, 46, 53, 69, 79, 92, 226, 303, 305, 306, 308, 314, 315, 316, 317, 318, 319, 320 equipment, 96, 98 erosion, 8 esophageal cancer, 276 esophagus, 256 , 217, 225 ETA, 152, 154, 156, 157, 158, 164 ethanol, 100, 110 ethical issues, 32 etiology, 45, 82, 90, 109, 141 euchromatin, 195, 198, 202 euphoria, 288, 289, 294, 295, 297 euploid, 21 Europe, 305 evacuation, 319 evidence, xi, 31, 32, 33, 45, 55, 56, 68, 75, 79, 83, 122, 127, 134, 135, 161, 162, 169, 170, 171, 181, 185, 199, 216, 254, 255, 256, 271, 272, 274, 284, 288, 293 evolution, viii, 2, 3, 6, 7, 9, 19, 21, 23, 24, 28, 39, 40, 42, 44, 71, 77, 78, 107, 124, 126, 190, 298 examinations, 28, 31, 32 excision, 56, 232, 286 exclusion, 202, 256 executive functions, 30, 290 exons, 105 exploitation, 122 exposure, 90, 150, 151, 249 expressed sequence tag, 136 expression, viii, ix, xi, 12, 13, 15, 16, 18, 19, 45, 50, 51, 55, 56, 59, 60, 61, 62, 65, 70, 71, 72, 74, 75, 78, 80, 81, 83, 84, 85, 86, 90, 91, 92, 100, 101, 104, 105, 106, 112, 113, 114, 122, 123, 124, 126, 127, 129, 130, 131, 133, 134, 137, 138, 139, 140, 141, 142, 143, 145, 146, 147, 149, 150, 151, 153, 155, 157, 158, 160, 161, 162, 163, 164, 165, 195, 196, 200, 201, 203, 209, 210, 211, 212, 213, 214, 215, 216, 217, 223, 224, 232, 234, 235, 239, 240, 241, 242, 243, 245, 246, 247, 249, 251, 253, 254,

329

255, 256, 260, 262, 263, 264, 265, 266, 272, 273, 274, 275, 276, 277, 278, 279, 284 external environment, 22 extraction, 97, 100, 259 extrapolation, 290

F factor analysis, 78 failure, 85, 135, 175, 254 false positive, 17, 208 family, xi, 15, 94, 106, 124, 134, 150, 152, 197, 200, 212, 215, 216, 217, 232, 240, 241, 244, 246, 253, 255, 273, 277 family members, xi, 134, 253 fatigue, 286 fatty acids, 217, 246 feces, 288 feelings, 284, 286 females, 200, 221, 288, 312, 314 fibers, 20, 51, 120 fibroblast growth factor, 259 fibroblasts, 231, 250, 275 fibrosis, 39 films, 259 Finland, 62 first generation, 305 fixation, 97 flank, 131 flight, 130, 283 fluctuations, 292 fluid, 159, 170, 178, 292 fluorescence, 74, 101, 102, 105, 108, 109, 117, 210, 238 fluorine, 63 focusing, 42, 90, 91 foramen, 180 fractality, 21, 25 France, 1, 56, 275 freedom, 260, 270 frontal cortex, 290 frontal lobe, 54, 170, 283, 289, 290, 295, 298, 300, 310 functional imaging, 75 fungal metabolite, 226, 227

G gadolinium, 171, 311, 313 gait, 174, 285, 295 game theory, 18, 69

330

Index

gastritis, 93 gastroesophageal reflux disease, 93 GDP, 215 gel, 99, 100, 107, 129, 204, 236, 257, 268 gender, 92, 121, 182, 242, 255 gene, viii, ix, xi, 11, 12, 13, 14, 17, 18, 19, 26, 42, 47, 51, 52, 56, 57, 58, 59, 61, 63, 64, 68, 70, 71, 72, 74, 75, 77, 79, 81, 82, 83, 84, 85, 86, 87, 90, 91, 92, 100, 101, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 120, 122, 123, 124, 125, 127, 129, 130, 131, 134, 136, 137, 138, 139, 140, 141, 142, 143, 161, 162, 163, 164, 170, 185, 194, 195, 196, 197, 199, 200, 201, 202, 203, 204, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 223, 224, 228, 229, 230, 231, 232, 233, 234, 235, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 255, 257, 261, 262, 263, 264, 266, 271, 272, 273, 275, 276, 277 gene amplification, 109, 123 gene expression, viii, ix, 14, 17, 18, 19, 26, 51, 52, 57, 58, 61, 70, 75, 81, 82, 83, 87, 90, 91, 92, 100, 101, 104, 105, 106, 108, 112, 113, 115, 116, 120, 123, 125, 127, 129, 138, 141, 163, 195, 196, 197, 199, 200, 201, 208, 210, 211, 217, 231, 235, 241, 245, 246, 257, 275, 276, 277 gene promoter, 125, 202, 206, 214, 247 gene silencing, 59, 197, 199, 200, 202, 203, 210, 214, 216, 224, 228, 230, 243, 246, 255, 276 gene targeting, 131 gene therapy, 26, 72 gene transfer, 82, 83 generation, 160, 185, 213 genes, viii, x, xi, 10, 11, 12, 14, 15, 17, 18, 19, 56, 58, 61, 70, 71, 72, 76, 80, 81, 84, 90, 91, 100, 101, 102, 103, 104, 105, 106, 108, 109, 114, 115, 116, 123, 124, 126, 127, 129, 130, 131, 134, 138, 139, 140, 142, 194, 195, 196, 197, 199, 200, 201, 202, 203, 204, 206, 207, 209, 210, 213, 214, 217, 218, 219, 220, 221, 222, 223, 225, 227, 233, 234, 235, 238, 239, 240, 243, 244, 246, 247, 248, 254, 271, 272, 274 genetic defect, 204, 254 genetic factors, 16 genetic information, 104, 122 genetic marker, viii, 90, 124 genetic mutations, x, 194 genetics, 47, 62, 64, 68, 71, 115, 131, 142, 277 genome, xi, 109, 194, 195, 199, 200, 202, 204, 205, 206, 208, 210, 230, 233, 234, 236 genomic regions, 204

genotype, 10, 16, 85, 188 germ cell, 300 Germany, 193, 303, 305, 306, 319 gift, 256, 259 gingivitis, 93 gland, 256 glia, 11, 16, 120, 121, 130 glial cells, ix, 3, 10, 47, 119, 120, 121, 141, 163, 215 glioblastoma, ix, x, 8, 11, 12, 15, 49, 58, 66, 70, 83, 84, 85, 86, 101, 119, 122, 124, 126, 128, 134, 136, 138, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 155, 156, 157, 158, 164, 195, 211, 212, 213, 214, 216, 217, 228, 243, 245, 246, 247, 249, 283, 289, 307, 308, 309 glioblastoma multiforme, ix, 8, 58, 119, 122, 128, 136, 214, 216, 228, 249, 289 glioma, vii, viii, x, 1, 2, 3, 4, 9, 11, 13, 16, 17, 18, 20, 21, 22, 23, 26, 27, 30, 31, 35, 36, 40, 42, 43, 44, 46, 47, 48, 50, 51, 54, 55, 56, 57, 58, 61, 62, 63, 64, 66, 67, 68, 70, 71, 72, 74, 75, 78, 79, 80, 81, 82, 83, 84, 86, 87, 106, 116, 121, 123, 124, 125, 126, 127, 128, 129, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 158, 161, 163, 169, 170, 193, 211, 212, 214, 215, 217, 223, 224, 228, 229, 238, 243, 244, 306, 319 globus, 290 glucose, 18, 26, 48, 52, 217 , 217, 225, 249 glycerol, 98 goals, 104 grades, ix, 3, 4, 5, 11, 15, 55, 92, 106, 112, 113, 119, 121, 125, 128, 130, 144, 212, 224, 305 grading, 8, 9, 28, 50, 51, 52, 53, 64, 66, 68, 70, 77, 121, 122, 126, 132, 139 grants, 274, 319 granules, 111 groups, xi, 4, 5, 6, 26, 32, 34, 40, 42, 90, 92, 125, 127, 156, 168, 173, 175, 180, 181, 183, 195, 200, 216, 223, 224, 225, 227, 253, 255, 260 growth, vii, viii, ix, x, 1, 2, 3, 4, 7, 8, 10, 11, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 30, 32, 37, 39, 43, 44, 45, 46, 48, 50, 51, 55, 56, 58, 59, 60, 61, 62, 63, 65, 67, 69, 71, 72, 74, 75, 77, 79, 80, 81, 82, 84, 86, 87, 109, 121, 123, 124, 126, 128, 132, 138, 139, 141, 142, 145, 146, 147, 150, 151, 153, 155, 158, 161, 163, 164, 165, 169, 194, 211, 213, 215, 216, 217, 235, 243, 244, 245, 248, 254, 255, 266, 274, 277, 278, 289 growth dynamics, 45, 63

Index growth factor, x, 10, 13, 14, 15, 18, 45, 46, 50, 58, 59, 60, 65, 71, 74, 75, 77, 79, 82, 84, 109, 124, 126, 128, 132, 138, 139, 141, 142, 153, 163, 164, 165, 194, 216, 277, 278 growth hormone, 243 growth rate, vii, 1, 4, 20, 21, 22, 23, 24, 25, 26, 28, 43, 51 growth spurt, 21 guanine, 125, 213, 243 guidance, 27, 215, 244, 306, 307 guidelines, ix, 6, 119

H hallucinations, 283, 287, 289, 290, 293, 295, 296, 298, 299 harm, 287 HE, 232 head and neck cancer, 250 headache, 39, 94, 180, 285, 296 headache,, 94, 180 health, 137, 298 heart disease, 93 heat, 199 heating, 108 heavy metals, 97 hematoma, 67, 319, 320 hemiparesis, 170, 316 hemisphere, 2, 30, 65, 75 hemorrhage, 45, 94 hepatitis, 116 hepatocellular carcinoma, 245, 321 herpes, 62 herpes simplex, 62 herpes simplex virus type 1, 62 heterochromatin, 195, 197, 199, 230, 232 heterogeneity, ix, 9, 17, 25, 26, 31, 47, 51, 56, 60, 85, 119, 121, 122, 243 hippocampus, 285, 291, 295, 301 histogenesis, 8 histogram, 76 histology, x, 16, 23, 26, 31, 51, 53, 83, 131, 180, 182, 189, 193, 223, 224 histone, xi, 194, 195, 196, 197, 198, 199, 202, 203, 208, 209, 225, 226, 227, 230, 231, 232, 233, 234, 235, 248, 250, 251 HIV, 168, 170, 179, 185 hopelessness, 285 hormone, 282 hospitalization, 286

331

host, 18, 20, 22, 24, 28, 44, 107, 200, 279 hot spots, 124 human behavior, 300 human brain, ix, 50, 56, 60, 61, 62, 68, 69, 70, 71, 76, 77, 79, 139, 140, 145, 147, 159, 161, 162, 229, 238, 282 human genome, 123, 138, 195, 199, 204 human immunodeficiency virus, 168 hybridization, 47, 100, 105, 106, 108, 109, 115, 116, 128, 130, 236, 237, 275 hydrocephalus, 286, 295 hydrocortisone, 175, 177, 178, 179 hydrolysis, 105, 152, 160, 217 hyperactivity, 285 hypernatremia, 300 hypertension, 23, 94, 159 hypertrophy, 163 hypothalamus, 5, 288, 291, 299 hypothesis, 29, 54, 184, 202, 203 hypoxia, 13, 25, 71, 79, 150, 159, 217

I iatrogenic, 31 ICAM, 134 ideas, 283 identification, ix, 60, 61, 62, 90, 91, 94, 101, 104, 105, 106, 113, 114, 123, 128, 129, 131, 132, 139, 161, 183, 204, 206, 246 identity, 212, 217, 299 illumination, 96 image analysis, 143 imbalances, 108, 109 immersion, 97 immigrants, 299 immune reaction, 134 immune response, 15, 134, 135, 142 immune system, 169, 199 immunity, 14 immunization, 140, 143 immunodeficiency, 186, 187 immunoglobulin, 170 immunohistochemistry, viii, xi, 31, 80, 90, 98, 109, 114, 254 immunoreactivity, 60, 63, 80, 113 immunotherapy, 85, 133, 135, 141, 142 implementation, 178 imprinting, 200, 202, 203, 233 impulsive, 289 in situ hybridization, 74, 108, 109

332

Index

in vitro, 12, 14, 16, 17, 29, 50, 61, 64, 69, 72, 76, 105, 116, 125, 133, 134, 150, 153, 160, 161, 163, 179, 216, 227, 255, 259, 260, 266, 272, 273, 279 incidence, vii, 2, 3, 7, 33, 59, 62, 72, 131, 136, 137, 146, 168, 169, 170, 179, 180, 181, 182, 184, 185, 291 inclusion, 93, 197 India, 78 indication, 28, 30, 32, 176, 305, 318 indicators, 61, 94, 248 indices, 9, 21, 31, 37, 51, 60, 69, 80, 273 indolent, vii, 1, 43 induction, 83, 150, 151, 154, 173, 177, 199, 211, 216, 227, 231, 255, 279 industrialized countries, 136 inefficiency, 18 infancy, 130 infection, 131, 318 inflammation, 134, 202, 255, 273 inflammatory cells, 133 inflammatory disease, 169 influence, 28, 33, 42, 48, 51, 74, 77, 116, 133, 203, 228, 277, 285 informed consent, 94 inhibition, 15, 19, 21, 61, 149, 151, 155, 158, 160, 161, 162, 201, 211, 216, 227, 234, 235, 238, 239, 245, 250, 255, 273, 279 inhibitor, 83, 90, 159, 160, 165, 210, 216, 226, 228, 229, 245, 248, 249, 250, 251, 258, 259, 271, 272 inhomogeneity, 5 initiation, 123, 228 injury, 50, 65, 318 inositol, 50 input, 105 insertion, 123, 162, 286, 295 instability, 10, 11, 18, 81, 202, 204, 212, 225, 227, 241, 248 institutions, 9, 32, 175, 180 instruments, 305, 319 integration, viii, ix, 2, 36, 48, 120, 290 integrity, 99 intellect, 187 intensity, 7, 8, 10, 94, 100, 132, 139, 260, 268, 306 intent, 98, 185 interaction, 14, 17, 18, 24, 44, 58, 90, 110, 164, 165, 197, 212, 215, 290 interactions, 48, 126, 133, 159, 198, 199, 203, 215, 255, 273, 277 intercellular adhesion molecule, 142

interest, 6, 9, 17, 24, 26, 27, 33, 38, 133, 135, 168, 172, 195, 203, 210, 213, 264, 319 interface, 11, 21, 34 interference, 101, 199, 225, 227, 232 interferon, 19, 134, 135, 217, 225, 286 interferons, 142 interpersonal relations, 285 interpersonal relationships, 285 interphase, 100, 108, 197 interpretation, 105, 108, 184 interrelationships, 3, 31 interval, 176, 177, 273 intervention, 36, 132, 173, 181, 284 interview, 93 intracranial pressure, 7, 33, 292 intraocular, 173, 186, 187 intravenously, 181 intuition, 42 involution, 87 iodine, 66 ionization, 130 ions, 120 ipsilateral, 30 iron, 97 irradiation, viii, 2, 12, 43, 44, 68, 69, 175, 177, 178, 179, 184, 185, 187, 188, 254 isochromosome, 204 isolation, 174, 236, 255, 257 isozymes, 152 Italy, 119

J Japan, 259, 274 justification, 184

K karyotype, 177 kidney, 158, 304, 316 kinase activity, 125, 277 kinetics, 17, 24, 44, 64, 67, 75, 78, 79 kinetochore, 227 knowledge, ix, 2, 5, 29, 32, 40, 42, 44, 90, 91, 104, 114, 120, 127, 129, 156, 274, 289

L labeling, 51, 60, 65, 66, 69, 71, 77, 78, 80, 82, 84, 111, 113, 204, 289 lactate dehydrogenase, 170, 183

Index land, 42 language, 6, 24, 29, 30, 36, 42, 43, 53, 54, 57, 58, 59, 68, 75, 76, 79, 82, 133 larynx, 93, 110 laser ablation, 316 lasers, 321 latency, 170 laws, 21, 25 lead, 2, 5, 8, 9, 10, 18, 25, 168, 203, 212, 214, 227, 254, 291, 292 learning, 29, 40, 42, 286 learning difficulties, 286 lesions, vii, viii, xi, 1, 24, 26, 29, 30, 31, 33, 36, 43, 49, 50, 55, 58, 60, 75, 79, 89, 90, 94, 117, 131, 144, 170, 171, 194, 195, 279, 282, 283, 290, 294, 305, 307, 309 leukemia, x, 105, 131, 167, 178, 183, 188, 189, 249, 251 life expectancy, 42 life span, 40, 42 lifetime, 42 ligands, 152, 153, 160, 278 likelihood, 174, 181 limbic system, 291, 299 limitation, 30, 66, 108 linear model, 260 links, 14, 108, 127, 233, 286 lipids, 27, 46 lipoma, 298 liquid chromatography, 129, 237 lithium, 288 liver, 39, 215, 236, 244, 304, 316, 319, 321 liver cancer, 215, 244 lobectomy, 314 local anesthesia, 35, 52 localization, ix, 66, 111, 119, 142, 159, 163, 198, 209, 211, 227, 238, 244, 273, 276, 306, 309, 312 location, xii, 9, 14, 33, 35, 73, 77, 86, 87, 108, 109, 111, 121, 124, 134, 207, 213, 281, 283, 285, 290, 291, 292, 293, 294, 295, 296, 297, 309 locus, 10, 126, 199, 208, 210, 232, 238, 240, 241 loss of appetite, 288 Louisiana, 253 lumbar puncture, 176, 188 lung cancer, 213, 234, 241, 248, 250, 311 lung disease, 93 lying, 7 lymph, 115 lymphatic system, 292 lymphocytes, 169

333

lymphoid, 134, 168 lymphoid organs, 134 lymphoma, x, 105, 167, 168, 169, 170, 171, 172, 173, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 220, 224, 247, 249, 251, 289, 294, 295, 300 lysine, 197, 198, 230, 231 lysis, 135, 140, 171, 174

M machinery, 195, 197, 199, 225, 227 Mackintosh, 190 macrophages, 112, 113, 132, 134, 147 magnesium, 250 magnetic resonance, 46, 49, 50, 51, 52, 55, 57, 60, 66, 69, 70, 71, 73, 74, 76, 82, 83, 122, 136, 170, 171 magnetic resonance imaging, 46, 49, 55, 69, 74, 83, 122, 136, 170 magnetic resonance imaging (MRI), ii magnetic resonance spectroscopy, 50, 51, 57, 60, 69, 70, 71, 73, 76, 82 major histocompatibility complex, 142 Malaysia, 300 males, 3, 221, 288, 312, 314 malignancy, ix, x, 4, 7, 10, 11, 22, 23, 32, 49, 50, 75, 84, 85, 90, 92, 101, 106, 112, 113, 114, 119, 120, 121, 129, 130, 144, 164, 167, 202, 214, 215 malignant growth, 10 malignant melanoma, 115, 276, 316, 318 malignant tumors, 194, 213, 242, 291 management, vii, x, xi, 1, 27, 29, 31, 46, 51, 53, 56, 62, 63, 65, 67, 68, 74, 76, 78, 79, 82, 84, 86, 94, 107, 136, 160, 167, 168, 186, 187, 193, 194, 229, 254, 287, 289, 292, 296 mania, 283, 285, 288, 290, 293, 297, 298 manic, 284, 285, 288, 293, 294, 295 manic episode, 284, 288 manic symptoms, 293, 295 manipulation, xi, 194 mapping, viii, 2, 3, 29, 35, 36, 47, 48, 53, 54, 55, 58, 63, 66, 68, 76, 79, 81, 85, 86, 115, 234, 301 marriage, 289 marrow, 115, 170 masking, 299, 300 mass, 8, 13, 24, 32, 44, 69, 94, 95, 96, 107, 108, 126, 129, 130, 131, 144, 168, 171, 237, 282, 283, 284, 285, 286, 287, 288, 292, 296, 317 , 107, 108, 129, 130, 144, 237

334

Index

matrix, 10, 13, 15, 16, 17, 18, 47, 48, 55, 58, 71, 73, 74, 82, 83, 84, 116, 122, 130, 133, 137, 142, 153, 163, 216, 255, 266, 278, 279, 304, 318 matrix metalloproteinase, 14, 55, 71, 74, 84, 163, 216, 266, 278 maturation, 13, 132, 135, 146, 213, 279 measurement, 20, 24, 26, 64, 76, 86, 115, 206 measures, 17, 23, 42, 175, 179 median, vii, 1, 2, 3, 4, 6, 22, 34, 38, 105, 135, 170, 171, 172, 173, 177, 181, 182 medication, 288, 293 medulla, 289 medulla oblongata, 289 , vi, x, xi, 46, 47, 193, 194, 204, 211, 213, 214, 215, 216, 224, 229, 234, 239, 240, 241, 243, 247, 253, 254, 255, 256, 259, 260, 261, 262, 263, 265, 266, 271, 272, 273, 274, 275, 277, 278, 279, 280 MEG, viii, 2, 3, 43 melanoma, 56, 116, 134, 140, 143, 157, 165, 256, 273, 276, 277, 278, 279 melting, 208, 210, 235, 238 melting temperature, 208, 210 membranes, xii, 258, 304 memory, 30, 36, 58, 200, 230, 284, 286, 287, 289, 294, 297, 301 men, 285, 287 meninges, 229 meningioma, 312, 313, 318, 319, 320 meningitis, 170 mental retardation, 93, 287 mental state, 48, 180 messenger ribonucleic acid, 158 messenger RNA, 51, 165 metabolism, 18, 27, 48, 64, 83, 129, 147 metabolites, 70 metabolizing, 19, 76, 107, 108, 147 metalloproteinase, 216, 245 metaphase, 108 metastasis, x, xi, 146, 193, 195, 202, 217, 224, 245, 254, 255, 256, 271, 272, 274, 275, 279, 280, 310, 311 metastatic disease, 254, 272 methodology, 256 methyl groups, 200, 207 methylation, xi, 11, 19, 124, 134, 163, 194, 195, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 216, 217, 223, 224, 225, 227, 228, 229, 230, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248

methylphenidate, 174 MHC, 133, 134, 140 mice, 17, 18, 46, 51, 53, 60, 65, 67, 81, 130, 135, 141, 148, 150, 161, 213, 215, 242 microarray technology, 100 microinjection, 274 microscope, 96, 97, 98, 100, 260, 308, 317 microwaves, 110 midbrain, 287 migration, 4, 10, 13, 14, 15, 16, 21, 22, 30, 32, 57, 59, 68, 101, 104, 132, 133, 137, 138, 141, 146, 147, 150, 152, 153, 158, 163, 212, 215, 244 minority, 182 mitogen, 152, 161, 164, 231 mitosis, 4, 163, 212, 240 mitotic index, 9, 77, 150 MMP, 216, 272 MMPs, 55, 216, 266, 272 mobility, 18, 146 mode, 32, 44, 125, 285 model system, 58, 79 modeling, 18, 24, 26, 46, 79, 81, 131, 164 models, 9, 17, 18, 21, 22, 25, 29, 57, 58, 60, 61, 64, 65, 66, 69, 70, 79, 105, 122, 130, 137, 139, 147, 148, 150, 210, 273, 279, 290 molecular beam, 21 molecular beam epitaxy, 21 molecular biology, vii, 1, 3, 31, 55, 62, 76, 104, 108, 123, 161 molecular weight, 257, 264 molecules, 10, 100, 133, 152, 154, 155, 157, 196, 197, 209, 215, 255 monitoring, 19, 25, 42, 44, 47, 75, 80, 84, 105, 116, 117 monoclonal antibody, 9, 63, 259, 264, 267, 274 monomers, 199 mood, 282, 283, 285, 286, 287, 288, 293 mood disorder, 288 morbidity, x, xi, 23, 63, 95, 96, 167, 171, 173, 183, 185, 253, 254 morphology, 18, 21, 122, 133, 138, 277 mortality, x, 23, 167, 182, 185, 254 motivation, 290, 301 motor control, 81 motor stimulation, 84 movement, 21, 83, 133, 273 MRI, 7, 8, 16, 20, 22, 24, 27, 28, 33, 34, 35, 36, 42, 43, 56, 64, 65, 68, 76, 80, 86, 94, 170, 283, 285, 286, 287, 288, 299, 308, 311, 312, 313, 315

Index mRNA, x, 16, 100, 104, 105, 106, 114, 115, 116, 126, 149, 155, 161, 162, 193, 211, 214, 224, 225, 240, 241, 255, 256, 260, 262, 263, 264, 265, 266, 272, 273, 274 MRS, 27, 66, 81, 83 mucosa, 159, 276 multidimensional, 138 multiple regression, 176 multiplication, 54 multipotent, 121 mutagen, 131 mutagenesis, 153, 154, 164 mutant, 19, 62, 72, 79 mutation, x, 11, 12, 61, 124, 126, 131, 160, 194, 203, 204, 216, 232, 233, 234, 238, 239, 242, 247 mutation rate, 203, 232 myelin, 57, 120, 215, 244 myofibroblasts, 154

335

neurosurgeon, 30 neurotoxicity, 174, 187 neurotransmitter, 142, 282 neurotransmitters, 120 neurotrophic factors, xi, 253, 255 next generation, 180 nightmares, 285 nitrogen, 97 NMR, 59, 60, 66, 81 non-neoplastic diseases, 93 normal development, 254 North America, 3 nuclear magnetic resonance, 83 nuclei, 109, 112, 113, 259, 267 nucleic acid, 97, 104, 117 nucleosome, 195, 197, 198, 202, 230, 232 nucleus, 195, 258 nutrients, 21 nystagmus, 289

N NAD, 199 nasopharyngeal carcinoma, 161 National Institutes of Health, 274 natural evolution, 5 nausea, 69, 94, 285, 295 necrosis, 27, 49, 58, 95, 96, 99, 112, 113, 122, 130, 292 needs, 3, 10, 21, 30, 135, 229 neglect, 284 neoangiogenesis, 7, 8, 10, 13 neocortex, 282, 290, 298 neoplasm, 73, 121, 132, 171, 288, 294 neoplastic tissue, ix, 56, 92, 119, 132 neovascularization, 13, 77, 87, 126, 146, 157, 163, 216, 255 nerve, xi, 120, 175, 253, 259, 278, 279, 320 nerve fibers, 120 nerve growth factor, xi, 253, 259, 278, 279 nervous system, x, 29, 58, 59, 64, 73, 79, 106, 115, 120, 134, 137, 140, 152, 167, 180, 181, 183, 190, 191, 195, 215, 239, 291 network, 29, 30, 50, 312 neural network, 140, 282 neural networks, 282 neuroblastoma, 105, 115, 212, 273, 277, 279 neurofibroma, 121 neuroimaging, 3, 26, 36, 92, 94, 168, 171 neurological disease, 301 neurons, 27, 82, 120, 121, 147, 163, 215, 244, 282

O obesity, 288 objective criteria, 39 observations, 22, 25, 26, 29, 72, 178, 183, 204, 272 obstruction, 292 oculomotor, 290, 314, 316, 318 oculomotor nerve, 314, 318 oedema, 150, 306, 316 oligodendroglioma, 8, 49, 65, 69, 72, 78, 83, 112, 113, 211, 217, 224 oligonucleotide arrays, 127 omission, 178 oncogenes, 10, 59, 123, 131, 202 oocyte, 274 operator, 97 optic nerve, 121, 162, 171 optimization, 31 organ, 40, 42 organelles, 244 organism, 100, 120, 195, 196 organization, viii, 2, 3, 29, 36, 94, 215, 230, 290, 298, 300, 301, 309 outline, viii, 90, 114, 210 ovarian cancer, 217 overproduction, 151 oxygen, 25

Index

336

P p53, 11, 12, 14, 15, 18, 19, 47, 51, 56, 57, 58, 60, 61, 63, 64, 66, 68, 70, 71, 72, 79, 82, 83, 85, 124, 126, 138, 139, 142, 143, 211, 234, 238, 239, 241, 242, 243 paclitaxel, 248 pairing, 232 palliative, x, 193 pancreatic cancer, 163 panic attack, 286, 290, 299 parameter, 17, 54, 182 paraphilia, 297 parasite, 251 parenchyma, xii, 6, 13, 21, 22, 34, 96, 98, 134, 287, 304, 307, 310, 314, 315, 316, 317, 318, 319 paresis, 133 parietal lobe, 284, 296, 310 particles, 111 passive, 6, 287 password, 94 pathogenesis, viii, 46, 60, 74, 83, 90, 139, 169, 195, 212, 234, 254 pathologist, 77 pathology, 6, 29, 45, 55, 60, 96, 106, 115, 121, 137, 161, 287 pathophysiology, 26, 58 pathways, viii, xi, 2, 5, 10, 11, 14, 18, 19, 36, 43, 54, 55, 58, 60, 63, 74, 80, 83, 84, 86, 89, 90, 101, 104, 122, 123, 127, 129, 130, 140, 141, 143, 152, 153, 154, 158, 195, 213, 238, 254, 292 pattern recognition, 129 PCR, viii, 12, 68, 90, 104, 105, 106, 107, 108, 112, 113, 114, 115, 116, 117, 154, 206, 207, 208, 209, 210, 229, 235, 236, 237, 257, 261, 262, 263, 264, 274, 278 pedal, 305, 317 pelvis, 170 peptidase, 161 peptides, 129, 130, 134, 135, 146, 147, 148, 149, 150, 151, 152, 154, 156 perforation, 312, 313, 316, 318 perfusion, 28, 43, 64, 86, 146 perinatal, 4 peripheral blood, 114, 115, 173, 174, 250 peripheral nervous system, 120, 121, 142 permeability, 74, 76, 146, 150, 152, 292 permit, 105 personal hygiene, 284 personality, 170, 283, 287, 288, 289, 290

perspective, 10, 44 PET, vii, viii, 1, 2, 3, 16, 26, 28, 31, 43, 49, 50, 52, 53, 59, 60, 61, 63, 70, 76, 78, 83 pH, 25, 57, 97, 152, 258 pharmacokinetics, 251 pharmacological treatment, 285 pharmacology, 85, 158 pharynx, 93 phenol, 100 phenotype, 4, 12, 13, 14, 16, 17, 18, 40, 42, 45, 65, 68, 74, 127, 163, 177, 195, 242, 255 phenylalanine, 260 phobia, 286, 295, 299 phosphorylation, 149, 161, 195, 197, 211, 217, 231, 271, 272, 273, 274 physiology, 10, 18, 21, 25, 29, 161, 301 pilot study, 27, 48, 187 pituitary tumors, 286 placebo, 285 placenta, 158 planning, 30, 35, 79, 171, 187 plasma, 229, 246 plasma membrane, 246 plasminogen, 14, 82 plasticity, 25, 29, 30, 36, 37, 40, 43, 44, 46, 49, 53, 65, 71, 76, 78, 301 platelet count, 176 plexus, 288, 295, 299 ploidy, 21 PM, 141, 144, 161, 191, 229, 241, 251, 275, 278 point mutation, 123, 126, 131, 207 polyacrylamide, 129 polymerase, xi, 9, 83, 104, 106, 114, 115, 116, 117, 170, 231, 232, 235, 237, 238, 254, 257 polymerase chain reaction, xi, 83, 104, 106, 114, 115, 116, 117, 170, 235, 237, 238, 254, 257 polymerase chain reactions, 104 polymorphism, 92, 107, 149, 162, 237, 238, 247 , 217, 225 pons, 287, 289, 296, 297 poor, vii, 5, 27, 102, 122, 136, 143, 146, 152, 170, 174, 176, 179, 182, 189, 195, 213, 215, 217, 228, 254, 256, 272, 275, 283, 284, 285, 288, 291, 292, 293, 294, 295, 297, 298, 309 population, 3, 6, 7, 13, 18, 20, 25, 59, 62, 98, 99, 110, 125, 127, 136, 140, 141, 146, 168 positron, 26, 47, 48, 52, 53, 58, 59, 64, 67, 69, 73, 77, 78, 82, 84 positron emission tomography, 47, 52, 59, 64, 67, 77, 82

Index potassium, 120, 143 power, 246 precipitation, 44, 100 precursor cells, 17, 123, 127 prediction, viii, 18, 28, 61, 80, 89, 90, 139, 235, 254 predictors, 49, 79, 92, 131 preference, 162 preparation, 177 pressure, ix, 22, 96, 111, 145, 146, 147, 152, 162, 170, 284, 291, 292, 304, 305, 308, 311, 313, 315, 316, 319 prevention, x, 167, 212, 217, 225 primary brain tumor, vii, viii, 2, 48, 69, 76, 80, 89, 90, 127, 136, 168, 186, 212, 242 primary tumor, 82, 204, 214, 216, 245, 256, 284, 292 principle, 100, 108, 259, 304 prior knowledge, 206 probability, 25, 43, 175, 180, 184 probe, 105, 108, 208, 258 production, x, 10, 25, 58, 145, 149, 150, 152, 153, 156, 160, 163, 256, 277, 282 prognosis, vii, viii, ix, x, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 25, 26, 28, 31, 32, 40, 42, 45, 51, 55, 65, 68, 72, 77, 79, 90, 92, 120, 122, 125, 136, 137, 138, 140, 142, 143, 174, 176, 179, 182, 193, 194, 213, 215, 272, 275, 276, 317 program, 100, 134, 182 proliferation, ix, 3, 4, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 25, 26, 31, 37, 48, 50, 51, 57, 58, 64, 65, 66, 70, 71, 75, 78, 83, 85, 101, 106, 112, 113, 119, 121, 122, 123, 126, 127, 129, 130, 132, 137, 146, 147, 149, 150, 152, 153, 154, 155, 156, 164, 195, 210, 211, 213, 215, 254, 255, 256, 260, 261, 266 promoter, 125, 138, 163, 165, 199, 201, 202, 203, 206, 210, 212, 214, 215, 216, 223, 224, 229, 234, 238, 239, 240, 241, 242, 243, 244, 245 prophylactic, 177, 180, 183, 185 prophylaxis, 175, 178, 179, 182, 185, 190, 191 proportionality, 20 prostate, 105, 107, 115, 140, 157, 160, 162, 163, 165, 237, 246, 249, 279 prostate cancer, 105, 165, 237, 249, 279 prostate carcinoma, 115 protease inhibitors, 258 protein family, 216, 217, 246 protein kinase C, 14, 161 protein kinases, 15, 277 proteinase, 100, 259

337

proteins, 14, 16, 18, 49, 62, 90, 101, 104, 124, 126, 129, 130, 133, 144, 149, 152, 153, 195, 196, 197, 198, 199, 202, 203, 210, 216, 227, 239, 240, 244, 264, 279 proteoglycans, 255 proteolysis, 13, 84, 86 proteomics, 28, 129, 130 protocol, 12, 44, 96, 100, 115, 178, 187, 188, 189, 235, 259, 260 proto-oncogene, 10, 12, 14, 57, 109 pseudopodia, 133 psychiatric disorders, 283, 289 psychiatric hospitals, 284 psychiatric illness, 283, 288 psychiatrist, 285, 286, 289 psychopathology, 282 psychoses, 93 psychosis, xii, 281, 286, 288, 295, 298, 299, 300 psychotherapy, 286 psychotic symptoms, 283, 284, 286, 296, 297 PTSD, 289 pulse, 189 pure line, 22 purification, 233, 255

Q quality control, 94 quality of life, viii, x, 2, 3, 30, 31, 34, 35, 39, 42, 44, 71, 74, 81, 193, 249, 254, 274 quantitative estimation, 208

R race, 176 radiation, 18, 26, 28, 47, 48, 49, 56, 59, 63, 64, 67, 71, 74, 76, 78, 79, 81, 82, 83, 96, 135, 136, 187, 195, 274, 285, 289, 294 radiation damage, 28 radiation therapy, 26, 47, 59, 63, 64, 67, 78, 79, 96, 135, 187, 195, 285, 289 radio, ix, 25, 32, 35, 43, 44, 119, 136 radiography, 283 radiotherapy, viii, 2, 25, 27, 28, 31, 33, 34, 35, 37, 38, 39, 40, 42, 43, 44, 45, 46, 48, 55, 59, 60, 63, 64, 65, 66, 67, 73, 74, 76, 78, 81, 83, 84, 135, 171, 172, 173, 174, 179, 186, 187, 189, 284, 286, 288, 293, 317 range, 27, 96, 100, 109, 121, 179, 199, 203, 225, 227, 305, 309, 310, 312, 314 reading, 36, 76

338

Index

reagents, 258 real time, 105, 107, 108, 115, 208 reality, 17, 81 , vi, xi, 10, 13, 15, 124, 132, 141, 142, 146, 147, 150, 151, 152, 153, 154, 155, 156, 158, 159, 160, 161, 163, 164, 165, 211, 244, 253, 254, 255, 264, 271, 274, 277, 278, 279 recognition, 51, 76, 110, 128, 135, 140, 229, 230 reconstruction, 86 recovery, 29, 30, 36, 43, 54, 59, 61, 65, 71, 82, 235, 236 recurrence, xii, 6, 7, 26, 28, 31, 33, 38, 42, 44, 45, 47, 49, 58, 60, 61, 114, 174, 180, 181, 182, 183, 191, 229, 278, 281, 292, 293, 298 red blood cells, 176 redistribution, 30 reduction, xii, 25, 27, 33, 34, 44, 171, 188, 199, 213, 216, 223, 257, 261, 304, 316, 317, 318, 319 redundancy, 5 refining, ix, 26, 40, 42, 90, 91, 114 reflexes, 284, 286 reflux esophagitis, 93 regeneration, 120, 162 Registry, 81 regression, viii, 2, 13, 171, 279 regrowth, 20 regulation, 12, 19, 47, 63, 76, 123, 126, 135, 147, 154, 159, 164, 195, 196, 198, 199, 202, 204, 212, 213, 214, 217, 228, 230, 231, 243, 244, 245, 248, 256, 271, 272, 274, 277, 280, 282, 291 regulators, 16, 120, 146, 197, 215 relapses, 174, 175, 179 relationship, 26, 47, 61, 64, 72, 75, 77, 142, 214, 224, 274, 275, 283, 291 relationships, 51, 121, 242 relevance, xi, 20, 80, 129, 139, 140, 194, 254, 273 reliability, 26, 28, 43 remission, 22, 168, 173, 175, 181, 186, 284, 285 remodelling, 29 renin, ix, 145, 146, 147, 148, 149, 151, 157, 159, 162, 163, 165 repair, xi, 81, 120, 125, 194, 197 reparation, 10, 11, 12 replacement, 72, 197, 286 replication, 131, 200, 213 repression, 197, 198, 199, 200, 201, 203, 213, 217, 228, 231, 233, 240 repressor, 199, 213, 230, 233 resection, x, 6, 23, 24, 27, 30, 32, 33, 36, 43, 47, 52, 53, 55, 63, 65, 67, 70, 72, 75, 85, 86, 96, 133,

171, 193, 194, 285, 287, 289, 305, 306, 308, 309, 317, 318, 319, 320, 321 residual disease, 63, 105, 116, 135 residues, 11, 197, 198, 199, 209, 213, 235 resistance, 14, 18, 19, 46, 48, 57, 68, 71, 74, 80, 81, 84, 107, 135, 152, 156, 157, 214, 242, 246, 318 resolution, xii, 25, 28, 50, 109, 116, 171, 281, 282, 293, 294, 295, 298 resources, 21, 90 respiratory, 286 responsiveness, 19 , 107, 108, 204, 208, 209, 237 , 107, 108 restructuring, 196 reticulum, 168 retina, 171 retinoblastoma, 124, 210 retrieval, 98, 110 retroviruses, 202 reverse transcriptase, 114, 115, 116, 257 ribose, 199, 231, 232 right ventricle, 295 risk, viii, x, 2, 3, 6, 7, 20, 23, 26, 31, 32, 33, 34, 35, 36, 37, 39, 40, 42, 43, 44, 46, 61, 65, 92, 108, 132, 136, 157, 161, 167, 173, 174, 175, 176, 177, 180, 181, 182, 183, 184, 185, 188, 190, 191, 193, 203, 225, 227, 229, 254, 255, 256, 277, 316, 318, 319 risk factors, 3, 35, 176, 177, 181, 182, 183, 185, 190, 191 RNA, ix, 96, 97, 99, 100, 104, 105, 116, 120, 135, 162, 195, 199, 225, 227, 232, 255, 257, 263 RNA splicing, 162 RNAi, 199 robustness, 17 rodents, 17, 135, 147

S safety, 16, 34, 37, 127, 178 sample, 95, 96, 98, 100, 101, 104, 106, 130, 175, 176, 224, 258, 259, 266, 267, 268 sampling, 8, 31, 59 satellite, 109, 120, 202, 316 scaling, 138 scarcity, 5 schizophrenia, 285, 286, 295 school, 286, 288, 295, 299 Schwerin, 305 , 113, 260

Index search, 69, 92, 106, 129, 206, 299 secrete, 10, 14, 149 secretion, 82, 147, 150, 152, 159, 275 seeding, 194, 266 segregation, 11 selectivity, 59 selenium, 237 self, 10, 13, 46, 71, 203, 283, 284, 293, 294, 297 senescence, 16 sensitivity, 18, 27, 28, 44, 59, 82, 87, 105, 130 separation, xii, 68, 108, 204, 206, 234, 304, 307, 309, 310, 314, 315, 317, 318, 319 sequencing, 136, 209, 235, 236 series, 5, 6, 7, 8, 9, 20, 22, 23, 24, 26, 32, 38, 42, 54, 58, 66, 68, 69, 106, 112, 113, 132, 155, 168, 171, 173, 181, 182, 203, 229, 284, 287, 289, 305 serum, 116, 149, 177, 214, 243, 256, 257, 259, 260 serum albumin, 257 sex chromosome, 124 sexual behavior, 289 shape, 22, 133 sharing, 15 shock, 199 short term memory, 284, 285, 289 short-term memory, 294, 295 side effects, 35, 37, 225, 227 signaling pathways, 16, 70, 76, 90, 152, 153, 254, 274 signalling, 153, 275, 277 signalling pathways, 153, 277 signals, 10, 120, 126, 158, 195, 255, 273, 274, 278 silver, 111 simulation, 87 Singapore, 298 sinus, 184 siRNA, 230 sites, 4, 5, 16, 22, 36, 55, 107, 108, 138, 169, 174, 181, 182, 183, 197, 202, 203, 204, 206, 207, 208, 209, 211, 223, 224, 234, 238 skin, 105, 182, 203, 279, 321 slit lamp, 170 smokers, 203 smoking, 94, 234, 241 smooth muscle, 85, 153, 158, 163 smooth muscle cells, 85, 153, 158, 163 social withdrawal, 283, 293, 297 sodium, 228, 246, 249, 250, 258 software, 258, 260 solid state, 22

339

solid tumors, 18, 45, 78, 115, 202, 212, 224, 228, 248, 249 somatic cell, 83, 131 somatization, 289 somatosensory function, 35 Southern blot, 104, 223 species, 188 specificity, 105, 110, 130, 161 spectroscopy, 27, 60, 74, 78, 83, 86 spectrum, 28, 127, 178, 187, 234, 242 speech, 36, 75, 284, 288, 289, 295 spinal cord, 4, 53, 180 spinal symptoms, 170 spindle, 212, 241 spine, x, 193 squamous cell, 110, 165, 235, 248, 283 squamous cell carcinoma, 110, 165, 235, 248, 283 stability, 17, 197, 200, 240, 262, 265 stabilization, viii, 2, 39, 96, 200, 212, 217 stages, 10, 17, 22, 28, 91, 121 standardization, 110 standards, 6, 100, 105 stellate, 121 sterile, 97, 305 steroids, 171, 286 stimulus, 7, 26, 290 strategies, vii, viii, x, 1, 2, 3, 31, 35, 40, 42, 47, 56, 90, 91, 107, 114, 131, 133, 135, 167, 195, 217, 225, 228, 229, 230, 248 stratification, 37, 42, 130, 277 stress, 19, 21, 22, 101, 153, 231 striatum, 36, 57, 290, 301 stroke, 29, 71, 76 stroma, 132, 154, 276 stromal cells, 14, 110, 153, 157 subjectivity, 9 substitution, 29, 197 substrates, 10, 14, 26, 156 subtraction, 51, 54 suicidal ideation, 287 suicide, 75 Sun, 80, 231, 241, 245 supervision, 174 supply, 132, 282 suppression, 18, 70, 164, 216, 217, 237, 238, 276 surgical intervention, ix, 36, 57, 119, 294, 295 surgical resection, 33, 36, 54, 56, 133, 136, 284, 287, 288, 293, 295 surveillance, 56

Index

340

survival, vii, viii, ix, x, xi, 1, 2, 3, 6, 7, 12, 17, 19, 23, 24, 26, 27, 31, 33, 34, 35, 40, 45, 46, 48, 49, 55, 56, 60, 61, 62, 63, 66, 67, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 84, 85, 86, 89, 90, 120, 125, 126, 127, 129, 135, 136, 137, 138, 139, 141, 145, 147, 151, 153, 156, 171, 172, 173, 174, 176, 180, 182, 183, 187, 189, 193, 214, 217, 228, 239, 253, 255, 256, 272, 273, 277, 279, 300 survival rate, xi, 7, 34, 72, 136, 173, 253 survivors, 274 susceptibility, 56, 74, 107, 130, 168 Switzerland, 136, 145, 274 symptom, 3, 7, 283, 287, 288, 289, 292, 293, 300 symptoms, vii, xii, 1, 94, 170, 171, 181, 182, 195, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 synaptic plasticity, 61 syndrome, 36, 54, 76, 174, 185, 186, 187, 228, 242, 249, 287, 299, 300 synthesis, 10, 49, 150, 159, 161, 164, 165 systems, 28, 69, 111, 131, 132, 152, 254, 261, 301

T T cell, 15, 135, 138 T lymphocytes, 14, 135, 140, 143, 169 targets, viii, 80, 90, 92, 100, 106, 107, 121, 131, 135, 198, 225, 227, 234, 241 taxonomy, 129 technetium, 86 technology, 104, 107, 109, 122 temperature, 208, 210 temporal lobe, 284, 286, 290, 294, 298, 299, 310, 314, 315, 318 temporal lobe epilepsy, 314, 315 TGA, 257 TGF, 13, 14, 15, 85, 150, 161, 163, 239 thalamus, 284 thallium, 72, 80 theory, 10, 127, 169 therapeutic agents, 157, 229, 250 therapeutic approaches, 92, 133, 178, 217, 225, 277 therapeutic interventions, 274 therapeutic targets, 107, 123, 130, 131 therapeutics, 132 therapy, x, xi, 6, 8, 27, 39, 48, 49, 53, 56, 57, 66, 68, 70, 71, 77, 78, 81, 82, 83, 84, 85, 90, 95, 96, 104, 125, 127, 136, 141, 142, 164, 165, 167, 168, 173, 174, 175, 177, 178, 179, 180, 181, 183, 184, 185,

186, 187, 189, 194, 195, 214, 217, 225, 227, 228, 229, 230, 248, 249, 253, 274, 285, 286, 293 thinking, 283, 284, 285, 292, 295 three-dimensional model, 158 threshold, 7, 135, 140 thymine, 209, 213 time, viii, xi, 2, 3, 4, 5, 6, 9, 10, 19, 20, 22, 23, 24, 27, 28, 30, 32, 35, 36, 37, 38, 40, 44, 48, 49, 54, 70, 75, 77, 80, 83, 90, 92, 94, 98, 101, 105, 106, 107, 110, 114, 115, 116, 117, 124, 127, 130, 133, 173, 176, 181, 183, 185, 197, 203, 208, 210, 214, 236, 238, 242, 254, 256, 260, 262, 263, 272, 278, 283, 284, 285, 286, 292, 294, 306, 309, 316, 321 timing, 31, 34, 38, 42, 67, 85, 143, 203 TIMP, 216, 243, 245 tissue, viii, ix, xii, 10, 15, 16, 19, 24, 28, 66, 76, 87, 90, 91, 95, 96, 97, 98, 99, 100, 101, 106, 108, 109, 110, 111, 112, 113, 116, 122, 125, 128, 131, 133, 134, 140, 142, 143, 145, 146, 147, 160, 162, 165, 171, 194, 200, 206, 211, 214, 215, 216, 217, 245, 259, 282, 291, 292, 303, 304, 307, 308, 309, 312, 316, 317, 318, 320, 321 tissue perfusion, 28 TNF, 50, 211 tobacco, 203 tobacco smoke, 203 toxicity, 34, 35, 39, 43, 81, 107, 135, 157, 171, 174, 183, 185, 195, 226, 274 traffic, 139, 140, 169 training, 53 traits, 195, 289 transcription, xi, 16, 83, 104, 106, 114, 115, 116, 117, 123, 135, 153, 194, 196, 197, 198, 200, 201, 202, 203, 213, 217, 225, 227, 230, 231, 233, 241, 271, 272, 278 transcription factors, 16, 106, 197, 227 transduction, 15, 16, 123, 129, 132, 152, 153, 158, 255, 273 transfection, 135 transformation, vii, viii, 1, 2, 5, 6, 7, 10, 11, 13, 16, 17, 23, 24, 25, 26, 27, 28, 30, 32, 33, 35, 42, 44, 46, 57, 89, 90, 101, 122, 127, 138, 169, 184, 202, 211, 212, 215, 225, 227, 245 transforming growth factor, 138, 164 transfusion, 176 transgene, 62 transition, 11, 61, 124, 243 transition mutation, 243 transitions, 203 translation, 16

Index translocation, 258 transmission, 120 transplantation, 131, 186 transversion mutation, 203, 234 trauma, 63, 173, 282, 304, 316 tremor, 286 trend, 24, 126, 178 trial, 6, 34, 37, 38, 39, 48, 49, 55, 63, 64, 74, 78, 81, 83, 135, 139, 143, 175, 177, 179, 181, 182, 184, 186, 187, 189, 225, 226, 248, 249, 250 triggers, 197 tropism, 45 , vi, vii, viii, ix, x, xi, xii, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 37, 39, 40, 42, 44, 47, 48, 49, 50, 53, 56, 59, 60, 61, 62, 63, 64, 67, 68, 69, 71, 72, 73, 77, 79, 82, 84, 86, 87, 90, 92, 95, 96, 99, 101, 105, 108, 109, 112, 113, 114, 115, 119, 120, 121, 122, 123, 124, 125, 126, 127, 129, 130, 132, 133, 134, 135, 136, 138, 139, 140, 141, 143, 145, 146, 147, 148, 149, 150, 151, 153, 154, 156, 161, 163, 164, 165, 168, 169, 171, 174, 180, 189, 193, 194, 202, 203, 204, 206, 210, 211, 212, 213, 214, 215, 216, 217, 223, 224, 225, 227, 234, 235, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 250, 253, 255, 260, 267, 271, 272, 273, 274, 275, 276, 278, 281, 282, 283, 284, 285, 286, 288, 289, 290, 291, 292, 293, 294, 295, 296, 298, 299, 300, 301, 321 tumor cells, x, 13, 22, 27, 42, 96, 112, 113, 122, 123, 133, 134, 135, 140, 145, 147, 149, 150, 151, 153, 156, 163, 174, 212, 213, 214, 215, 216, 273, 274 tumor growth, vii, 21, 22, 48, 63, 79, 86, 126, 130, 132, 150, 154, 165, 216, 255, 272, 273 tumor invasion, 64, 164, 215, 274, 276 tumor metastasis, 48, 271, 272, 276 tumor necrosis factor, xi, 253 tumor progression, ix, xii, 3, 13, 17, 18, 27, 56, 60, 79, 101, 119, 120, 130, 146, 153, 211, 242, 254, 274, 276 tumors, vii, viii, ix, x, xi, xii, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 26, 27, 28, 31, 32, 33, 34, 35, 37, 38, 39, 40, 42, 43, 45, 46, 47, 48, 50, 51, 52, 53, 55, 56, 58, 59, 60, 61, 62, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 90, 92, 93, 94, 96, 101, 104, 106, 108, 113, 114, 119, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 142, 143, 144, 145, 146, 149, 151, 153, 156, 157, 169,

341

185, 193, 194, 195, 203, 204, 210, 211, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 228, 229, 236, 237, 238, 239, 240, 242, 243, 247, 250, 253, 254, 255, 273, 275, 277, 279, 281, 282, 283, 288, 289, 290, 291, 292, 293, 298, 299, 300 turnover, 27 tyrosine, xi, 27, 61, 66, 124, 125, 132, 140, 142, 152, 165, 246, 253, 255, 258, 259, 277, 279

U UK, 281 ultrasound, 319, 321 underlying mechanisms, viii, 90, 101, 129 United Kingdom, 167, 180 United States, vii, 81, 136, 168, 179, 305 universality, 21 unmasking, 29, 55, 62, 235 urinary dysfunction, 174 urokinase, 14

V vaccines, 135 Valencia, 257 validation, 55, 69, 98, 104, 105, 128 validity, 288 values, 28, 101, 102, 261, 272 variability, 10, 25, 77, 104 variable, 10, 13, 24, 32, 107, 124, 201, 309, 310 variables, 9, 69, 78 variation, 20, 101, 116, 127, 197, 206, 208, 209, 212 vascular endothelial growth factor (VEGF), 216 vasculature, x, 13, 21, 48, 82, 113, 114, 122, 145, 146, 147, 148, 150, 151, 154, 157, 161, 282, 292 vasoconstriction, 147 vector, 131 VEGF expression, 13, 149 velocity, 21, 22 ventricle, 121, 284, 285, 286, 287, 288, 289, 295, 296 vertebrates, 196, 200, 215 vertigo, 289 vessels, xii, 13, 132, 150, 289, 304, 308, 310, 314, 316, 317, 319 violence, 295 viruses, 199, 202 vision, vii, 3, 29, 170 visual acuity, 170 visual area, 301

Index

342 visual field, 97 visual images, 301 visual stimuli, 290 visualization, 97, 98 voice, 288 vomiting, 69 voting, 56

working hours, 288 workplace, 300 World Health Organization, ix, 65, 75, 85, 115, 119, 121, 140 wound infection, 316 writing, 36

X W Wales, 242 water, 98, 162, 304, 319, 320, 321 weakness, 284 web, 71, 98, 232 weight loss, 283, 288, 294 white blood cells, 169 white matter, 2, 4, 14, 22, 47, 79, 81, 94, 194 wild type, 11, 83 wood, 304 word recognition, 30, 58 work, 10, 98, 228, 229, 272, 274, 283, 286, 288, 294, 295

xenografts, 17, 64, 69, 77, 87, 131, 243, 279 X-irradiation, 136

Y yeast, 199, 231 yield, 67, 69, 130, 172, 173, 223 young adults, 2, 122

Z zinc, 149, 212, 213, 217, 225