MOLECULAR GENETIC PATHOLOGY
MOLECULAR GENETIC PATHOLOGY Edited by
LIANG CHENG, MD Professor of Pathology and Urology Director of Molecular Pathology Laboratory Chief of Genitourinary Pathology Division Department of Pathology and Laboratory Medicine and Clarian Pathology Laboratory Indiana University School of Medicine, Indianapolis, IN
DAVID
Y. ZHANG, MD, PhD, MPH
Associate Professor of Pathology Director, Molecular Pathology Laboratory Department of Pathology Mount Sinai School of Medicine and the Mount Sinai Medical Center New York, NY
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Humana Press
Preface
We have had the opportunity to witness both the beginning and the subsequent growth of an exciting specialty that combines both pathology and medical genetics, a field commonly referred to as molecular genetic pathology. The birth of this specialty took place in 1988 when Kari Mullis developed a new DNA amplification technology called the polymerase chain reaction (PCR). Within a few years, this technology was no longer being used exclusively in research laboratories. The technique had found numerous new applications in clinical medicine as a tool for diagnosis and diseases monitoring. The use of PCR technology has greatly expanded the specialties of anatomic and clinical pathology and has increased the availability of genetic testing in the clinical setting. We expect that such advances as the completion of the Human Genome Project, the maturation of pharmacogenomics, the growth of proteomics, and the rapidly growing field of molecular genetic pathology will lead to a new era of personalized and customized patient care. More recently, the American Board of Pathology (ABP), in conjunction with the American Board of Medical Genetics (ABMG), established a new subspecialty, molecular genetic pathology. Fellowship training for molecular genetic pathology is approved by the Accreditation Council for Graduation Medical Education (ACGME). Many pathologists and medical geneticists are applying for advanced training in this growing subspecialty. Training in molecular pathology is also becoming a required element in pathology residency curricula. To meet these demands, a team of more than 50 leading experts has compiled this quick reference book for medical students, general practitioners, medical technologists, pathologists, and medical geneticists . We also hope that residents or fellows who are training in pathology and medical genetics will find this book helpful in their preparation for board examinations . Molecular Genetic Pathology contains two parts. Part I covers general molecular genetic pathology and technology, including principles of clinical molecular biology, principles
of clinical cytogenetics, diagnostic methodology and technology, tissue microarrays and biomarker validation, laser capture microdissection, clinical flow cytometry, conceptual evolution in cancer biology, clinical genomics in oncology, clinical proteomics, clinical pharmacogenomics, clonality analysis in surgical pathology, fluorescence in situ hybridization (FISH), conventional cytogenetics for hematology and oncology diagnosis, instrumentation, genetic inheritance and population genetics, and genetic counseling . Part II provides disease-based information, including prenatal diagnosis, familial cancer syndromes, molecular testing for solid tumors, molecular pathology of the central nervous system, molecular virology, molecular bacteriology, mycology and parasitology, molecular testing for coagulopathies, molecular hemoglobinopathies, molecular diagnostics of lymphoid malignancies, molecular diagnostics of myeloid leukemias, HLA system and transfusion medicine (molecular approach), molecular forensic pathology, gene therapy, ethical and legal issues in molecular testing, and quality assurance and laboratory inspection. Each chapter begins with a detailed Table of Contents for easy reference. Assembling this diverse guidebook has truly been a team effort, cutting across many traditional specialty boundaries . We are most grateful for all the contributors who made this project possible. Our special thanks go to Mr. Ryan P. Christy from the Multimedia Education Division of the Department of Pathology at Indiana University, who has edited the illustrations for the book. We would like to thank the staff at Humana Press/Springer, including Ms. Mary Jo Casey, Mr. Paul Dolgert, Mr. Richard Hruska, and Mr. David Casey for their assistance in the development and editing of this text, and in particular Ms. Amy Thau, without whose outstanding work this book would have been an impossible achievement.
Liang Cheng, MD David Y. Zhang, MD, PhD, MPH
v
...
Contents
Preface Contributors
v ix
11
Part I General Sections 1 Principles of Clinical Molecular Biology Shaobo Zhang, Darrell D. Davidson, David Y. Zhang, Jodi A. Parks, and Liang Cheng 2 Principles of Clinical Cytogenetics Stuart Schwartz
1
33
3 Diagnostic Methodology and Technology Josephine Wu, Tao Feng, Ruliang Xu, Fei fe, Bruce E. Petersen, Liang Cheng, and David Y. Zhang 65 4 Tissue Microarrays and Biomarker Validation Martina Storz and Holger Moch
133
5 Laser Capture Microdissection Matthew Kuhar and Liang Cheng
141
6 Clinical Flow Cytometry Magdalena Czader
155
7 Conceptual Evolution in Cancer Biology Shaobo Zhang, Darrell D. Davidson, and Liang Cheng 185 8 Clinical Genomics in Oncology Hugo M. Horlings and Marc van de Vijver 9 Clinical Proteomics David H. Geho, Virginia Espina, Lance A. Liotta, Emanuel F. Petricoin, and Julia D. Wulfkuhle
10 Clinical Pharmacogenomics Catalina Lopez-Correa and LawrenceM. Gelbert
209
231
Clonality Analysis in Modem Oncology and Surgical Pathology Liang Cheng, Shaobo Zhang, Timothy D. Jones, and Deborah E. Blue
241
261
12 Fluorescence In Situ Hybridization (FISH) and Conventional Cytogenetics for Hematology and Oncology Diagnosis Vesna Najfeld
303
13 Instrumentation Bruce E. Petersen, Josephine Wu, Liang Cheng, and David Y. Zhang
365
14 Genetic Inheritance and Population Genetics Tatiana Foroudand Daniel L. Koller
393
15 Genetic Counseling Kimberly A. Quaid and Lisa J. Cushman
405
Part II Disease-Based Sections 16 Molecular Medical Genetics Lisa Edelmann, Stuart Scott, and Ruth Kornreich
415
17 Prenatal Diagnosis Nataline Kardon and Lisa Edelmann
441
18 Familial Cancer Syndromes Michelle P. Eliefj, Antonio Lopez-Beltran, Rodolfo Montironi, and Liang Cheng ..... 449
vii
Contents llIiJltEii
19 Molecular Testing for Solid Tumors Neal I. Lindeman and Paola Dal Cin
467
20 Molecular Pathology of the Central Nervous System Eyas M. Hattab and Brent T. Harris
497
21 Molecular Virology Josephine Wu, Mona Sharaan, and David Y. Zhang
533
22 Molecular Bacteriology, Mycology and Parasitology Mona Sharaan, Josephine Wu, Bruce E. Petersen, and David Y. Zhang
viii
689
28 Molecular Forensic Pathology P. Michael Conneally and Stephen R. Dlouhy
703
and Clinical Applications 717
581
623
30 Ethical and Legal Issues in Molecular Testing Kimberly A. Quaid
731
31 Quality Assurance and Laboratory Inspection Carol L. Johns and Liang Cheng
737
637
25 Molecular Diagnostics of Lymphoid Malignancies Francisco Vega and Dan M. Jones
rr
27 The HLA System and Transfusion Medicine: Molecular Approach S. Yoon Choo
Kenneth Cornetta
24 Molecular Hemoglobinopathies Jodi A. Parks, Tina Y. Fodrie, Shaobo Zhang, and Liang Cheng
21M I
675
29 Gene Therapy: Vector Technology
23 Molecular Testing for Coagulopathies Veshana Ramiah and Thomas L. Ortel
lU l U
26 Molecular Diagnostics of Myeloid Leukemias C. Cameron Yin and Dan M. Jones
Appendix
Liang Cheng and Shaobo Zhang 655
m1ttt lllTJrr_wn
Index
751 767
Contributors
DEBORAH E . BLUE, MD Assistant Professor of Pathology Associate Director, Molecular Pathology Laboratory Department of Pathology and Laboratory Medicine and Clarian Pathology Laboratory Indiana University School ofMedicine Indianapolis, IN LIANG CHENG, MD Professor of Pathology and Urology Director ofMolecular Pathology Laboratory Chief, Genitourinary Pathology Division Department of Pathology and Laboratory Medicine and Clarian Pathology Laboratory Indiana University School of Medicine Indianapolis, IN S.
Yo ON CHOO, MD Associate Professor of Pathology and Medicine Director of HLA Laboratory, Associate Medical Director of Blood Bank Departments of Pathology and Medicine Mount Sinai School of Medicine and the Mount Sinai Medical Center New York, NY
P. MICHAEL CONNEALLY, PhD Distinguished Professor Emeritus, Medical and Molecular Genetics and Neurology Department of Medical and Molecular Genetics Indiana University School of Medicine Indianapolis, IN KENNETH CORNETIA, MD Joe C. Christian Professor and Chairman Department ofMedical and Molecular Genetics Indiana University School ofMedicine Indianapolis, IN LISA 1. CUSHMAN, PhD Certified Genetic Counselor Department of Medical and Molecular Genetics Indiana University School of Medicine Indianapolis, IN
MAGDALENA CZADER, MD, PhD Assistant Professor of Pathology Director of Clinical Flow Cytometry Laboratory Department of Pathology and Laboratory Medicine Indiana University School of Medicine Indianapolis, IN PAOLA DAL ON, PhD Associate Professor of Pathology Cytogenetics Laboratory Department of Pathology Brigham and Women's Hospital and Harvard Medical School Boston, MA DARRELL D . DAVIDSON, MD, PhD Assistant Professor of Pathology Department of Pathology and Laboratory Medicine Indiana University School of Medicine Indianapolis, IN STEPHEN R. DLOUHY, PhD Associate Research Professor Department ofMedical and Molecular Genetics Indiana University School of Medicine Indianapolis, IN LISA EDELMANN, PhD Assistant Professor Department of Genetics and Genomic Sciences Director, Molecular Cytogenetics Co-Director, Genetic Testing Laboratory Mount Sinai School of Medicine and the Mount Sinai Medical Center New York, NY MICHELLE P. EUEFF, MD Staff Pathologist Diagnostic Pathology Services, Inc. Nampa, ID
JltW
ix
Contributors mUI
VIRGINIA ESPINA, MS Research Professor The Center for Applied Proteomics and Molecular Medicine George Mason University Manassas, VA TAO FENG, MS, MP (ASCP) Research Assistant Department of Pathology Mount Sinai School of Medicine and the Mount Sinai Medical Center New York, NY TINA Y. FODRIE, BS, MT, MP (ASCP) Supervisor, Department of Molecular Pathology Indiana University School of Medicine and VA Medical Center Indianapolis, IN TATIANA FOROUD, PhD Professor ofMedical and Molecular Genetics Department of Medical and Molecular Genetics Indiana University School of Medicine Indianapolis, IN DAVID H. GEHO, MD, PhD Associate Director of Imaging Merck and Company, Inc. West Point, PA LAWRENCE M. GELBERT, PhD Research Advisor Eli Lilly and Company Indianapolis, IN BRENT T. HARRIS, MD, PhD Assistant Professor of Pathology Department of Pathology Dartmouth Medical School Lebanon, NH EYAS M. HATIAB, MD Associate Professor of Pathology Department of Pathology and Laboratory Medicine and Clarian Pathology Laboratory Indiana University School of Medicine Indianapolis, IN
_ _ _ _ _.....
HUGO M. HORLINGS, MD Department of Pathology The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital Amsterdam, The Netherlands CAROL L. JOHNS, PhD Supervisor and Technical Coordinator Clarian Molecular Pathology Laboratory Indiana University School of Medicine Indianapolis, IN DAN M . JONES, MD, PhD Professor of Pathology Department of Hematopathology Medical Director, Molecular Diagnostics Laboratory The University of Texas M. D. Anderson Cancer Center Houston, TX TIMOTHY D . JONES, MD Staff Pathologist Department of Pathology Floyd Memorial Hospital and Health Services New Albany, IN NATALINE KARDON, MD Associate Professor Director of Prenatal Cytogeneti cs Laboratory Department of Genetics and Genomic Science s Mount Sinai School of Medicine and the Mount Sinai Medical Center New York, NY DANIEL L. KOLLER, PhD Research Assistant Professor Department of Medical and Molecular Genetics Indiana University School of Medicine Indianapolis, IN RUTH KORNREICH, PhD Research Assistant Professor ofHuman Genetics Co-Director Genetic Testing Laboratory Department of Human Genetics Mount Sinai School of Medicine and the Mount Sinai Medical Center New York, NY MATIHEW KUHAR, MD Resident Department of Pathology and Laboratory Medicine and Clarian Pathology Laboratory Indiana University School of Medicine Indianapolis, IN ... Jjl...
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Contributors NEAL I. LINDEMAN, MD Associate Pathologist, Clinical Chemistry Associate Pathologist, Molecular Diagnostics Brigham and Women 's Hospital Assistant Professor of Pathology Harvard Medical School Boston , MA
JODI A. PARKS, MD Visiting Lecturer, Clinical Chemistry Department of Pathology and Laboratory Medicine and Clarian Pathology Laboratory Indiana University School of Medicine Indianapolis, IN BRUCE E. PETERSEN, MD Molecular Genetic Pathology Fellow Department of Pathology Mount Sinai School of Medicine and the Mount Sinai Medical Center New York, NY
LANCE A. LIOTIA, MD, PhD Director, The Center for Applied Proteomics and Mole cular Medicine Professor of Life Scien ces George Mason University Manassas, VA
EMANUEL F. PETRICOIN, PhD Professor of Life Sciences Director, The Center for Applied Proteomics and Molecular Medicine Chair, Department of Molecular and Microbiolog y George Mason University Manassas, VA
ANTONIO LOPEZ-BELTRAN, MD, PhD Professor of Pathology Department of Pathology and Surgery Cordoba University Medical School Cordoba, Spain CATALINA LOPEZ-CORREA, MD, PhD Principal Research Scientist Eli Lilly and Company Indianapolis, IN
KIMBERLY A. QUAID, PhD Professor of Medical and Molecular Genetics Department of Medical and Molecular Genetics Indiana University School of Medicine Indianapolis, IN
HOLGER MOCH, MD Professor and Chairman Institute of Surgical Pathology Department of Pathology University Hospital Zurich Zurich, Switzerland
VESHANA RAMIAH, MD Hematology/Oncology Fellow Duke University Medical Center Durham, NC
RODOLFO MONTIRONI, MD, FRCPath Professor of Pathology Institute of Pathological Anatomy and Histopathology Polytechnic University of the Marche Region (Ancona) School of Medicine United Hospitals Ancona, Italy
STUART SCHWARTZ, PhD, FACMG Professor of Human Genetics Department of Human Genetics University of Chicago Chicago,IL
VESNA NAJFELD, PhD Professor of Pathology and Medicine Director, Tumor Cytogenetics, and Oncology, Molecular and Cellular Tumor Markers Mount Sinai School of Medicine and the Mount Sinai Medical Center New York, NY THOMAS L. ORTEL, MD, PhD Associate Professor of Medicine and Pathology Hemostasis and Thrombosis Center Duke University Medical Center Durham , NC
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STUART SCOTT, PhD Clinical Molecular Genetics Fellow Department of Human Genetics Mount Sinai School of Medicine and the Mount Sinai Medical Center New York, NY MONA SHARAAN, MD Molecular Genetic Pathology Fellow Department of Pathology Mount Sinai School of Medi cine and the Mount Sinai Medical Center New York, NY
AU
XI
Contributors MARTINA STORZ, BS
Directorof TMA Core Facility Institute ofSurgical Pathology Departmentof Pathology University Hospital Zurich Zurich, Switzerland
RULIANG XU, MD, PhD
Associate Professor of Pathology Departmentof Pathology New York University School of Medicine New York, NY FEI YE, PhD
MARC VAN DE VIJVER, MD, PhD
Professor of Pathology Department of Pathology The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital Amsterdam, The Netherlands
Assistant Professor Departmentof Pathology Mount Sinai School of Medicine and the Mount Sinai Medical Center New York, NY
C. CAMERON YIN, MD, PhD FRANCISCO VEGA, MD, PhD
Assistant Professor of Pathology Departmentof Hematopathology The University of Texas M. D. Anderson Cancer Center Houston, TX
Assistant Professor of Pathology Departmentof Hematopathology The University of Texas M. D. Anderson Cancer Center Houston, TX DAVID Y. ZHANG, MD, PhD, MPH
JOSEPHINE WU, DDS, CLSp(MB), CLDir
Assistant Professor Departmentof Pathology Mount Sinai School of Medicine and the Mount Sinai Medical Center New York, NY JULIA D. WULFKUHLE, PhD
Research Professor The Centerfor Applied Proteomics and Molecular Medicine George Mason University Manassas, VA
RID
xii
Associate Professorof Pathology Director, Molecular Pathology Laboratory Departmentof Pathology Mount Sinai School of Medicine and the Mount Sinai Medical Center New York, NY SHAOBO ZHANG, MD
Associate Research Professor Departmentof Pathology and LaboratoryMedicine Indiana University School ofMedicine Indianapolis, IN
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Part I General Sections
1
Principles of Clinical Molecular Biology Shaobo Zhang,
MD,
Darrell D. Davidson, MD, PhD, David Y. Zhang, Jodi A. Parks, MD, and Liang Cheng, MD
MD, PhD, MPH,
CONTENTS I. Deoxyribonucleic Acid (DNA) Overv iew Types of DNAs DNA Repli cation DNA Mutation DNA Mutation and Disease Factors Related to DNA Aberrations DNA Repair Mechanisms
II . Genes Overview Gene Components Functional Categories of Genes Cancer- Related Genes Regulation of Gene Expression Signal Tran sduction
III. Chromosomes Overview Chromatin
1-4 1-4 1-5 1-7 1-8 1-9 1-9 1-12
1-13 1-13 1-13 1-13 1-14 1-14 1-19
1-19 1-19 1-19
Chromosomes
IV. RNA and Proteins Overview Types of RNA Ribosome and Ribozyme mRNA Processing Protein Translation
V. Mitochondrial DNA Overview mtDNA Inheritance Characteristics of mtDNA Mitochondrial Genes and Gene Expression mtDNA Replication mtDNA Damage, Mutations, and Repair Mitochondrial Disease
VI. Suggested Reading
1-19
1-22 1-22 1-23 1-23 1-24 1-27
1-28 1-28 1-28 1-28 1-28 1-29 1-30 1-31
1-32
3
1-4
Molecular Genetic Pathology
DEOXYRIBONUCLEIC ACID (DNA)
Overview
- Nucleotide is made up of a phosphate group, a pentose sugar (deoxyribose), and a nitrogenous base (Figure 1)
• Definition - DNA is a large nucleic acid polymer arranged in chromosomes for storage, expression and transmission of genetic information - The genetic information is encoded by a sequence of nucleotides • Components of DNA - Bases are molecules containing carbon-nitrogen rings in DNA • Purines: adenine (A) and guanine (G) have two joined carbon-nitrogen rings • Pyrimidines: thymine (T) and cytosine (C) have one carbon-nitrogen ring - Nucleoside is made up of a five-carbon sugar (deoxyribose) and a base - Deoxyribose is the same sugar found in RNA, but with oxygen removed from the 2' carbon position
Adenine
Guanine
• The phosphodiester bond • The phosphate group is bond to the nucleoside at the hydroxyl group of the 5' carbon atom of deoxyribose • Phosphodiester bonds are strong covalent bonds between phosphate groups connecting the 5' carbon of one deoxyribose to the 3' carbon of the next deoxyribose of the adjacent nucleotide nucleotide (Figure 2) • The phosphodiester bond determines DNA chain polarity (ends designated as either 5' or 3') • DNA sequence refers to the order of the nucleotides in a DNA strand, which code for unique sets of genetic information, both proteins and regulatory segments
Cytosine
Thymine
N j'N=C ~) NH2
I
HOf>°""I ~H HO H
'\ N
HOf> 0",,1
~H HO
Cytidine
2-Deoxyribose
N
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Adenosine
I Nucleoside
Fig. 1. There are four bases in DNA: Adenine (A), guanine (G), thymine (T), and cytosine (C). Adenine and guanine are purines and thymine and cytosine are pyrimidines. Deoxyribose is the sugar in DNA. The carbon atoms are numbered as indicated. Note there is no oxygen on site 2 of deoxyribose. A nucleoside molecule is composed of a base and deoxyribose. When a phosphate group is added to nucleoside, the complex becomes a nucleotide . Nucleotides are the basic building blocks of DNA.
4
Principles of Clinical Molecular Biology
1-5
5 CH2
Base
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o
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Fig . 2. 3'5' Phosphodiester bonds joint by the unit of the repetitive sugar-phosphate chain. Each nucleotide is linked by the 3' carbon atom of upstream ribose to the 5' carbon of the downstream ribose. Phosphodiester bonds are central to all life on earth, as they make up the backbone of DNA and RNA strands in every organism.
- The deoxyribonucleotides in DNA differ only in the bases they carry, so the DNA sequence is denoted by a base sequence (e.g., -ATTGCAT-) - Base sequence is presented from 5' to 3' - DNA strands are pairs of complementary molecules, which entwine each other in an antiparallel direction - Two strands of DNA wind around each other to form a double helix (Figure 3) • Deoxyribose-phosphate backbone is on the exterior of the DNA double helix • The interior of the DNA is formed by paired bases attached to each other by hydrogen bounds. G (Guanine) pairs with C (Cytosine) via three hydrogen bonds, and A (Adenine) pairs with T (Thymine) via two hydrogen bonds inside the double helix. Note that the three hydrogen bonds joining G to C (GC bond) are stronger than the two hydrogen bonds joining A to T (AT bond) (Figure 4) - DNA has two DNA chains; one is oriented 5' ~3' while the other strand is oriented 3' ~5' direction (antiparallel) • Sense is a DNA strand that could be transcribed. Sense strand has a sequence similar to its RNA transcript • Antisense is the complimentary strand of sense. Antisense works as template for the RNA transcript
• A DNA fragment appears to have a unique function, either structural , regulatory, or coding
Types of DNAs • Single copy DNA is a specific DNA sequence that is present only once in the genome • Repetitive DNA is a DNA segment with a specific DNA sequence that is repeated multiple times in the genome • Moderately repetitive DNA refers to 10-10 5 copies of the sequence per genome - Moderate repeated DNA is found primarily in noncoding sequences • Highly repetitive DNA describes DNA sequence present in greater than 105 copies per genome - Highly repeated DNA is found primarily in centromere and telomere regions as tandem repeats • Tandem repeat DNA contains a variable number of short DNA sequences repeated many times in series . The number of repeats is unique to each individual , and can be used for relationship testing - The tandem repeat pattern may vary from one base repeats (mononucleotide repeat) to several IOOO-bp repeat sequences
5
1-6
Molecular Genetic Pathology
3'end 5'
3'
3' end
3'
5'
5'end
Fig. 3. Human genomic DNA contains two polynucleotide chains wound around each other to form a double-stranded helix . The two chains are "antiparallel,' one running 5'-3' and the other running 3'-5' direction . The DNA strands are synthesized and read out by RNA polymerase in the 5'-3' direction . The purine or pyrimidine attached to each deoxyribose project s into the center of the helix. Base A pairs with T and a G pairs with C through hydrogen bonds in the central axis.
- These segments of DNA are satellite DNA because of the experimental observation that they often form a minor satellite band near the major centrifugation fraction when DNA is separated by density gradient - Clusters of such repeats are scattered on many chromosomes. Each variant is an allele that is inherited co-dominantly - Megasatellite DNAs are tandem repeat DNA segments with a length greater than 1000 bp (1 kbp) repeated 50-400 times - Satellite DNAs comprise about 15% of human DNA. The repeated sequence ranges from 5 to 170 bp and the complex is about 100 kbp in length - Minisatellite DNAs are repeated sequence s ranging from 14 to 500 bp in length . The repeat complex is 0.1-20 kbp in length. Minisatellite DNA is present in telomere region
6
- Microsatellite DNAs are sequences <15 bp in length that repeat 10-100 times without interruption. There are approx 200,000 microsatellite loci in the human genome (Table 1) • Loss of heterozygosity (LOH) in a cell represents the loss of one parent's contribution of DNA to a cell 's genome, often in microsatellite regions • It often indicates the presence of tumor-suppressor gene loss around the microsatellite locus • LOH can arise through deletion, nonreciprocal DNA transfer, mitotic recombination, or chromosome loss • LOH is often used to analyze the clonal origin of cancer-associated loci • Microsatellite DNA loci are useful markers for the detection of LOH
Principles of Clinical Molecular Biology
1-7
• When parents' contributions of certain microsatellite loci are of different size, these microsatellite loci are informative - Microsatellite instability at critical loci is a marker for malignancy or premalignant genetic change (see details in Chapter 7) • Mitochondrial DNA (mtDNA) (see Mitochondrial DNA)
DNA Replication • Double-stranded DNA (dsDNA) is exactly duplicated prior to cell division so that each daughter cell is endowed with an exact replica of the parent cell DNA • DNA replication occurs during S (synthesis) phase of the cell cycle
H I N
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Fig. 4. The double helical structure of DNA is largely due to hydrogen bonding between the base pairs linking one complementary strand to the other. Hydrogen bonds are noncovalent, weak bonds between electron donors and recipients. There are two hydrogen bonds between A and T and three hydrogen bonds between G and C, thus the bonds between G and C are stronger than between A and T.
- Cell cycle refers to a cycle of events in a eukaryotic cell from one cell division to the next; it consists of Go' G I' S, G 2, and M phases (Figure 5) - Semiconservative replication means that each DNA molecule consists of one original and one newly synthesized chain (Figure 6) • DNA Polymerases - DNA polymerases are enzymes involved in DNA replication. Eukaryotic cells have five different DNA polymerases • DNA polymerase
a and 8 replicate nuclear DNA
• DNA polymerase ~ and repair • DNA polymerase (mtDNA)
E
are involved in DNA
y replicates mitochondria DNA
• Multiple replications means that replication begin s at multiple sites within a DNA strand and proceeds bidirectionally from each origin (Figure 7)
Table 1. Major Characteristics of Repetitive DNA
Form of DNA
Length (bpJ
- The replication apparatus at each origin forms a bubble and extends toward both ends of the DNA molecule until it meets another bubble - The leading strand is the DNA chain that is synthesized continuously in the 5'-3' direction
Number of repeats
• Synthesis of the leading strand is catalyzed by DNA polymerase 8 - The lagging strand is the DNA chain that is synthesized as a series of short fragments, known as Okazaki fragments, polymerized in the 5' ~3' direction also (Figure 8)
Single copy
Vary
Single copy
Moderately repetitive
Vary
10-105
Highly repetitive
Vary
>105
• The newly synthesized DNA fragments will eventually meet and ligate to create an intact strand
Megasatellite
>1000
50-400
Satellite
5-170
500-2000
Minisatellite
14-500
7-40
Microsatellite
<15
10-100
• The lagging strand is synthesized by DNA polymerase a • The lagging strand polymerizes from 5' to 3' at the nucleotide level but overall growth by ligation of Okazaki fragments is in the 3' ~5' direction
Tandem repeat
7
1-8
Molecular Genetic Pathology
M PHASE mitosis
(nuclear division)
S PHASE (DNA replication) Fig. 5. The cell cycle, or cell-divi sion cycle, is the series of events in a eukaryotic cell between one cell division and the next. The cell cycle consists of four phases , G I' S, Go' and M phase. Go is a period in which cells exist in a quiescent state. Go' G l' G2, and S phase are collectively known as interpha se. Cells in Go phase are resting cells unable to divide without a signal to re-enter the cell cycle. DNA synthesis occurs during S phase, which is followed by a short G 2 phase . Mitosi s and cytokinesis together are defined as the M (mitotic) phase, during which the mother cell divides into two daughter cells .
DNA Mutation • DNA mutation is a permanent change in the genetic material sequence • Most mutations are found in noncoding sequences • Single base pair substitution (point mutation) involves a single nucleotide, which is replaced with another nucleotide - Point mutation is the most common form of mutation - It happens most commonly in non-coding sequences - It is also the most frequent type of mutation associated with tumor suppressor gene mutation • Transitions are the mutations that substitute a different purine for a purine or a pyrimidine for a pyrimidine • Transversions are mutations that substitute a different purine for a pyrimidine or a pyrimidine for a purine • Synonymous (silent) mutation is a single base pair substitution yielding a different codon that still codes for the same amino acid • Missense mutation is a single base pair substitution that results in a different codon and a different amino acid • Nonsense mutation is a single base pair substitution that converts a codon specifying an amino acid into a stop codon
8
- Deletion is an irreversible mutation in which one or more nucleotides are removed from the DNA sequence • The deletion will cause a shift of the reading frame • A one base deletion, for example, will shift all codons left, altering the amino acids for which they code • Insertion is a mutation that adds one or more nucleotides to the DNA sequence - An insertion in the coding region of a gene may cause a shift in the reading frame - An insertion alters splicing of messenger RNA (mRNA) (splice site mutation) • Amplification increases the dosage of genes located within a locus by inserting multiple copies of the chromosomal region or by promulgating fragments of DNA containing the locus outside the chromo somes. Proteins produced from amplified genes are generally increased • Loss of heterozygosity (LOH) is a DNA alteration in which one allele from one parent's contribution is lost, either by deletion or a recombination event • The most frequently observed gene associated with LOH in sporadic cancer is p53
Principles of Clinical Molecular Biology
1-9
Fig. 6. Semiconservative DNA duplication occurs when DNA replicates, each parental chain is used as template for synthesis of a complimentary daughter chain. Newly formed duplex strands contain one parental and one daughter chain as indicated by color scheme.
DNA Mutation and Disease • DNA mutations cause errors in protein sequences, creating partially or completely non-functional proteins • DNA mutations give rise to offspring that carry the mutation in all their cells - Human beings with a single allele mutation will transmit the mutation to half of the progeny
- If both alleles are mutated, all progeny will inherit the mutation
Factors Relatedto DNA Aberrations • Spontaneous chemical reactions - Spontaneous chemical reactions cause single base pair substitutions or single base pair deletion through the following processes • Tautomerism-base change by repositioning a hydrogen atom
• Depurination-loss of a purine base (A or G) • Deamination-changes a normal base to an atypical base • Change from C~U • Spontaneous deamination of 5-methycytosine (irreparable) • Change from A~HX (hypoxanthine) • Transition-a purine changes to another purine, or a pyrimidine to a pyrimidine • Transversion-a purine becomes a pyrimidine, or a pyrimidine becomes a purine • Induced mutations - Chemical mutagenesis can modify bases and cause either interstrand or intrastrand cross-linking. Common chemical mutagens include: • Nitrosoguanidine (N-methyl-N'-nitrosoguanidine) • Hydroxylamine (NHPH)
9
1-10
Molecular Genetic Pathology
t
Multiple DNA replication sites
-- t -- -....---.....
....---.....
....---.....
DNA fragment extension
:..------- ----~:..---------------': :::---..---: :::---..---:
t
New strands synthesised
+ Fig. 7. Multiple replications are an efficient way to synthesize chromosomal DNA. DNA synthesis begins at many locations and proceeds bidirectionally from each location . Eventually the replication bubbles merge and the DNA fragments are ligated to form two daughter DNA strands. Each of the newly synthesized double strands consists of one parental and one newly synthesized chain (semiconservative). 5'
5'
3'
•
"sf ~\,s,
3' Lagging strand
3'
•'\
•
"~ ~s
5' Leading strand
5'
3'
'\
3' 5'
\' 5
Fig. 8. The DNA double helix is unwound by the enzyme helicase before synthesis of a new DNA chain begins. A DNA polymerase (shown in green) binds to the strand and moves along the strand assembling the leading strand (fragment inside the left fork). The lagging strand is synthesized in discontinuous polynucleotide segments called Okazaki fragments . A series of Okazaki fragments are linked by DNA ligase to form the lagging strand. In eukaryotic cells the leading and lagging strands are synthesized by DNA polymerase 0 and a, respectively. • Base analogs (e.g., bromodeoxyuridine) only mutate DNA when the analog is incorporated during S phase in replicating DNA • Alkylating agents (e.g., cyclophosphamide) mutate both replicating and non-replicating DNA. The alkylating agent transfers an alkyl group, often to the N7 position of guanine
10
• Polycyclic hydrocarbons are converted within cells to highly reactive epoxy compounds that react with DNA (e.g., benzpyrenes found in internal combustion engine exhaust and cigarette smoke) • DNA intercalating agents insert themselves between the stacked bases at the center of the DNA strand (e.g., ethidium bromide)
Principles of Clinical Molecular Biology
• DNA cross-linkers cause both interchain covalent bonds and stable bonds between the DNA strands and nuclear proteins (e.g., platinum) • Oxidative damage caused by oxygen radicals accelerating hydroxylation of guanine to 8-hydroxyguanine, causing a G:C to A:T transversion - Radiation • Ionizing radiation can cause individual base lesions, cross-linking, or strand breakage, sometimes mediated by oxygen radicals • Ultra violet radiation causes covalent bonding between adjacent cytosine and thymine bases creating pyrimidine dimers - Viral mutagenesis involves a DNA virus, RNA or retrovirus integrating all or part of its sequence into the human genome • An episome is a DNA molecule separate from the chromosomal DNA and capable of autonomous replication. It is the common status of viral particle s during viral infection • Integration means that DNA fragments of viral origin have become inserted into chromosomal DNA • Epstein-Barr virus has been associated with lymphoproliferative disorder in immunocomprornised patients (such as post-transplantation) • Human herpesvirus 8 has been associated with Kaposi's sarcoma, Castleman's disease, body cavity lymphoma (primary effusion lymphoma), and multiple myeloma • Human papillomavirus has been associated with premalignant and malignant transformation of the uterine cervix • Inborn errors of metaboli sm - Inborn error s comprise a large class of genetic diseases involving metabolic disorders - The majority of inborn errors are due to single gene defects that code for enzymes to convert intermediary metabolites
DNA Repair Mechanisms • DNA repair include s a collection of processes through which a cell identifies and corrects damage to its DNA • DNA repair is essential to cell survival • If DNA damage is irreparable then programmed cell death (apoptosis) should ensue • Failure to correct molecular lesions in gamete-forming cells leads to progeny with congenital mutation s • Single-strand damage repair - Direct repair is an enzyme-catalyzed reaction that directly reverses DNA damage. No template is needed to correct an altered base back to its natural state - Base excision repair (BER) removes a damaged base and replaces it with a normal base (Figure 9)
1-11
5' I
3"
I
I
I
If1
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!!
I
I
I
I 3'
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+ 5' TTCTG 3'
~ ~
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3,11 f 1 W y 1 f Y5'
DNA polymerase ~ 5' and ligase
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3'
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I 3'
A G T C C AAGACA T C A G G I
I
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Fig. 9. A single base in DNA may be chemically mutated, for example, by deamination or alkylation , causing incorrect base pairing (X), and consequently, incorrect codons in the DNA. BER is initiated by DNA glycosylases linking particular types of chemically altered bases to the desoxyribose-phosphate backbone. Thi s mutated base is excised as a free base , generating sites of base loss called apurinic or apyrimidinic (AP) sites. The AP sites are substrates for AP endonucleases. The ribose-phosphate backbone is then removed from the DNA by an exonuclease called deoxyribophosphodiesterase (dp-diesterase). Then the DNA polymerase and a ligase catalyze incorporation of a specific deoxyribonucleotide into the repaired site, enabling correct base pairing. - Mismatch repair removes nucleotides, which are mispaired with the corresponding base on the complementary chain, usually by DNA polymerase errors. This repair process can also remove up to 30 base insertions (Figure 10) Translation synthesis temporarily replaces the conventional polymerase at a DNA lesion by one of a group of specialized polymerases that can replicate damaged DNA. It is better for the daughter cell to inherit a point mutation than to have a significant part of a chromosome deleted, causing framshift
11
1-12
Molecular Genetic Pathology
Human mismatch repair
____x~ __ .=x- - - -
3'- - - - - - ...JA '-- - - - - -5' S
~
V
1
Excision
3'- -5'- -
-
-
A
---.J
'--
V
-
-
-
-
Fig. II . Homologous recombination repairs a double-stranded break. It allows the precise replacement of a sequence from one allele with a sequence from the homologous allele. The breaks in dsDNA use a homologous dsDNA molecule as a template . Homologous recombination requires a homologous sequence to guide the repair.
- 5' 3'
1
A '-
3" 5'-
---.J -
DNA synthes is
Nonhomologous end joining
-----3' 5'
~---;.____--------5'
/
3'
Fig. 12. NHEJ can also repair double-strand breaks in DNA. NHEJ directly ligates the breaks without a homologous template. NHEJ typically utilizes short homologous DNA sequences,
1
termed microhomologous, to guide the repair but still results in some DNA sequence information being lost or "spliced out."
3' -- - - - - - - - - - - - - - -5' 5'
3'
Fig. 10. Mismatch repair is a cellular procedure for recognizing and repairing insertion, deletion or misincorporation. The DNA damage is repaired by excising the mis-incorporated base or segment and synthesizing a new stretch of DNA is synthesized to replace the excised segment. This process involves more than just the mismatched nucleotide itself and can lead to the removal and synthesis of a significant piece of DNA. Also there are multiple excisionrepair systems in a single cell type .
• Double-strand break repair - Homologous recombination uses an intact homolog sequence as a template for repair of a broken DNA strand (Figure 11) - Non-homologous end joining (NHEJ) links the ends of two double-strands without needing for a homologous template and regardless of sequence similarity. Just as some information is lost when splicing a movie film or cassette tape, NHEJ introduces mutations that are minimized by various failsafe mechanisms (Figure 12)
GENES Overview
Gene Components (Figure 13)
• Genes are DNA sequences that encode heritable biologic characteristics
• Promoter is a DNA fragment to which RNA polymerase binds to initiate transcription
- The human genome is divided into two categories, nuclear and mitochondrial genome • DNA in the human nuclear genome encodes about 30,000-40,000 different genes, much lower than previous estimates of around 100,000 genes before completion of the genome map • DNA in the human mitochondrial genome encodes 37 genes
12
- Core promoter directs the basal transcription complex to initiate the transcription of the gene • TATA box is a short sequence located within the promoter of most genes - TATA box has a core 5'-TATAAA-3' sequence - The TATA box is usually found as the binding site of RNA polymerase II
Principles of Clinical Molecular Biology
1-13
Polyadenation signal
IStop codon I ~
I
Enhancer
I
Fig. 13. A gene consists of both coding and non-coding sequences. The coding sequence (open reading frame , ORF) extends from a start codon to a stop codon. Introns are non-coding sequences that will be spliced out after transcription. 5' untranslated region (UTR) is a part of mRNA located between cap site and start codon. 3' untranslated region is also a part of mRNA following coding sequence. A promoter and different regulatory motifs are located up stream of a gene. Enhancer or silencer may be located upstream or downstream of the gene it regulates .
• CCAAT Box (CAT box) is located at -75 and serves as a modulator for the basal transcription - CCAAT box has a core 5'-CCAATC-3' sequence - CCAAT box is the binding site of nuclear factor I (NF-I) and CCAAT box binding factor (CBF) • GC box is also called Sp I box. It has consensus sequences GGGCGG and is found within 100 bp from the transcription initiation site - GC boxes serve as a modulators to the basal transcription of the core promoter • CpG sites are region s of DNA with a high frequency of phosphodiester-linked cytosine-guanine pairs . The "r" in CpG indicates that a normal phosphodiester bond between nucleosides gives the the CG sequence direction - CpG island s are located near or within 40% of mammalian gene promoters - The genes with CpG islands are expressed if the CpG islands are not methylated • Enhancers are DNA sequences that when bound by certain factors increase transcription levels of genes - Unlike promoters, enhancers do not have to be within or near the genes they act on, or even located on the same chromosome • Silencer is a DNA sequence that can bind regulators of transcription called repressors. The binding of repressor prevents RNA polymerase from initiating transcription - When repressor is bound to target DNA, RNA synthesis is decrea sed or fully suppressed • An exon is any region of DNA within a gene that encodes a protein. Exons of many eukaryotic genes interleave with segments of non-coding DNA (intron s). Mature mRNA contains only sequentially linked exons
• Introns are sections of non-coding DNA located between exons, which are transcribed into RNA but are spliced out to form mRNA • Open reading frame (ORF) is the sequence of DNA or mRNA molecule from the start codon (ATG) to a stop codon (TAA, TAG, or TGA). An open reading frame codes for amino acid codons that can be translated into a protein • Boundary elements (insulator elements) are regions of DNA that mark the 5' and 3' ends of a gene • Gene expression is the process by which a gene 's DNA sequence is converted into the structures and functions of a cell - Protein-coding genes are translated into proteins - Non-protein coding genes code for RNAs, (e.g., ribosomal RNA [rRNA] genes, and transfer RNA [tRNA] genes), which usually have a structural , regulatory or catalytic role
Functional Categories of Genes • Housekeeping genes are genes that are transcribed at a relatively constant level and remain unaffected by environmental conditions - Housekeeping gene product s are necessary for cell maintenance • Since their expression is typically unaffected by experimental conditions, they may be used for normalization of other gene expression levels in the cell • Housekeep ing genes often lack the CCAAT and TATA boxes - Actin and glyceraldehyde 3-phosphate dehydrogenase are example s of housekeeping genes commonly used as control s for mRNA quantitation
13
1-14
Molecular Genetic Pathology
• Facultative genes are transcribed only when needed • Inducible gene expression is either responsive to environmental changes or dependent on the stage of the cell cycle • Pseudogenes are multiple copy genes characterized by defective copies , mostly truncated, of a functional gene - Pseudogenes arise from gene duplication or retrotransposition • RNA genes transcribe mRNA as their end products without protein translation
Cancer-Related Genes (Table 2) • Tumor-suppressor genes prevent cell overgrowth (neoplasia) - They are involved in cell cycle control, cell differentiation, and apoptosis - Tumor-suppressor gene products generally promote genomic stability - Tumor-suppressor gene inactivation mechanisms include point mutation, deletion, and epigenetic inactivation of the gene • Proto-oncogenes are normal genes that cause a malignant phenotype either because of mutation or increased expression . Proto-oncogenes code for proteins to regulate cell growth and differentiation - A proto-oncogene becomes an oncogene when mutated, inappropriately expressed or over expressed, transforming the cell by unregulated growth and differentiation • Oncogene - Oncogene products include • Growth factors bind receptors on the cell surface to stimulate cell proliferation or to control differentiation • Receptors are proteins on the cell surface, within the cytoplasm or in the cell nucleus for binding specific molecules (ligands) to initiate a cellular response • Protein kinases chemically add phosphate groups to specific amino acids of substrate proteins. This process usually results in a functional change of the target protein resulting in changed enzyme activity, altered cellular location, or modified association with other proteins
14
virus (Src-family), SYK-Zeta-chain associated protein kinase 70 (Syk-ZAP-70 family), and Bruton's tyrosine kinase (BTK family) oncogenes belongs to the cytoplasmic tyrosine kinases group. The bcr-abl transcript (fusion gene of the Philadelphia chromosome in chronic myelocytic leukemia (CML) is also a tyrosine kinase . The bcr-abl fusion gene kinase activates mediators of the cell cycle regulation system, leading to a clonal myeloproliferative disorder • Regulatory GTPases are a large family of enzymes that bind and hydrolyze GTP (guanosine triphosphate) existing in GTP-bound and -unbound states. They play important roles in the following cellular processes: • Signal transduction at the intracellular domain of transmembrane receptors • Protein biosynthesis at the ribosome • Control of differentiation during cell division • Translocation of proteins through membranes • Transport of vesicles within the cell • Ras oncogene produces a small regulatory GTPase important as a molecular switch for a variety of signal pathways. Ras controls such processes as cytoskeletal integrity, cell proliferation, adhesion, apoptosis, and migration
• Cytoplasmic serine/threonine kinase phosphorylates the hydroxyl group of serine or threonine . The Raj kinase, and cyclin-dependent kinases belong to the serinelthreonine kinase family • Adaptor proteins are small accessory proteins, which lack intrinsic enzymatic activity but bind signal transduction pathway components, driving the formation of active protein complexes • Transcription factors mediate the binding of RNA polymerase to DNA and initiation of transcription. A transcription factor may work to either stimulate or repress transcription of a gene
Regulation of Gene Expression • Regulation of gene expression controls the amount and appearance agenda of a gene's functional product - All steps of gene expression can be modulated - Regulation of gene expression is the basis for cell differentiation, diversity, and adaptation • Cis-action factors are short regulatory sequences located within the promoter or in the vicinity of a gene's structural portion. Cis-sequences facilitate the transcription of adjacent polypeptide-encoding sequences
• Receptor tyrosine kinases are membrane-bound enzymes to transfer a phosphate group from ATP to a tyrosine residue in a protein. The tyrosine kinasebinding hormones and growth factors are generally growth-promoting and mitogenic agents, such as epidermal growth factor receptor (EGFR)
• Trans-action factors bind to the cis-acting sequences to control gene expression
• Cytoplasmic tyrosine kinases are non-receptor tyrosine kinases (TK) to regulate many cellular processes, such as inducing gene of Rouse sarcoma
• Enhancer is a short region of DNA that upregulates transcription levels of genes. Enhancer sequences are active when bound to trans-action factors
Principles of Clinical Molecular Biology
1-15
• Response element is a short sequence of DNA within the promoter of a gene that can bind to a specific hormone receptor complex and regulate transcription of genes subject to that hormone
Signal Transduction • A signal transduction pathway is a sequence of enzymes and second messengers by which a receptor communicates with the cell nucleus • The signal transduction pathway "translates" the receptor ligand message at the surface into a cellular response in the nucleus
- abl and ras are signal transducers • Transcription factors
- A transcription factor is a molecule that initiates transcription of DNA in the eukaryotic nucleus - Transcription factors interact with promoter or enhancer sequences either by binding directly to DNA or by interacting with other DNA-bound proteins - myc is an example of a transcription factor that activates expression of many genes by binding to consensus sequences • Programmed cell death regulators are molecules that prevent apoptosis . Activation of these regulators leads to overgrowth of abnormal cells - bcl-2 is an example of a programmed cell death regulator that governs mitochondrial outer membrane permeability and suppresses apoptosis
CHROMOSOMES Overview
Telomere
-e:
• A chromosome is an enormous macromolecule into which somatic DNA is packaged in eukaryotic cells. Three billion base pairs of nucleotides (a complete set of DNA) are divided among 46 chromosomes, each containing many genes, regulatory elements, and intervening nucleotide sequences (Figure 14)
1 p
Centrome re
• Chromosomes are found only in the eukaryotic nucleus and can be seen only during nuclear division • During most of the life cycle, the genetic material occupies areas of nuclei in the form of chromatin, and individual chromosomes cannot be distinguished • In eukaryotes, the basic function of the chromosome is to package and compress the DNA, exposing specific genes for transcription during certain phases of the cell life span
q
Chromatin • Chromatin is the form of genetic material existing during interphase of eukaryotic cells and is made up of DNA and protein
Telomere
-e:
Fig. 14. Structure of a typical human chromosome.
• Chromatin can be seen with the light microscope after staining with nuclear stains • Chromatin is a packaged state of DNA in a small volume to strengthen the DNA, to allow mitosis and meiosis, and to serve as a mechanism for expression control • Chromatin functions as a gene regulator - The changes in chromatin structure are effected mainly by methylation (DNA and proteins) and acetylation (proteins) - Euchromatin is a loosely packed form of chromatin that is involved in active transcription or regulation and is lightly stained by nuclear stains
- Heterochromatin is a darkly stained and tightly packed form of DNA. Its major biologic characteristic is that it is not transcribed • Chromatin composition - Histones are the major chromatin binding proteins. They act as spools around which DNA winds • Histones playa role in gene regulation • Histones H2A, H2B, H3, and H4 form octamers (two of each) with a cylindrical shape (Figure 15)
15
1-16
Molecular Genetic Pathology
• The centromere divides the chromosome into four arms • The two equal short arms are designated "p" (petite) • The two equal long arms are designated "q" (follows p in the Latin alphabet) • Telomere: (see Chapter 7, Telomere section) • Chromosome grouping - Chromosomes are numbered and grouped according to their morphologic characteristics
Fig. 15. Nucleosomes are the fundamental repeating subunits of all eukaryotic chromatin. They package DNA into chromosomes inside the cell nucleus and control gene expression . The DNA winding around the nucleosome core particle consists of about 146 bp of dsDNA wrapped in 1.65left-handed superhelical turns around complexes of the four histone proteins known as the histone octames. The DNA hanging between two nucleosome cores is typically 55-bp long and is known as linker DNA.
- Chromosomes are numbered according to their relative sizes from largest to smallest. The position of the centromere determines chromosome grouping • Group A have nearly equal p and q arms whereas group E have the centromere almost at the telomere - Chromosome identification is confirmed by the banding pattern unique to each chromosome (Figure 16) • Chromosome number - Human cells contain 46 chromosomes (23 from each parent) including 22 pairs of autosomes and one pair of sex chromosomes - The number of chromosomes doubles during cell division
• When DNA winds 1.65 times around a histone octamer a nucleosome results • Each nucleosome contains 146 bp • The nucleosomes are stacked and further coiled into a 30-nm fiber, which makes up the chromosome residing in the cell nucleus • Linker DNA is the DNA hanging between two nucleosomes, typically 55-bp long • Histone-DNA interaction regulates gene expression. Acetylation of histone modulates gene expression and leads to transcription activation. The extent of interaction between histone and DNA is affected by the degree of histone acetylation • Histone acetylation is the process in which charged lysine side chains are acetylated, leading to reduced affinity between histone and DNA. After acetylation, RNA polymerase and transcription factors have better access to the promoter
Chromosomes • Chromosome structure - Each chromosome has two short arms (p), two long arms (q), one centromere, and four telomeres • The centromere is the constricted region of a chromosome, which has a special sequence and structure for attachment to the spindle filament during M-phase and for separation of chromosomes during mitosis (Figure 14)
16
• Meiosis is a process allowing one diploid cell to divide in a special way into four haploid cells in eukaryotes • Mitosis is the process by which a cell separates its duplicated chromosomes into two identical sets of chromosomes • Ploidy is the number of homologous sets of chromosomes in a cell • Haploidy (monoploidy) is the number of chromosomes in the gamete of an individual (23 in a human) . Haploid chromosomes have only one short and one long arm • Diploidy is the normal state of chromosomes in a cell, with two copies of each chromosome, one from each parent. The two chromosomes in a pair are said to be homologous • Polyploidy is the state of cells with extra chromosomes beyond the basic set of paired chromosomes • Aneuploidy is a condition in which the number of chromosomes is abnormal owing either to extra or missing chromosomes. The number of chromosomes in an aenuploide cell cannot be a multiple of the haploid set (Figure 17) • Monosomy is a type of aneuploidy with at least one missing parental chromosome (Figure 18) • Trysomy is a type of aneuploidy with one extra chromosome added to a pair of homologous chromosomes
1-17
Princ iples of Clinical Molecular Biology
Human Genome and Chromosomes
Genome size: 3.2x10 9 nucleotides 22 autosomes and 2 sex chromosomes Diploid (2N): (1-22) x 2 + XV/XX = 46 (\J
35,000 genes
150
2
~y millionnucleotide pairs
13
o
x
Fig. 16. Chromosomes are grouped according to their relative size, the position of their centromere, and banding patterns .
• ..~ , ' I'·
--... '\ ,I ~ 1' ·, - > ~
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., ., '. . .. .
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Fig. 17. Aneuploidy is a condition in which the number of chromosomes is not a multiple of the haploid set due to gaining or losing chromosomes. The figure shows a fluorescent probe chromosome painting of tumor cell chromosomes featuring a series of chromosome gains and loses.
Fig. 18. Monosomy is a type of aneuploidy with loss of one chromosome from a pair in the cell's diploid chromo some set. Fluorescence in situ hybridization shows the loss of one chromosome in these cells indicated by having only one signal present in each nucleus.
17
1-18
Molecular Genetic Pathology
• Structural alterations of chromosomes (see details in Chapter 2) - Inversion occurs when a chromosome segment is flipped end to end. Inversion is designated by the symbol inv Reciprocal translocation is a chromosomal rearrangement caused by the interchange of chromosome segments between non-homologous chromosomes . Reciprocal translocations are denoted by the symbol t followed by parentheses showing the exchanged chromosome breakpoints separated by a semicolon Isochromosome is a chromosome which has lost one set of its arms, either p or q, and replaced them with an exact copy of the other arms. Isochromosomes thus have four identical arms , either p or q. Isochromosome is denoted by the symbol i
- Ring chromosome is a chromosome that is formed when the telomeres have been lost, and the ends of arms fuse together to form a ring. A ring chromosome is denoted by the symbol r • Fragile sites are chromosome regions that are poorly connected to the rest of the chromosome - Fragile sites are often rich in CGG or CGC repeats and are inherited like a gene and break away frequently - Double chain breaks in fragile sites lead to the loss of genetic material - Fragile sites are especially prone to breakage when cells are cultured under conditions that inhibit DNA replication or repair Selected tumors with chromosomal anomalies (Table 2)
Table 2. Selected Tumors with Commonly Found Chromosomal Anomalies Tumor
Common chromosome anomalies
Genes involved
Epithelial tumors Basal cell carcinoma
9q22 .3
PTCH
Clear cell renal carcinoma
3p25-26
VHL
Translocation renal cell carcinoma
t(X; I)(p 11.2;q21)
PRCC-TFE3
t(X; 17)(p11.2-q25)
ASPL-TFE3
t(X ;I)(p 11.2;p34)
PSF-TEFJ
Papillary renal cell carcinoma
Gain 7, 17, loss Y, and 4
-
Hereditary papillary renal cell cancer
7q31
c-MET
Breast cancer
Iq
-
17q21
BRCA1, Her-2/neu
13ql2
BRCA2
del(l6q)
-
del(l7p)
TP53
12p
Ras
3pl4
FH1T
5q21-22
APC
18q21
DCC, SMAD4
del(3p)
FH1T
13q
RB
9p21
P16
17p
TP53
Colorectal cancer
Lung cancer
(Continued)
18
Principles of Clinical Molecular Biology
1-19
Table 2. (Continued) Tumor Prostate cancer
Bladder transitional cell carcinoma
Common chromosome anomalies
Genes involved
t(21 ;2 1)(p22.2;q22.3)
TMPRSS2-EGR
del(8pI2-2l)
NKX3.1
lq24
HPCI
Xq27-28
HPCX
Xqll
AR
del(lOq24)
PTEN
Trisomy 7
-
Loss ofY
-
gain 3,7, 17,del(9p21)
P53, Pl6
(UroVysion panel) Medullary thyroid carcinoma
lOql1.2
RET
Papillary thyroid carcinoma
IOqll-q13
RET
inv(1)
NTRKI-TPM3 (TRK)
Mesothelioma
del(3p21)
CTNNBI
Ovarian papillary cystadenocarcinoma
t(6;14)
-
Granulosa cell tumor and Brenner tumor
trisomy 12
-
i(12p)
-
i(12p)
-
12p overrepresentation
-
del(1 lpI3)
WTI
Alveolar soft-part sarcoma
t(X; 17)(p II ;q25)
TFE3-ASPL
Alveolar rhabdomyosarcoma
t(2; 13)(q35;q 14),
PAX3-FKHR,
t(1; 13)(p36;q 14)
PAX7-FKHR
Clear cell sarcoma (melanoma of soft part)
t(12 ;22)(q 13;q12)
EWS-ATFI
Dermatofibrosarcoma protuberans and giant cell fibroblastoma
t(17 ;22)(q22 ;q13)
COLlAI-PDGFB
Myxoid chondrosarcoma
t(9;22)(q22 ;q12)
EWS-CHN
Lipoma
t(3; 12)(q27;q 13)
HMGIC-LPP
Lipoblastoma
8q rearrangement
-
Myxoid liposarcoma
t(12;16)(q13 ;pll)
CHOP-FUS
t(12;22)(q 13;q12)
EWS-CHOP
Ring chromosome 12
-
Testicular germ cell tumors
Wilm's tumor
Soft tissue tumors
Well-differentiated liposarcoma
(Continued)
19
Molecular Genetic Pathology
1-20
Table 2. (Continued) Tumor
Genes involved
Common chromosome anomalies t(l i ;22)(q24;q 12),
EWS-FLl l,
t(21;22)(q22;q 12)
EWS-ERG
Desmoplastic small round cell tumor
t(li ;22)(p 13;q 12)
EWS-WTl
Synovial sarcoma
t(X;18)(pll ;qI I)
SIT-SSXl
t(X;20 )
-
Infantile fibrosarcoma and congenital mesoblastic nephroma
t(l2;l5 )(p 13;q25)
ETV6-NTRK3
Inflammatory myofibroblastic tumor
t( I ;2)(q22 ;p23)
TPM3-ALK
t(2;I9 )(p23 ;p 13)
TPM4-ALK
Gastrointestinal stromal tumor
4qll -2l
c-kit exon II
Hemangiopericytoma
t(l2;19)
-
Uterine leiomyoma
t(l 2;14)(q l3 -I5 ;q24.I )
HMG1C
Endometrial stromal sarcoma
t(7;17)(p l5-p2I ;q 12-q2I )
JAZFl-JJAZI
Leiomyosarcoma
del(l p)
-
Pleomorphic adenoma
t(3;8)(p I2 ;q 12)
FGFR1-FlM
Aneury smal bone cyst
I7p rearrangement
-
Desmoplastic fibrobla stoma and fibroma of tendon sheath
t(2; II )
-
Melanoma
del(9p21)
CDKN2
I2qI4
CDK4
del(22q)
-
Acoustic neuroma
22qI2.2
NF2
Schwan noma
deI(22qI3)
NF2
Meningioma
del(22q II-q 13)
NF2
Mono somy 22
-
Trisomy 7 Monosomy 10, 22 t(lO ;19)(q24;qI3)
(7pII )
del(lOq)
PTEN and DMBTl (l Oq)
i(l7q)
-
del( 17p13.2)
-
Trisomy 8
-
2p del( Ip31-32)
N-Myc amplification
Ewing's sarcoma/primitive neuroe ctodermal tumor
Neural/neuroendocrine tumors
Gliobl astoma multiforme
Medulloblastoma
Neuroblastoma
EGFR gene amplification
(homogenous staining region and double minute s) (Continu ed )
20
Principles of Clinical Molecular Biology
1-21
Table 2. (Continued)
Tumor
Common chromosome anomalies
Genes involved
del(lp36)
TP73
del(l9q13)
PEG3
Retinoblastoma
del(l3qI4)
RB
Pheochromocytoma
del(22q13)
SLCI
del(lp11-36)
RIZI
t(8; 14)(q24;q32),
c-myc-IgH
t(8;22)(q24 ;qll)
c-myc-IgL
t(2;8)(p 12q24)
c-myc-IgK
Follicular lymphoma
t( 14;18)(q32;q21)
IgH-BCL2
Mantle cell lymphoma
t(lI;14)(qI3;q32)
IgH-BCLl (cyclin 01)
llq
ATM mutation or deletion
del(l3q14)
-
Trisomy 12
-
del(l7p)
-
Mucosa-associated lymphoid tissue (MALT) lymphoma
t(ll; 18)(q21;q21) Trisomy 3
API2-MALTl
Diffuse large cell lymphoma
t(l4;18)(q32;q2l)
IgH-BCL2
t(3; 14)(q27;q32)
BCL6-1gH
Anaplastic large cell lymphoma
t(2;5)(p23;q35)
NPM-ALK
Lymphoplasmacytic lymphoma
t(9;14)(p13;q32)
PAX5
Myelodysplastic disorder
del(5q)
-
Trisomy 8
-
Monosomy 7
-
del(7q)
-
del(l7p)
-
del(20q)
-
Chronic myelogenous leukemia
t(9;22)(q34;q11)
BCR-ABL
Acute myelogenous leukemia (AML)-M2
t(8;2l )(q22;q22)
AMLl-ETO
Acute promyelocytic leukemia -M3
t(l5 ;17)(q22;q2l)
PML-RARa
AML-M4eo
inv(l6)(p13;p22)
MYHII-CBFB
AML-M4,M5
t(l1 ;19)
MLL-ENL
llq23
MLL
Oligodendroglioma
Lymphomas/leukemia Burkett's lymphoma
(Continued)
21
Molecular Genetic Pathology
1-22
Table 2. (Continued) Tumor
Common chromosome anomalies
Genes involved
AML-M5
t(9;II)(p22 ;q23)
MLL-AF9
PediatricAML
translocation involving IIq23
MLL-AF9
AML-M7
t(l ;22)
-
Pre-B ALL
t(l ;19)
E2A-PBXl
B-ALL
t(l2 ;21)
TEL-AMLl
InfantileALL
t(4;II )
AF4-MLL
ALL
t(l2;21) (most common translocation in ALL)
TEL-AMLl
Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL)
del (l3q) del (llq) del (l7p)
-
trisomy 12q
-
t(lI ;14)(q13;q32)
-
Monosomy or partial deletion of 13 (I3q)
IgH-BCLl (cyclin Dl)
t(lI ;14)(qI3;q32)
-
Multiple myeloma
19H-BCLl (cyclin DI)
RNA AND PROTEIN Overview • What is RNA? - RNA is a single-stranded nucleic acid polymer consisting of nucleotide monomers - The five-carbon sugar in RNA is ribose (contains a 3' hydroxyl group) inste ad of deoxyribose -
Bases in RNA are A (adenine), G (guanine), C (cytosine), and U (uracil), which takes the place ofT (thymine) in DNA
-
RNA folds back on itself to form hair pin or loop structures via intramolecular hydrogen bonds (Table 3)
• What is protein? - Proteins are chains of amino acids joined by peptide bond s - The peptide bond forms when the carboxyl group of one amino acid residue is joined to the amino group of the next amino acid residue • Synthesis of RNA in cell s - In transcription a DNA sequence is enzymatically copied by an RNA polymerase in a process analogous to DNA chain duplication. In RNA
22
transcription, A from DNA template determines U in RNA instead of T (Figure 19) - RNA polymerases are a group of nucleotidyltransferases that polymerize ribonucleotides in accordance with the information present in DNA • RNA polymerase I transcribes genes encoding ribosomal RNA (rRNA) • RNA polymerase II transcribes genes encoding proteins (mRNA) and certain small nuclear RNAs (snRNA) • RNA polymerase III transcribes genes encoding transfer RNA (tRNA) and other small RNAs (5SRNA in ribosomes) • Primase catalyzes the synthesis of the short RNA primers on single -stranded DNA templates used by DNA polymerase to initiate the synthesis of Okazaki fragments on the lagging strand • Prima se is an essential enzyme in all wellcharacterized systems of DNA replication because no DNA polymerase can initiate DNA synthesis without an RNA primer • The enzyme is required for synthesis of the lagging strand
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Principles of Clinical Molecular Biology
• mRNA constitutes about 5% of the total cellular RNA
Table 3. The Differences Between DNA and RNA
mRNA components • Exons are sequences in mRNA that encode an amino acid sequence
DNA
RNA
Bases
A,G,C,T
A,G,C,U
Strand
Double stranded
Single stranded
Structure
Antiparallel helix
Hairpin and loops
Sugar
Deoxyribose
Ribose
Location
Nuclear or mitochondrial
Nuclearor cytoplasmic
Lifetime
Long
Short
Process
Transcription
Translation
Types
Nuclear DNA, mtDNA
mRNA, tRNA, rRNA, miRNA, siRNA, ribozyme
• Introns are non-coding sequences between exons, which are spliced out of mRNA by exonucleases prior to translation (see mRNA Processing section and Gene Components section) • Codons are a set of 3-base "words" conveying genetic information to be translated into amino acid sequences • Polyadenylation is the covalent linkage of 50-250 adenosine ribonucleotide units to the 3' end of mRNA at the polyadenylation signal (AAUAA). This structure is known as the polyadenosine (poly-A) tail
RNA
~ DNA
-
-
-
polymerase
-
3' ...L.L.............L.L........... _
• Poly (A) stabilizes mRNA and protects the mRNA molecule from exonucleases • Poly-A is important for transcription termination and for export of the mRNA from the nucleus to the cytoplasm
Coding strand
5' -
• Adding Poly (A) is one of the steps for producing mature mRNA required for translation
-
-
-
- 3' 5'
~.LLL.LJ..LL.J...
• 5' UTR (5' untranslated region) is a particular section of mRNA located between the cap site and the start codon at the 5' end
• It is not translated • It affects the mRNA stability • It regulates gene expression in response to iron
Direction of polymerization
mRNA
•
Transcription bubble
Fig. 19. Transcription is the process through which a DNA sequence is copied by RNA polymerase II to produce a complementary RNA chain. The RNA polymerase II proceeds along one chain of DNA moving in the 3' ~5' direction and assembles ribonucleotides into a strand of RNA. Synthesis of the mRNA product continues in the 5' ~3' direction until it reaches the stop codon.
Types of RNA • mRNA is transcribed from a DNA template to carry sequence information from the nucleus to the cytoplasm • Transcription is the process of copying sequence information from DNA to RNA. Translation is the process of converting mRNA sequence information into a protein chain
• It facilitates the initiation of translation
• 3' UTR (3' untranslated region) is a particular section of mRNA located between the stop codon and the poly-A tail at the 3' end • It contains a polyadenylation signal sequence, usually AAUAAA, or a slight variant
• It is a binding site for proteins that may affect the mRNA stability or local concentration in the cell • It contains binding sites for microRNAs (miRNAs) • tRNA is an amino acid-specific adaptor molecule containing an anticodon at the 3' end of an RNA molecule containing three hairpin loops, one of which binds a particular amino acid - tRNA constitutes about 10% of the total cellular RNA - tRNA transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation (Figure 20) - Anticodon refers to the unit in a tRNA molecule made up of the three nucleotide sequence that is complementary to the three bases of the codon
23
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Molecular Genetic Pathology
5'
AUG
-----TrAGG -CUC-~~~
- A U G - U G G 7 r 3'
UAC
N terminus Met
~~ A r g - Leu
+c}eUo Trp
Fig. 20. tRNA transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has a site for amino acid attachment and a three-base region called the anticodon that recognizes the corresponding three-base codon region on mRNA via complementary base pairing.
- tRNA molecules are encoded by RNA genes of nuclear and mtDNA
Ribosome and Ribozyme • rRNA is the RNA component of a ribosome - rRNAs in eukaryotes include 5S, 5.8S, 18S, and 28S RNA subcomponents essential for ribosome structure and function - rRNA is the most abundant and stable RNA species in the cell and constitutes about 50% of the total cellular RNA - Ribosomes are the protein manufacturing machinery of all living cells • Ribosomes are composed of rRNA and ribosomal proteins • Ribosomes translate mRNA into polypeptide chains • Ribozyme is an RNA molecule that catalyzes a chemical reaction - Natural ribozymes catalyze their own cleavage or the cleavage of other RNAs - They also catalyze the aminotransferase reaction of the ribosome - Ribozyme consists of a conserved catalytic core motif which is required for trans-cleavage of a phosphodiester bond within an RNA target - Ribozyme-based cancer therapy uses specially designed ribozymes to knock down oncogene mRNA
(Figure 21) • Non-coding RNA - Many RNA genes encode RNA that is not translated into protein • The human nuclear genome contains about 3000 unique RNA genes (<10% of total gene number) • The human mitochondrial genome contains 24 RNA genes, including genes for 28S and 18S rRNA subunits of mitochondrial ribosomes. The other
24
Cleavage site mRNA 5'--NNNNNNNNUHNNNNNNNN - - 3'
!
I I I I I I I I I
I I I I II I I
3' NNNNNNNNA NNNNNNNN 5'
I
Substratebinding arms
A A
C
u
GA
G U C_G AG "C-G Catalytic G-C core G-C
A
AA
G
Fig. 21. The figure illustrates an mRNA (in red) and a ribozyme (in black) with secondary structures of substrate-binding arms, catalytic core, and cleavage site. A ribozyme is an RNA molecule that catalyzes a chemical reaction. Ribozymes function by binding to the target RNA and cleaving the phosphodiester backbone at a specific cutting site. Five classes of ribozymes have been described based on their unique characteristics in the sequences as well as by their threedimensional structures. The binding arms of a ribozyme include sequences complementary to the target RNA. Ribozymes recognize a target via interactions between the binding arms of the ribozyme and the mRNA. Ribozymes can cleave any RNA substrate that matches the binding sequence and contains an NUH triple, N is any nucleotide and H could be A, C, or U.
mitochondrial RNA (mtRNA) genes are for tRNA molecules - miRNA (see Chapter 7, MicroRNA section) • Double-stranded RNA (dsRNA) - dsRNA is RNA with two complementary strands - dsRNA forms the genetic material of some viruses - In eukaryotes, it acts as a trigger to initiate the process of RNA interference - dsRNA is an intermediate step in the formation of siRNAs
Principles of Clinical Molecular Biology
1-25
7' - methylguanosine I
5' - end of mRNA
I HO
OH
.M
H2N(:):N\ N
N
o
f
~H
r> r> r>
S'
:~2~CH20 I
(=0N
I
Purine base
Ribose
5' - 5' Triphosphate linkage 3
·0 CH 2
Pyrimidine base
N
o
Ribose
/NX:: ~. \
)
CH2 N N
t7 I I I
Purine base
Ribose
OH
I
Fig. 22. Capping adds a guanosine nucleotide to the 5' end of mRNA via a 5'-5' triphosphate linkage followed by guanosine methylation. The process of 5' capping is critical to create mature mRNA, which is then able to undergo translation. Capping stabilizes the mRNA in the process of protein synthesis so that truncated proteins are not produced by ribonuclease digestion of mRNA during protein synthesis. Capping is a highly regulated process occuring in the nucleus.
• Catalytic RNA - Catalytic RNA catalyses chemical reactions Catalytic RNA cuts and ligates other RNA molecules, for example when introns are spliced out of mRNA - Catalytic RNA can also catalyze peptide bond formation by the ribosome
- It is critical in regulation of mRNA export from the nucleus - It prevents the mRNA from degradation by exonucleases - It promotes protein translation - It promotes 5' intron excision
mRNA Processing
• Polyadenylation (see mRNA in types of RNA section)
• Capping is the process adding a guanosine nucleotide to the 5' end of mRNA via a 5'-5' triphosphate linkage followed by guanosine methylation (Figure 22)
• mRNA splicing is the process of removing introns from the primary transcript and joining the exons via an intermediate usually described as "lariat" structure (Figure 23)
25
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Molecular Genetic Pathology
A DNA Exon1
2
t
3
Transcript ion Primary Transcription Pre-mRNA
M~r
Intron Looping
RNA Splicing
mRNA Translation
t
Prote in
B I","",
5'
G A\ ?.-/O ?
O-P-O" 5'
b
3'
3'
---+ 3'
o-p- o
o-p-o \
O"-P-O
b
5'N
5'C\ ? 2/A
5,
5'
b-
? O"-P -O 0< b 3'
3'
5'
---+ 3'
5'
b-
0
+ 0" O- PI - 0
b
Fig. 23. RNA splicing is a modification of the molecule after transcription, in which introns of precursor messenger RNA (pre-mRNA) are removed and exons are joined. Splicing only occurs in eukaryotes. In panel A the exons are shown in colored boxes and introns are shown in lines. After transcription the introns form loops following the GU-AG rule, the loops are removed and the exons are joined to form mature mRNA. Panel B shows the two-step biochemical process of RNA splicing . Both steps involve transesterification reactions occur between approximated RNA nucleotides.
• The intron generally starts with GT and ends with AG - A"lariat" structure is formed by 5' G of the intron joining in a 2', 5'-phosphodiester bond to an adenos ine near the 3' end of the intron Two transe sterifications are needed for splicing to occur between RNA nucleotides, forming an excised intron loop (the "lariat" structure) and an exon-to-exon joined mRNA segment • First, the 2' OH of a specific branch-point nucleotide within the intron that is defined during spliceosome assembly performs a nucleophilic attack on the first nucleotide of the upstream intron at the 5' splice site forming the lariat intermediate
26
• Second , the 3' OH of the released 5' exon performs a nucleophilic attack at the last nucleotide of the intron at the 3' splice site, thus joining the exons and releasing the intron lariat • The function of the lariat structure is to form a stable hairpin , excise the intron and join the exons - Mutations that affect mRNA splicing include the 5' and 3' splicing sites • The intron generally starts with GT and ends with AG. If the initial G changes to A, the site is cut at the upstream intron but the reaction stops and ligation fails. A trunc ated mRNA molecule is the result of this error
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Principles of Clinical Molecular Biology
End of mRNA (3' end) ....
e
Start of mRNA
lIIIIIIIIII'~
(5',od)
-KJ--h--'r-~;-~~/
\
Incoming ribosomal subunit s
Growing polypeptide
Complete polypeptide
-
Fig. 24. Translation occurs in the cytoplasm where ribosomes are located . Translat ion is the process that converts an mRNA sequence into a chain of amino acids that form a protein . Translation proceeds in four phases: activation, initiation , elongation, and termination. The amino acid to be added is bonded by its carboxyl group to the 3' OH of the tRNA by an ester bond . Initiation involves the small subunit of the ribosome binding to 5' end of mRNA with the help of initiation factors and other proteins . Elongation occurs when the next aminoacyl-tRNA in line binds to the ribosome along with GTP and an elongation factor. Synthesis is terminated when the polypeptide meets a stop codon.
• If the 5'-2' sequence is incorrect, then 3' cutting does occur and the intron is not excised • Other mutations block the reaction altogether • Alternative splicing means that a primary mRNA transcript from one gene may be spliced at different locations within the sequence to produce different mature mRNA molecules and therefore to produce different proteins - Alternative splicing of pre-mRNAs is a powerful and versatile regulatory mechanism for quantitative control of gene expression and functional diversification of proteins - It contributes to major developmental decision s as well as to fine tuning of gene function • RNA editing describes the molecular process by which information content in mRNA is altered through a chemical change in the base makeup - U editing is achieved by deamination of cytosine to uracil. Through this editing process the codon CAA (Gly) is changed to UAA (stop codon) - A-I editing modifies adeno sine (A) to inosine (I), which serves in a codon as if it were guanine (G) during protein translation
Protein Translation • Protein translation occurs in the cytoplasm where ribosome s reside • The ribosome travels down the mRNA one codon at a time, and the amino acids are added one by one (Figure 24)
• During protein translation mRNA is read in the 5' ~3' direction until the ribosome reaches a stop codon - Protein translation begins at a start codon (AUG) - Protein translation ends at a stop codon (UGA, UAG. and UAA) - Protein is made from N-terminal (amino terminus) to C-terminal (carboxyl terminus) • Genetic code is the set of rules by which information encoded in genetic material (DNA or RNA sequences) is synthe sized into protein (amino acid sequences) in living cells • Protein translation relies on aminoacyl-tRNA that carries a specific amino acid and recognizes the corresponding codon in mRNA by anticodon base pairing - Ribosomes move along an mRNA molecule in the 5' ~3' direction, adding amino acid residues via aminoacyl-tRNA to the protein chain • The peptide bond is formed between two amino acid residues. The amino group from one amino acid reacts with the hydroxyl group of another amino acid at its carboxyl acidic end, releasing a molecule of water. This reaction is facilitated by a ribozyme-containing enzyme (Figure 25) • Post-translational modific ation is the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis for many proteins - Pronase may remove amino acids from the amino end of the protein
27
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Molecular Genetic Pathology
-
OH
0Y
OH
I
CH2
• Protein phosphorylation is the most important regulatory event for enzymes, receptors and signal transduction molecules
SH
I
CH2
• Many enzymes and receptors are switched "on" or "off' by phosphorylation and dephosphorylation
CH2
• Phosphorylation is catalyzed by various specific protein kinases, whereas dephosphorylation is catalyzed by phosphatases
7I 7 I '-------, 7 I H - N - C - C - N - C - C - : ~ : - N - C - C -OH I
H
II
0
I
H
II
0
~
~ _J
I
H
Phosphorylation adds phosphate groups to a protein
II
0
-
H20
Post-transcriptional modification may also involve addition of functional groups • The chemical nature of protein is changed by adding groups, such as acetate, lipid or carbohydrate
Fig. 25. A peptide bond is formed between two amino acids by the carboxyl group of one amino acid reacting with the amino group of the other amino acid, releasing a molecule of water (H 20 ). Polypeptides and proteins are chains of amino acid residues held together by peptide bonds.
• Protein structure is also changed by forming disulfide bridges between cystine or cysteine residues
MITOCHONDRIAL DNA
Overview
• mtDNA contains only a few non-coding intergenic regions
• mtDNA is a double-stranded circular DNA located in the mitochondrial matrix
• The size and number of mtDNA
mtDNA Inheritance • mtDNA is inherited entirely from the mother • Sperm carries the father's mtDNA in its tail, which is lost during fertilization • mtDNA inheritance is non-Mendelian, because Mendelian inheritance presumes that half the genetic material of an embryo derives from each parent • mtDNA passes unchanged from mother to offspring by this mechanism
-
Human mtDNA is 16,569 bp in length
-
Human cells typically contain thousands of copies of mtDNA, several copies per mitochondrion
Mitochondrial Genes and Gene Expression • mtDNA genes mtDNA encodes for 37 genes • There are 13 peptide coding genes, including the following peptides • Transcription factor A
Characteristics of mtDNA
• The mtRNA processing ribonuclease P
• The two strands of mtDNA have significantly different compositions from nuclear DNA and from one another (Table 4) - The heavy (H) strand is rich in purines (adenine and guanine) - The light (L) strand is rich in pyrimidines (thymine and cytosine)
• The transcription termination factor
• mtDNA is highly conserved, so it is useful for phylogenetic study • 80% of mtDNA encodes for functional mitochondrial proteins, and therefore most mtDNA mutations lead to functional anomalies • Mutations in nuclear DNA may also have a wide array of effects on mtDNA replication • mtDNA is devoid of introns
28
• The mitochondrial peptides are synthesized on mitochondrial ribosome • The heavy strand encodes the 2 rRNAs, 12 polypeptides, and 14 tRNAs • The light strand encodes 1 polypeptide, and 8 tRNAs • mtDNA expression - Transcription initiation sites of mtDNA • The promoters of Hand L strands (termed PH and PL ) are both located in the D-Ioop region and 150 bp apart (see mtDNA replication at the end of this section) (Figure 26) • Heavy-strand transcription starts at nucleotide 561 • Light-strand transcription starts at nucleotide 407
Principles of Clinical Molecular Biology
1-29
Table 4. The Differences Between Nuclear DNA and mtDNA Nuclear DNA
mtDNA
Location
Nuclear
Mitochondria matrix
Size
3200 Mb
16.6 kb
Structure
Antiparallel double helix, linear
Double strand, circular
lntron
Present
Absent
Non-coding sequence
Many non-coding sequences (98%)
Few non-coding sequences (7%)
Transcription
Monogenic
Multigenic
Copy number
One setpercell
Hundreds to thousand percell
Number percell
46
Several thousands
Associate proteins
Histone and non-histone
Largely protein free
Encoding genes
30,000
37
Transcription
Individual gene transcription
Bulk-transcription for whole strand
Codon
Universal codon
Mitochondrial codon (see Table 5)
Inheritance
Mendelian, from both parents
Maternal
• Transcription of mtDNA starts from the promoters in the D-Ioop region and continues in opposing directions for the two strands around the circle to generate large multigenic transcripts • Transcription initiation in mitochondria involves three types of proteins • The mtRNA polymerase (POLRMT) • Mitochondrial transcription factor A (TFAM) • Mitochondrial transcription factors BI and B2 (TFBIM, TFB2M) • POLRMT, TFAM, and TFBIM or TFB2M assemble at the mitochondrial promoters and begin transcription - Promoters of mitochondrial gene expression • Heavy strand I (HI) promoters initiate transcription of the entire heavy strand • Heavy strand 2 (H2) promoters initiate transcription of the two mitochondrialrRNAs • Light strand (L) promoter initiates transcripts of the entire light strand • Mitochondrial mRNA - Mitochondrial mRNAs are small molecules - Full-length transcripts are cut into functional tRNA, rRNA, and mRNA molecules - Mitochondrial mRNA lacks a 5' cap structure - Mitochondrial mRNAs lack both a 5' and a 3' UTR
- The first codon specifies N-formylmethionine and is located at or very near the 5' end • Regulation of mtDNA expression - mtDNA expression depends on a large number of proteins encoded by nuclear DNA - The regulatory proteins are synthesized in the cytosol and enter the mitochondria via specialized pores - mtDNA replication is regulated by only one regulatory region controlling both the heavy and the light strands • Post-transcriptional modification of mitochondrial mRNA - Mitochondrial mRNAs are processed by mitochondrial ribonuclease (mtRNase)cleavage of the transcript - The light strand may produce either short transcripts, which serve as primers for mtDNA replication, or a long transcript for peptide and tRNAproduction - The production of primer occurs by processing of light-strand transcripts with the mtRNAse P - The Hand L strand mRNA molecules are polyadenylated by a mitochondrial poly(A) polymerase, imported from the cytosol • Mitochondrial mRNA translation - The protein components necessary for mitochondrial translation, including ribosomal proteins, tRNA synthetases, ribonucleases, initiation, and elongation factors, are all encoded by nuclear genes
29
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Molecular Genetic Pathology
... ...
...
Fig. 26. mtDNA is DNA located in a mitochondrion. In human beings, 100% of the mtDNA contribution to an embryo is inherited from the mother. Human mtDNA is present at 100-10,000 copies per cell, with each circular molecule consisting of 16,569-bp coding for 37 genes: 13 peptides (red), 22 tRNAs (black), and two rRNAs (yellow). H strand encodes two ribosomal RNAs, 12 peptides, and 14 tRNAs, whereas L stand encodes one peptide and eight tRNAs. Each mtDNA consists of a heavy (H) strand, which is rich in adenines and guanines, and a light (L) strand, which is rich in thymine and cytosine. There are no introns in mtDNA. Transcription of H strand originates from two closely located promoters in the D-Ioop region, a triple-stranded structure, shown as PH and PL' The origin and direction of transcription of Hand L strands is shown by arrows at PH and PL' The mtRNA transcripts come from both heavy and light chains. The mitochondrial genes lack introns and are transcribed in full length then processed to the natural products. Origins 0H and 0L are the beginning points for replication of mtDNA.
- Mitochondrial protein synthesis and DNA replication are thus under nuclear regulatory control - Mitochondrial translation is bacteria-like both in its sensitivity to antibiotics that act on the ribosome, and in its use of N-form ylmethionyl-tRNA for initiation - Mitochondrial ribosomes are smaller than those found in the cytosol, and have a sedimentation coefficient of 55 S instead of the den ser 80 S sedimentation coefficient for cytosolic ribosomes or 70 S coefficient for bacterial ribosomes
mtDNA Replication • mtDNA replication is an asynchronou s process, which begins at the origin of the H strand mtDNA replication is controlled by chromosomes in the nucleu s based on how many mitochondria the particular cell needs at that time - When the replication apparatu s meets the origin of the L strand, it is forced into a single-strand configuration
30
by the extending daughter H strand, and L-strand replication begins at this point RNA derived from the L-strand promoter serves as a primer for H-strand DNA replicat ion • The D-Ioop (displacement loop) is a I I23-ba se stretch of DNA, often triple-stranded, which contains sites for DNA-binding protein s that control mtDNA replication and transcription - The D-Ioop contains the promote rs for both the H- and L-strand transcripts - mtDNA replication causes the D-Ioop to move along the heavy strand as mtDNA polymera se-y produce s a complimentary replica strand • Heavy-strand DNA replication begins at the D-Ioop and proceed s in a 5'-3' direction until returning to the origin of replication • DNA polymerase y begins in the reverse direction to produce a complimentary replica of the light
Principles of Clinical Molecular Biology
strand when replication of the heavy strand reaches the light strand replication origin (OL) • Two identical double-strand mtDNA molecules are the result of this process • When mitochondria have enough copies of mtDNA, sufficient mitochondrial proteins, and adequate surface area, a nuclear protein may permit the mitochondrion to divide by fission into two daughter mitochondria
mtDNA Damage, Mutations, and Repair • mtDNA damage - mtDNA is susceptible to insult by all the same processes that damage nuclear DNA - mtDNA is especially susceptible to insult by reactive oxygen species, which are prevalent in mitochondria • Because mtDNA is not bound to histones, it is exposed to damage caused by free oxygen radicals produced by electron transfer during oxidative phosphorylation of the respiratory chain • mtDNA also undergoes the same types of mutation as nuclear DNA including spontaneous modifications and replication errors • mtDNA mutations - The rate of mutation in mtDNA is calculated to be about 10 times greater than that of nuclear DNA - The mtDNA mutations may be either acquired or inherited - Several different mutations of mtDNA may present clinically as the same disease - Large deletions and duplications of mtDNA increase with age • This may account for some aging processes in oxygen-dependent organs, such as brain, kidney, muscle, and heart • Mutant electron transfer proteins may release more oxygen-free radicals into the mitochondrial matrix, accelerating the aging process in some cases of Alzheimer's and coronary artery disease - There are hypervariable segments (HVI and HV2) located at base 57-372 and base 16,024-16,383, respectively. The rate of mutation in these regions is significantly higher than in the rest of mtDNA • mtDNA repair - mtDNA does not code for any DNA repair proteins - Proteins from the cytosol under nuclear control enter the mitochondrion through specialized membrane pores - Recent evidence has suggested that mitochondria have enzymes to proofread mtDNA and fix mutations owing to free radicals - Evidence for nucleotide excision repair, direct damage reversal, mismatch repair, and recombinational repair mechanisms have also been found in mitochondria (see DNA Repair section)
1-31
- As with nuclear DNA repair, the ability of mitochondria to repair DNA damage declines with age
Mitochondrial Disease • Mitochondrial diseases result from failures of the mitochondrial specialized compartments for oxidative phosphorylation, which are present in every cell of the body except red blood cells - About one in 4000 children in the United States will develop a mitochondrial disease by the age of 10 years - 1()(){)-4000 children per year in the United Sates are born with some type of congenital mitochondrial disease - Mitochondrial diseases may either be observable at birth, or symptoms may not be seen until late adulthood - Heteroplasmy refers to a phenomenon in which the number of mutant versus wild-type mitochondria varies from cell to cell and from tissue to tissue When a tissue reaches a certain ratio of mutant to wild-type mitochondria, a disease becomes manifest - Mitochondrial disease may be caused either by mtDNA mutations (acquired or inherited) or by mutations in nuclear DNA coding for mitochondrial components - Types of mutations • Homoplasmic: similar distribution of mtDNA mutation in all tissues • Heteroplasmic: variable distribution of mtDNA mutation in different cells or tissues • Typical symptoms of mitochondrial disease include - Loss of muscle coordination, muscle weakness Neurologic problems, seizures Visual and/or hearing problems Developmental delays, learning disabilities Heart, liver, or kidney disease Gastrointestinal disorders and severe constipation Diabetes Increased risk of infection Thyroid and/or adrenal dysfunction Autonomic dysfunction Neuropsychologic changes characterized by confusion, disorientation, and memory loss • The diagnosis of mitochondrial disease is problematic There is no reliable and consistent means of diagnosis Evaluating the patient's family history is essential - Diagnosis may require one of the few physicians who specialize in mitochondrial disease - Diagnosis can be made by blood DNA testing and/or muscle biopsy but neither of these tests is completely reliable • Mitochondrial code is similar to the universal code with four exceptions highlighted in Table5
31
1-32
Molecular Genetic Pathology
Table 5. Genetic Code of mtDNA UUU Phe
UCU Ser
UAUTyr
UGUCys
UUC Phe
UCC Ser
UAC Tyr
UGC Cys
UUA Leu
UCA Ser
UAA STOP
UGA Trp (STOP)
UUGLeu
UCG Ser
UAG STOP
UGGTrp
CUU Leu
CCU Pro
CAU His
CGU Arg
CUC Leu
CCC Pro
CAC His
CGCArg
CUALeu
CCA Pro
GAAGln
CGAArg
CUGLeu
CCG Pro
CAGGln
CGGArg
AUU lie
ACUThr
AAU Asn
AGU Ser
AUC lie
ACCThr
AACAsn
AGC Ser
AUA Met (lie)
ACA Thr
AAA Lys
AGA STOP (Arg)
AUG Met
ACGThr
AAG Lys
AGG STOP (Arg)
GUUVal
GCUAla
GAU Asp
GGUGly
GUCVal
GCCAla
GACAsp
GGG Gly
GUA Val
GCAAla
GAAGlu
GGAGly
GUGVal
GCGAla
GAGGlu
GGGGiy
() indicatedthe universal code. Bold markedthe different mitochondrial codons.
SUGGESTED READING Anderson S, Bankier AT, Barrell BG. Sequence and organization of the human mitochondrial genome . Nature 1981;290:457--465.
Lewis JD, Tollervey D. Like attracts like: getting RNA processing together in the nucleus . Science 2000;288:1385-1388.
Blackburn EH. Structure and function of telomeres. Nature 1991;350: 569-573.
Lindahl T, Wood RD. Quality control by DNA repair. Science 1999;286: 1897-1905.
Brenner S, Jacob F, Meselson M. An unstable intermediate carrying information from genes to ribosomes for protein synthesis . Nature 1961;190:576-581 .
Moore MJ. From birth to death: the complex lives of eukaryotic mRNAs.
Science 2005;309:1514-1518.
Buratowski S. Mechanisms of gene activation. Science 1995;270:1773-1774.
Noller HF. RNA structure : reading the ribosome . Science 2005;309: 1310-1514.
Darnell JE Jr. Variety in the level of gene control in eukaryotic cells.
Qiu J. Epigenetics: unfinished symphony. Nature2006;441 :143-145.
Nature 1982;297:365-371. Eschenmoser A. Chemical etiology of nucleic acid structure . Science 1999;284:2118-2124. Felsenfeld G, Groudine M. Controlling the double helix. Nature 2003 ;421:448--452. Friedberg EC. DNA damage and repair. Nature 2003;421:436-440. Grunstein M. Histone acetylation in chromatin structure and transcription.
Nature 1997;389:349-352. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000 ;100:57-70. Horn PJ, Peterson CL. Molecular biology. Chromatin higher order folding-wrapping up transcription. Science 2002;297:1824-1827. Ionov Y, Peinado MA, Malkhosyan S, et al. Ubiquitous somatic mutations in simple repeated sequences reveals a new mechanism for colonic carcinogenesis. Nature 1993;363:558-561.
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Rajagopalan H, Lengauer C. Aneuploidy and cancer. Nature 2004 ;432:338-341. Redon R, Ishikawa S, Fitch KR, et al, Global variation in copy number in the human genome. Nature2006;444:444--454. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science 200 1;293:1089-1092. Rouse J, Jackson SP. Interfaces between the detection, signaling, and repair of DNA damage . Science 2002;297:547-551. Sharp PA. Splicing of messenger RNA precursors . Science 1987;235: 766-771. Watson JD, Crick FH. Molecular structure of nucleic acid; a structure for deoxyribose nucleic acid. Nature 1953;171:737-738. WoltTeAP, Matzke MA. Epigenetics: regulation through repression . Science 1999;286:481 ~86 .
2 Principles of Clinical Cytogenetics Stuart Schwartz,
PhD, FACMG
CONTENTS I. Historical Overview HistoricalAspects Chromosome Structure Methodology Cell Culture G-Banding Fluorescence In Situ Hybridization Indications for Cytogenetic Studies
II. Autosomal Abnormalities Types of Abnormalities Polyploidy Triploidy Tetraploidy Aneuploidy Trisomy 21 Trisomy 18 Trisomy 13 Aneuploidy-Causes Non-Disjunction
III. Structural RearrangementsIntrachromosomal Intrachromosomal Rearrangements Deletions Ring Chromosomes Isochromosomes Insertions Duplications Inversions
2-2 2-2 2-2 2-2 2-2 2-4 2-5 2-9
2-9 2-9 2-9 2-9 2-10 2-10 2-1 0 2-11 2-11 2-12 2-13
IV. Structural RearrangementsInterchromosomal Interchromosomal Rearrangements Reciprocal Translocations Robertsonian Translocations
V. Contiguous Gene Syndromes Definition Microdeletions Prader-Willi Syndrome Angelman Syndrome MiIler-Dieker Syndrome Velocardiofacial Syndrome Langer-Gideon Syndrome Aniridia-Wilms TumorAssociation Smith-Magenis Syndrome William Syndrome Rare Deletions Duplication Syndromes
VI. Sex Chromosome Aberrations 2-14 2-14 2-14 2-16 2-16 2-17 2-17 2-18
Sex Determination Pseudoautosomal Region Sex Chromosome Abnormalities Turner Syndrome Klinefelter Syndrome Lyon Hypothesis Significance
VII. Suggested Reading
2-19 2-19 2-19 2-19
2-22 2-22 2-22 2-23 2-23 2-23 2-23 2-24 2-24 2-24 2-24 2-24 2-25
2-25 2-25 2-26 2-27 2-27 2-28 2-30 2-31
2-31
33
2-2
Molecular Genetic Pathology
HISTORICAL OVERVIEW
Historical Aspects • Ages of cytogenetics - I-Dark ages <1952 - II-Hypotonic 1952-1958 - III-Trisomy 1959-1969
Telomere
Centromere -
]
Short Arm (p arm)
- IV-Banding era 1970-1976 - V-High resolution 1976-1988 - VI-Molecular cytogenetics 1988 till present • I92 I-Painter (48 chromosomes) - Chromosome number stayed at 48 chromosomes from 1921 to 1956 • 1959-1960 Trisomies found for a number of chromosomal syndromes - I959-Lejeune et al.-Down syndrome -
I959-Ford et al.-Turner syndrome 1959-Jacobs and Strong-47,XXY
-
1959-Jacobs et al.-47,XXX I96Q-Patau et al.-trisomy 13
-
196Q-Edwards et al.-trisomy 18 196Q-Nowell and Hungerford-Phi
• Lymphocyte cultures developed in 1960 - Short-term culture technique developed-lymphocyte cultures - Effective due to phytoheamaglutinin • Stimulates cell in Go to undergo cell division - Previously cytogenetic analysis done on either fibroblasts or bone marrow • Chromosome banding - Prior to I97Q-everything solid stained - 197Q-Caspersson developed quinacrine banding - 1976-Yunis-initiated high-resolution studies - I988-Pinkel/Ward-independently introduce fluorescence in situ hybridization (FISH) • Multiple tissues can be used for chromosomal analysis - Lymphocyte cultures - Bone marrow - Solid tumors - Fibroblasts Amniotic fluid - Chorionic villi
Chromosome Structure • A chromosome consists of a primary constriction (centromere) connected to both a short arm (p arm) and a long arm (q arm) (Figure 1)
34
Long Arm (q arm)
Telomere Fig. I. An image showing both chromosome structure and morphology. • Based on the placement of the centromere the chromosome could be one of the following - Metacentric - Submetacentric - Acrocentric • An acrocentric chromosome has both satellites and stalks on its short arm • Chromosomes can be classified into seven groups - Group A (l-3)-metacentric - Group B (4,5)-submetacentric - Group C (6,7,8,II,X)-metacentric (9,10,12)submetacentric - Group D (l3-15)-acrocentric - Group E (16)-metacentric (17,18)-submetacentric - Group F (l9,20)-metacentric - Group G (21,22,Y)-acrocentric • A karyotype is the particular chromosome complement of an individual as defined by the number and morphology of their chromosomes
Methodology Cell Culture • Chromosome cultures - An understanding of the cultures for chromosome analysis is based on a good knowledge of both the cell cycle and mitosis Cell cycle (Figure 2) • This is the period between successive mitosis that lasts between 16 and 24 hours • During interphase the chromosomes are thin and extended
Principles of Clinical Cytogenetics
2-3
• Each chromo some become s attached to a centriole by microtubule s • The chromosomes are easily visible, maximally contracted, and resembles an X in its configuration • Anapha se • The centromere of each chromosome divides longitudinally • Two daughters separate to opposite poles • Telophase • Chromatids-independent chromosomes • Two groups of daughter chromosomes enveloped in a new nuclear membrane • Cell cytoplasm also separates • Two new daughter cells - Suspension culture s • In most cases for routine analysis peripheral blood culture is used as this is the most readily available Fig. 2. A diagram of the cell cycle illustrating the G l ' S, and Gz phases along with the stages of mitosis. • DNA replication occurs and chromatid is replicated to become two chromatids • The chromosomes begin to conden se in preparation for the next mitotic division - Mitosis (Figure 3) • This is the proces s of somatic cell division during which the nucleus divides • Each chromosome divides into two daughter cells • One chromosome segregates into each of two daughter cells • The number of chromosomes in the nucleus remain s unchanged • Mitosis consist s of five major steps and lasts for about 1-2 hours • Interphase • The chromosome s begin to conden se • Mitotic spindle begins to form • Two centrioles form • Microtubules radiate and move toward opposite poles • Prometaphase • Nuclear membrane disintegrates • Chromosomes spread around cell • Each become s attached at its centromere to a microtubule of the mitotic spindle • Metaphase • The chromosome become s oriented along the equatorial plate
• This can be obtained by venipuncture, finger stick, or heal stick • Blood must be collected in an anticoagulant (e.g. heparin ) • Blood can be kept at room temperature or at 4°C and set up in culture several days later if transport of the sample is slow • Blood cannot be frozen • The blood can either be set up by macromethods where the white blood is separated out or it can be set up as whole blood by micromethods • The white blood cells are stimulated to go from Go to G[ by the use of phytohemagglutinin, which is a T-cell antigen • Cells are harvested to obtain metaphase spreads by utilizing colcemid or colchincine • Colcemid is a mitotic inhibitor • It prevents formation of mitotic spindles and prevents cells from entering anaphase • During the harvesting protocol the cells are treated with a hypotonic solution (usually NaCI) • With the spindle apparatus gone, the chromo somes are held together by cytoplasmic membrane s • The hypotonic solution causes the cells to swell and disperse the chromosomes due to a concentration gradient between the cytoplasm and hypotonic solution • The final step of the harvesting procedure is to treat the cell s with a fixative, usually 3: I methanol :acetic acid • This treatment removes water from the cells • It enhance s the morphology of the chromosomes and its ability to pick up stain
35
2-4
Molecular Genetic Pathology
Interphase
Prophase
46 Chromosomes
Chromosomes doubled (92)
Prometaphase
Nucleus dissolve and microtubules attach to centromeres
Metaphase
Chromosomes align on metaphase plate
Anaphase
Chromosomes pulled apart
Telophase
Cell division begins
Cytokinesis
Two daughter cells formed each with 46 chromosomes
Fig. 3. An illustration of the stages of mitosis showing the movement of chromosomes.
G-Banding • Chromosome banding
• It can be accomplished by either pretreatment by a proteolytic enzyme (such as trypsin) or chemical (e.g., acetic saline solution)
- Allowed each chromosome to be individually identified • Ideograms (diagrammatic representation) of each chromosome allows identification of each region and subregion of the chromosome - G-banding • This is the most common type of permanent stain, making discrimination of bands easy
36
- Q-banding • Initial banding, described by T. Caspers son • Researcher at Karolinska Institute (Sweden) and consultant to Cancer Research Foundation in Boston • Authority on fluorescence and interferometry
Principles of Clinical Cytogenetics
• Used alkylating agent to fluoresce molecule, might cross-link guanine • Fluorescent bands visible after staining with quinacrine mustard, quinacrine dihydrochloride, or similar compound • The occurrence of bands related to AT region s, which show brightness due to fluorochromes, which intercalates in DNA • Increased fluore scence with runs of AT bases • Protein s can modify fluorescence • GC-rich regions-quenches fluorescence • G-dark and Q-bright band s similar - R-banding (reverse) • This banding is the reverse of G- and Q-banding • Staining is usually accompli shed by denaturation by heat - C-banding • This banding stains constitutive heterochromatin; therefore it stains the centromeric and pericentromeric regions of chromosomes
Fluorescence In Situ Hybridization • General information - FISH • AlIows for the identification of sequences (unique or repetitive ) on metaphase or interphase chromosomes - Utilization of FISH • Identification of deletions • Identification of marker chromosomes • • • • • • • • •
Identification of duplications Identification of subtle translocations Characterization of chromosome structure Phenotypelkaryotype correlations Interphase cytogenetics Characterization of rearrangements Quantification of mosaicism Gene mapping Replication analysis
• Evolutionary studies • Material s for FISH - DNA FISH probe s • Chromo some- specific sequences (library-paint) (Figure 4) • Identification of translocations, markers , and duplications • DNA-repetitive sequences (a -, p-, classical satellite or telomeres) • Centromeric probe s-identification of aneuploidy and marker chromosomes
2-5
• Telomeric probes-identification of deletion s and cryptic translocations • Unique sequences---cosmids, YACs (Yeast Artificial Chromo somes), BACs (Bacterial Artificial Chromosomes), fosmid clone s • Microdeletion probes-identification of specific syndromes (Figure 5) • Single-copy probes-e-derection of present or absence of specific DNA • Fusion probes-i-detection of specific translocations in leukemia • Labeled by nick translation or random priming or polymerase chain reaction • Types of celIs that can be used for FISH - Lymphocytes - Bone marrow - Amniocytes (cultured or uncultured) - Chorionic villus material - Fibroblasts - Blood smear - Buccal smear - Paraffin sections - FISH analysis can be done on either metapha se chromosomes or interphase celIs • Advantages of metapha se analysis - Can visualize all of the chromosomes - Can characterize position of signal on chromo somes - "Gold standard" of analysis • Drawbacks of metapha se analysis - Often limited number of spreads available - Morphology may be compromised and affect hybridization - Requires metaphases to be present • Advantages of interpha se analysis (Figure 6) - Increased number of cells to examine - AlIows for investigation of nuclear organization - Can save what might have been a failure due to lack of metaphases - Replication studies - AlIows study of non-dividing celIs • Disadvantages of interphase analysis - Cannot appreciate chromosomes - Uncertainty in the form of inefficient hybridization - Limited in the number of probes/experiment • FISH protocol - Slide with unbanded cells fixed - DNA probe labeled - Most are now directly labeled with fluorophore - Cot-l DNA suppres sion of extraneous DNA - Slide dehydrated in ethanol
37
2-6
Molecular Genetic Pathology
Fig. 4. Picture of FISH with a chromosome 14 paint demonstrating extra material on a derivative 17.
Fig. 5. Picture of FISH with a TUPLEl probe elucidating a microdeletion of 22ql1.21.
38
2-7
Principles of Cl inical Cytogenetics
Fig. 6. Picture of interphase FISH with locus specific probes on chromosomes 13 and 21 demonstrating three signals indicating the presence of trisomy 21.
- DNA target dehydrated with heat and formam ide
- Comparative genomic hybridization
- DNA probed denatured
• Control DNA is labeled red
- Cold ethanol used to "fix" single strands
• Test DNA is labeled green
- Probe applied to slide - Cover slip and seal slide
• They are equally mixed and hybridized to normal chromosomes • The red:green ratio is analyzed by computer software and detects gains and/or losses of material from the test DNA
- Slide and probe hybridized overnight (4-20 hours) at 37°C - Excess probe washed off - Slide counterstained with DAPI (4' ,6-diamidino-2phenylindole) or propidium iodide - Visualized with fluorescent microscope - Image captured • Other FISH technologies
- Subtelomeric probes-detection of abnorm al phenotype (Figure 8) • Increase in causes of mental retardation detected with subtelomeric probes
- Fiber FISH
• Associated with loss of subtelomeric/telomeric segments
- Primed in situ labeling (PRINS)
• Cryptic telomeric rearrangements detected
- Reverse painting
• Individuals with unexplained MR (mental retardation) (3%) explained by use of telomeric probes
- Spectral karyotyping or M-FISH (Figure 7) • Combinatorially or ratio-labeled probes
- BAC arrays
• Are used to create a distinct "color" for each chromosome • Chromosomes are studied simultaneously • Computer software detects the probes • Pseudocolors the chromosomes for analysi s
• Utilizes BACs produced during human genome project
• Especially useful for complex rearrangements
• Arrayed on filter (1 Mb or less interval)
• Next logic step in cytogenetics? • Combines current technology with genomic studies
39
2-8
Molecu lar Genetic Pathology
Fig. 7. Picture of m-FISH demonstrating that the extramaterial on 15p originated from a Y chromosome.
Fig. 8. Picture of FISH with telomere locus probes for chromosome 18 (p arm telomere-green; q arm telomere-red) demonstrating the a deletion in 18q.
40
Principles of Clinical Cytogenetics
• Detected by comparative genomic hybridization • Can detect very subtle changes • Also can be studied using quantitative SNP array
Indications for Cytogenetic Studies • Why are chromosomes studied ? - There are a number of indications with respect to why chromosomes are studied • Prenatal diagnosis • Confirmation or exclusion of chromosomal syndrome (e.g., trisomy 21) • Unexplained psychomotor retardation with/without dysmorphic features
2-9
• Monogenic disorders associated with mental retardation and/or dysmorphic features • Abnormalities of sexual differentiation and development • Infertility • Recurrent miscarriages or stillbirths • Parents • Fetus • Neoplastic conditions • Leukemia • Lymphoma • Solid tumors
AUTOSOMAL ABNORMALITIES Types of Abnormalities
Polyploidy
• Numerical - Polyploidy
• Numerical changes-polyploidy - Haploid (n)
- Aneuploidy • Interchromosomal-abnormalities involving more than one chromosome - Reciprocal translocations - Robertsonian translocations • Intrachromosomal - Deletion - Inversion - Ring - Duplication • Numerical abnormalities - Polyploidy • Multiple of the haploid number (n) - Aneuploidy • Not a multiple of the haploid number • Loss or gain of particular chromosome - Mixoploidy • Two or more cell lines which differ in chromosome number - Mosaic • Two or more different cell lines derived from a single zygote - Chimera • Two or more cell lines that originate from different zygotes
- Diploid (2n)
Triploidy • Triploid (3n) • 2/3 of human triploids arise by fertilization of a single egg by two sperm • Fertilization is between a normal haploid gamete and a diploid gamete • Involvement of a diploid sperm most common occurrence • 20% of spontaneously aborted fetuses are triploid • Phenotypic features - Voluminous placenta • Hydatidiform changes - Craniofacial dysmorphology • Deformed skull • Ocular anomalies • Palatal abnormalities • Severe micrognathia • Low-set poorly folded ears - Neck, thorax, and abdomen • Diastasis recti • Omphalocele (occasional) - Limbs are usually deformed • Syndactyly-toes and fingers
41
2-10
Molecular Genetic Pathology
• Phenotypic features - Newborn period • Hypotonia • Sleepy • Excess nuchal skin
2
4
3
5
- Craniofacial abnormalities • Brachycephaly
6
13
• Epicanthal folds
7
.8
14
15
10
9
~
16
,. <>
19
20
21
11
12
• Upward slanting palpebral fissures
"17
18
• Small ears Limbs abnormalities
X
Y
• Single palmer crease
•
22
• Protruding tongue
• Small middle phalanx of the fifth finger Fig. 9. G-banding karyotype demonstrating trisomy 21.
• Wide gap between first and second toe - Cardiac abnormalities • Atrial and ventricular septal defect
- Often severe malformations are seen • Cerebral malformations, including holoprosencephaly - Cardiac, digestive, kidney, and internal genitalia malformations are often seen
• Common atrioventricular canal • Patent ductus arterio sus - Other abnormalities • Anal atresia • Duodenal atresia
Tetraploidy
• Short stature
• Tetraploidy (4n) • Tetraploidy is most often due to the failure of the first cell division of a zygote
• Strabismus
• 2-3 % of all fertilized eggs are polyploid - Majority of these are spontaneously lost • 6% of spontaneously aborted fetuses are tetraploid • Some polyploidy pregnancies do result in livebirths ; these die very early during the first few hours or days
Aneuploidy • Numerical changes-aneuploidy Aneuplo id changes • Nullosomic (2n - 2) • Monosomic (2n - I) • Trisomic (2n + I) • Tetrasomic (2n + 2) • Double trisomy (2n + I + I)
Trisomy 21 • • • •
42
Trisomy 21-Down syndrome (Figure 9) Frequency, at birth, is approximately 1/650-1/700 The sex ratio is approximately 3 males/2 females The most frequent cause leading to trisomy 21 is advanced maternal age
• Natural history of trisomy 21 - Broad range of intellectual ability • IQ between 25 and 75 Social skills • Well advanced skills • Happy and very affectionate Adult height , approximately 150 em (4 ft II in.) Life expectancy-good (-60 years) • Except for severe cardiac anomaly • Early death in 10-20% of the cases • Life expectancy approximately 9 years in 1930 40% of the individuals have significant heart defect • Atrioventricular canal defects - 5% of the individual s have serious gastrointestinal anomalies • Duodenal stenosis is the most common - Children with Down syndrome have a 15-20 fold increase risk of leukemia • Overall this is a frequency of approximately 1% - Most affected adults develop Alzheimer disease • All >35 years old develop it
Principles of Clinical Cytogenetics
• Overlap of phenotypic feature s with the general population - Most features in trisomy 21 can be found in individual s in the general population - The phenotypic feature s are especially important when all of the features are grouped together • For example-single palmer crease
• Limbs abnormalities • Clenched hands • Overlapping fingers • Absent distal flexion creases • Rocker bottom feet • Genitalia abnormalities
• In 50 % of the cases of Down syndrome
• Cryptorchidism
• Seen in 2-3%-general population
• Clitoral hypotrophy
• Trisomy 21-chromosome findings - 95% are due to an extra free trisomy 21 (resulting from a meiotic error) - 3-4% result from a Robert sonian translocation -
2-11
1-2% result from mosaicism (resulting from a mitotic error)
• Trisomy 21-recurrence risk - This risk is related to maternal age • Approximately 11100-1/200
Trisomy 18 • Trisomy IS-Edward syndrome - The frequency of this syndrome is about 115000-I/SOOO - The sex ratio at birth is approximately 4 femalell male - Overall survival of affected individuals • 30% die within first month • 50% die within 2 months • 90% die within I year - Phenotypic information • General features • Many pregnancies demonstrate postmaturity with delivery at 42 weeks • Low birthweight with severe growth retardation • Hypoplasia of skeletal muscle • Newborns can be wither hypotonic or hypertonic • Affected individual s have severe mental retardation • Craniofacial dysmorphology • Dolichocephaly • Protuberant occiput • Protuberant nose • Short palpebral fissures • Low set malformed ears • Micrognathia • Neck, thorax, and abdomen abnormalities • Short neck, exces s skin • Short sternum • Narrow pelvis
• Hypoplasia of the labia majora • Other malformations are seen in >95% of patients including : • Cardiac malformations o This is often responsible for death • Gastrointestinal malformations • Renal malformations
Trisomy 13 • Trisomy 13-Patau syndrome - The frequency of this syndrome is about 1/10000-1115000 Overall survival of affected individuals • 30% die within first month • 50% die within 2 months • 90% die within I year - Phenotypic information • General features • Newborns have failure to thrive • Seizures • Newborns are usually hypotonic • Affected individuals have severe mental retardation • Craniofacial dysmorphology • Holoprosencephaly • Microcephaly • Micropthalmia • Iris colobomata • Cleft lip andlor palate • Hemangiomas • Neck, thorax, and abdomen abnormalities • The last rib is either hypoplastic or absent • Limbs abnormalities • Hexadactyly • Rocker bottom feet • Genitalia abnormalities • Cryptorchidism • Scrotal abnormalities
43
2-12
Molecular Genetic Pathology
Meiosis
Reduction Division
Similar to Mitotic Division
I
I
\
MI
I
Mil
Each gamete has half the normal number (n=23) of chromosomes after meiosis
Fig. 10. A diagram of normal meiosis demonstrating normal segregation of chromosomes. • Clitoral hypertrophy • Bicornuate uterus and double vagina • Other malformations seen • Cardiac malformations • Digestive malformations • Ocular malformations o Micropthalmia o
Anopthalmia
• Visceral malformations • Cerebral malformations o Holoprosencephaly • Urinary malformations o Polycystic kidneys
Aneuploidy-Causes • Causes of aneuploidy - Non-disjunction • Can be either meiotic or mitotic
44
• Failure of paired chromosomes to separate (disjoin) at meiosis I • Failure of paired sister chromatids to disjoin at meiosis II or mitosis • Conjoined chromosomes/chromatids migrate to one pole • The other pole has no chromosome - Anaphase lag • Failure of incorporation of a chromosome into one of the daughter nuclei following cell division • Occurs due to delayed movement of the chromosome during anaphase and chromosome is subsequently lost - Meiosis (Figure 10) • This is the process where the diploid count is halved • From 46 to 23 chromosomes (and becomes haploid) • Occurs only at the final division of gamete maturation • This is a two-step process and involves two cell divisions
Principles of Clinical Cytogenetics
- Meio sis I • Referred to as the stage of reduction division, because the chromosome number is halved • Propha se I • Homologous chromosomes pair • Crossing over (recombination) occurs between non-sister chromatids • Prophase I is relatively lengthyconsisting of five stages o
Leptotene
o
Zygotene
o Synaptonemal complexes are formed o
Pachytene
o Pairs of homologous chromosomes o Bivalents formed o Crossing over occur s o
Diplotene
o Chromosomes separate o The chromosomes are attached by chiasma o
Diakenesis
o Separation of the chromo somes proceed s • Metaphase I • Chromosomes are attached to spindle • Anaphase I • Chromosomes separate and go to opposite poles • Telophase I • Two new daughter cells are formed - Meio sis II • This is essentially similar to mitotic division • Each chromosome (pair of chromatids) • Becomes aligned along equatorial plate • Forms two new daughter gametes • What are the consequences of meiosis? - Two major objecti ves are achieved • The diploid number of chromosomes is halved • Haploid • Meiosis provides extraordinary potential for generat ing genetic diversity • Compari son between mitosis and meiosis - Location • Mitosis: all tissues • Meiosis: only in testis and ovary - Product s • Mitosis: diplo id somatic cells • Meiosis: haploid sperm and egg cells - DNA replication and cell division • Mitosis : normally one round of replication per cell division
2-13
• Meiosis: only one round of replication (in meiosis I); but two cell divisions - Length in prophase • Mitosis: short (- 30 minutes in human cells) • Meiosis: long and complex in meiosis I; can take years to complete - Pairing of homolog s • Mitosis: none • Meiosis: yes (in meiosis I) - Recombination • Mitosis: rare and abnorm al • Meiosis: normally at least once for each pair of homologs - Relationship between daughter cells • Mitosis : genetically identical • Meiosis: different (recombination and independent assortment of homolog s) • What are the causes of anueploid? - Anueploidy results from non-disjunction • Failure of chromo somes to separate normally during cell division • In either meiosis or mitosis - Parental origin of non-disjunction • Origin of non-disjunction determined by parental polymorphisms • Chromosomal heteromorphisms have been used • DNA markers used now o For example, microsatellite markers • Origin of non-disjunction • Trisomy 21 o Maternal-88% of time o Paternal-8% o Mitotic-3 % • Trisomy 13 o Maternal-95 % o Paternal-5 % • Trisomy 18 o Maternal-89% o Paternal-O% o Mitotic-II %
Non-Disjunction (Figure 11) • Two major causes of non-di sjunction - Advanced maternal age • Primary oocyte can remain in a state of suspended inactivity for up to 50 years • Stays in dictyotene stage • Well-documented association between advanced maternal age and non-di sjunction • No association with advanced paternal age
45
Molecular Genetic Pathology
2-14
Nondisjunction Meiosis II
Nondisjunction Meiosis I
@ / @ iill Errorhere
~
(])
/ \ Trisomy
/
Meiosis I
/ \
Meiosis II
CD Trisomy
Monosomy
Monosomy
Normal
Normal
Trisomy
Trisomy
Chromosome Constitution of Zygote after Fertilization . Fig. II. An example of non-disjunction resulting in monosomic, euploid, and trisomic gamete s. - Altered recombination
10,------------------,
- Association of advanced maternal age and increased risk of non-disjunction (Figure 12)
"'Ol
€E
.-
0 '0
CD~
Ole:
>>.
Maternal age
Risk of non-disjunction
:J (/) 5 -e:
~~
riio
20 year old
1/1500
25 year old
1/1350
30 year old
11900
35 year old
11400
40 year old
1/100
45 year old
1/30
~.J::
Q):!:
a.::
o 25
30
40
35
45
Age
Fig. 12. A maternal age curve showing the increase in the percent of livebirths with Down syndrome with increasing maternal age.
STRUCTURAL REARRANGEMENTS-I NTRACHROMOSOMAL • Interchromosomal - Reciprocal translocations
- Inversion - Dicentric, acentri c
- Robertsonian translocations • Intrachromosomal (Figure 13) -
46
Deletion Ring Isochromosome Duplication Insertion
Intrachromosomal Rearrangements Deletions (Figure 14) • Involves loss of part of a chromosome Results in monosomy of that segment of the chromosome - Large deletions will be incompatible with survival to term
Principles of Clinical Cytogenetics
2-15
Terminal Deletion
-
-
Interstitial Deletion
Duplication
Ring
Isochromosome
Fig. 13. Diagrammatic representation of several different structural abnormalities including a terminal deletion, inter stitial deletion, duplication, isochromosome, and ring .
-
Any deletion of a loss of >2 % of the haploid genome will usually be lethal
• Types of deletions (I) - Deletions visualized under the microscope • Wolf-Hirschhorn syndrome • Cri-du-Chat syndrome - Microdeletions, contiguous gene deletions • Prader-Willi syndrome • Velocardiofacial syndrome • Types of deletions (II) - Terminal deletion • Single break • Acentric terminal fragment lost
-
Del(4p), del(5p), del(9p), del(ll p), del(ll q), del(l3q), del(l8p), and del(l8q)
-
Wide variability in phenotypes
5p Deletion • Deletion (5p)-general - Cri-du-Chat syndrome Delet ion-short arm of chromosome 5 (pI4pI5) -
- First reported-Lejeune et al. in 1963 • Deletion (5p)-phenotype - General • Cry-mewing of a kitten • Craniofacial dysmorphism
• Telomeres? • May be reattached or reformed
• Microcephaly • Moon-like face
- Interstitial deletion
• Hypertelorism
• Two breaks • Telomere retained • Interstitial acentric fragment lost • Deletions-phenotypes - Deletions seen in most chromosome arms - Certain deletions are seen more frequently
Frequency-1I45,OOO-1I50,OOO
• Micrognathia • Malformations rare -
Larynx • Laryngealmalasia, laryngeal stridor • Distinctive cry
47
Molecular Genetic Pathology
2-16
• Defects in closure of scalp • Cleft lip and/or palate • Coloboma • Cardiac defects (SO%) -
Mental retardation • Very pronounced • IQ <20
- Cytogenetics finding s
• De novo deletion-90% of cases • Parental mosaicism or translocation •
10% of case s
• Deletions-nomenclature - Interstitial deletion: • 46,XX ,del(S)(p 14p IS) - Terminal deletion: • 46,XX ,del(S)(pI4)
Ring Chromosomes • Breaks occur in each arm of the chromosome • Two "sticky" end s • Reunites as a ring Normal chromosome
Deleted chromosome
• Two distal chromosome fragments lost • Have been found for all human chromosomes
Fig. 14. Diagrammatic repre sentation of an interstitial deletion. -
Mental retardation
• Ring s--effects - If autosomal • Effect s can be serious
• Usually severe • IQ often <20 • Frequency of disorder higher among individuals with mental retardation (I.S/ I000)
• Depends on amount of material deleted • Difficult to make phenotype/karyotype correlations - Sex chromosome • reX), r(Y)
4p Deletion • Deletion (4p)- general Wolf-Hirschhorn syndrome
-
- Often unable to complete mitotic division s
Deletion-short arm of chromosome 4 (p 16)
-
Leads to mosaici sm
Frequency-1I4S,00D-lIS0,000
-
Loss of ring
First reported-Wolf et al. in 1965
-
Double rings
-
Extra ring s
• Delet ion (4p)- phenotype - General • Severe growth retardation • Severe mental retardation - Craniofacial dysmorphism • Microcephaly • "Greek warrior helmet" - Genitalia abnormalities (male and female) - Malformations
48
• Rings-s-cytogenetics
• Rings-nomenclature - 46,xx,r(4)(p 14q34)
Isochromosomes • Loss of one arm with dupli cation of the other arm • Isochromosome-formation - Originally thought to be due to centromere misdivision • Centromeredividestransversely ratherthan longitudinally
2-17
Principles of Clinical Cytogenetics
Regions inserted
Regions to be inserted
Normal chromosome (7)
Chromosomes after insertion
(18) Fig. 15. Diagrammatic representation of a balanced insertion .
- More recent studies • Most breakpoints not in the centromere
• Shift Either inserted straight or inverted
• FISH o
Dicentric-two centromeres present
• Isochromosome-phenotype - For most (but not all) autosomal chromosomes • Not viable • i(l8q)-viable • Monosomy and trisomy for either chromosome arm - i(9p), i(18p), i( 18q), and i(12p) • These are viable when accessory chromosomes - Sex chromosomes • i(Xq)-see 2-27 • Isochromosome-nomenclature - 46,XX,i( 18)(pII)
Insertions (Figure 15) • Insertions involve three breaks - Two breaks to delete segments - One to reinsert segment - Insertion involves transfer to another chromosome • Will discuss with translocations - When in the same chromosome
Duplications (Figure 16) • Can be de novo or familial - Familial • Due to abnormal segregation of familial balanced rearrangement • Most duplications are due to abnormal segregation of parental translocation, insertion, or inversion - De novo-many types • Tandem • Inverted tandem • Isochromosome • Accessory chromosome
Inversions (Figure 17) • Two break rearrangement involving a single chromosome in which a segment is reversed in position (i.e., inverted) • Two types - Paracentric
49
Molecular Genetic Pathology
2-18
Duplication Regions to be duplicated """'\----'
Paracentric Inversion
NormalChromosome 7
Pencentric Inversion
Fig. 17. Diagrammatic representation of both a paracentric and a pericentric inversion.
Segregation • Inversion-segregation
Normalchromosome
Duplicated chromosome
- During meiosis inverted chromosome will pair by forming an inversion loop • If crossing over occurs in inversion loop
Fig. 16. Diagrammatic representation of a duplication.
• Unbalanced gametes • Lead to duplications and deletions - Pericentic inversion-segregation • Small inversion
- Pericentric • Frequency: 1/100-1/1000
• Less likely for crossing over to occur • Greater degree of imbalance if crossing over
• Inversions-types - Pericentric
• More likely to spontaneously abort
• Involves both chromosome arms and includes the centromere - Paracentric • Involves only one of the chromosome arms and does not include the centromere - Inversions-phenotype • Balanced rearrangement which rarely causes problems in carriers
• Large inversion • More likely for crossing over to occur • If crossing over occurs-smaller imbalance • More likely for livebirth - Pericentric inversion-risk • Balanced pericentric inversion
• 5-10% risk for having a liveborn child with a viable imbalance
• Unless breakpoint is in an important functional gene • Can lead to significant chromosome imbalance in offspring with important clinical consequences
o
•
If ascertained due to a previous abnormal child
1% risk If ascertained due to a history of multiple spontaneous abortions
o
STRUCTURAL REARRANGEMENTS-I NTERCHROMOSOMAL
Interchromosomal Rearrangements Reciprocal Translocations • Definition - Frequency-lIIOOO livebirths - Involves two chromosomes
50
• An interchromosomal rearrangement - Involves breaking and exchanging segments - New morphologically different, but recognizable rearrangement of genetic material - Different types of translocations
2-19
Principles of Clinical Cytogenetics
Translocation Formation
• Ascertainment dependent • Previous child with unbalanced translocation
• Formation - Mutual breakage and exchange of material
o
- Exchange may not be mutual
• Previous multiple sabs
- Terminal translocation
o
• Most common type of translocation involving terminal regions • Less common type of rearrangement involving exchange of interstitial regions inserted into chromosomes - Cryptic translocation • Translocation not seen with typical banding analysis • Usually need FISH to visualize • Inheritance - Familial
- De novo
Translocation Segregation • Meiotic segregation - Translocations do not disjoin as normal homologous pairs of chromosomes - Forms a quadrivalent in pachytene stage of meiosis • Chiasma keeps the configuration together during meiosis • Types of segregation Alternate segregation
-3-4% risk
• Other ascertainment o
- Insertional translocation
-20% risk
-7%risk
• Unbalanced segregants-risks (II) - If ascertainment is because of multiple sabslower risk • Larger translocation exchange segments involved • Unbalanced segregants usually not viable If ascertainement because of a previous unbalanced child-higher risk • Smaller translocation exchange segments involved • Increased viability of unbalanced segments • Translocation-types - Most translocations not similar - Leads to difficulties in counseling Usually use overall group risks based on ascertainment One exception: • t(11;22)(q23;q 11.2) • Model for 3:I segregation • 3: 1 disjunction - Usually one acrocentric chromosome involved - Disparity of length of chromo somes involved
Adjacent-l
- Best example-t(11;22)(q23;ql1.2)
• Segregation involving non-homologous centromeres Adjacent-2
- Short interstitial region between the centromere and point of breakage - Most frequently maternal transmission - t(11;22)(q23;q 11.2)-risks
• Segregation involving homologous centromeres 3:I disjunction • Outcomes of segregations Alternate or adjacent-I segregation • Most frequent types of segregation • Can have normallbalanced or unbalanced segregants • Dependent of chiasma placement - Adjacent-2 or 3:1 segregation • Will always lead to unbalanced segregants • Unbalanced segregants - Both monosomic and trisomic segments present • Makes phenotype/karyotype correlations difficult • Counseling difficult but imperative • Unbalanced segregants-risks (I) - Overall an -11-12% risk of unbalanced segregant - Empiric data from prenatal diagnosis studies
• • • •
Empiric information based on > I00 individuals Risk of unbalanced segregants (5-6%) Most common-47, +der(22)t(11;22)(q23;ql1.2) Increased risk of pregnancy loss-35 %
• Overall risk for recognized pregnancy >40 % • Parental translocation-segregation risks Alternate and adjacent-l segregation most commonly observed • Frequency depend s on distance between the centromere and breakpoint and relative length of the chromo some arms - Adjacent -2 segregation-large imbalances present • Results in non-viable gametes or early embryonic deaths - 3:1 segregation • Commonly observed if one translocation product is a small acrocentric
51
2-20
Molecular Genetic Pathology
- Risks • Empiric risks often used • Best to obtain individual family histories • Depends on type of translocation and viability of unbalanced products • Familial pattern for inherited translocation with unbalancedsegregants may be distinctive - Once a rearrangement is found in proband • Study parents • Study other family members including siblings
De Novo Translocations • Serious clinical implications • Unbalanced translocation - Obvious clinical implications • Need to delineate abnormality • FISH • Balanced translocation - Incidence of mental retardation • Increased in de novo rearrangements • 20% of translocations in newborns-de novo • 55% of translocations in MR-de novo 3/1 000 individuals with MR with de novo translocations - Incidence of congenital anomalies • Increased in de novo rearrangements • Balanced de novo translocations-why clinical findings? Possible position effect • Genes turned on/off in rearranged chromosome Breakage within a gene • For example-Duchenne musculardystrophy - Subtle deletion/duplication • FISH, molecular studies, and array studies - Uniparental disomy • Prenatal diagnosis translocation 6-9/10,000 amniocentesis • Determine if parent has a rearrangement • If de novo determine paternity - If de novo • 8% risk of phenotypic abnormality • Based on empiric data • Perform high-level ultrasound
Robertsonian Translocations (Figure 18) • Definition - Centric fusion-Robertson described in 1916
52
• Most common structural abnormality • Frequency-l/lOOO livebirths • Involves two acrocentric chromosomes • Differenttypes of rearrangements • Homologous chromosomes • Non-homologous chromosomes
Translocation Formation • Formation - Not a centric fusion of acrocentric chromosomes - Arise from pericentromeric exchanges • Within short arms, stalks, and satellite regions - Location of breakage can vary • Most occur within proximal satellite III DNA - Dicentric vs monocentric Robertsonian translocations • Translocations involving homologous chromosomes - 10% of all Robertsonian translocations - Involves homologous chromosomes • For example, rob(l3;13) (qlO;qlO) - Are they isochromosomes? • Studies revealing homozygosity of molecular markers suggests that they are isochromosomes - Most are monocentric - Segregation • Leads to trisomies or monosomies • Non-homologous translocations - 90% of all Robertsonian translocations - Involves non-homologous chromosomes • For example, rob(13;14) (qlO;qlO) - Preferential involvement of specific types • For example, rob(l3 ;14) (qlO;qlO); rob(l4 ;2l) (qlO;qlO) seen most frequently - Most are dicentric • Ascertainment-overview - Couples with multiple spontaneous abortions - Unbalanced probands - Translocation trisomy 13 or 21 - Prenatal/newborn studies - Neoplasia • Ascertainment-multiple spontaneous abortions - There is an increase in rob(l3 ;14) over other types of translocations - There is an increase in homologous translocations • Ascertainment-unbalanced proband - Predominance of rob(l3 ;14) and rob(l4;21) over other types of translocations • Ascertained through trisomy 13 and 21 offspring
Principles of Clinical Cytogenetics
2-21
14
15
14/15 Robertsonian Translocation
Fig. 18. Diagrammatic representation of the formation of a Robertsonian translocation.
Poss ible Gametes
~I
Normal
7 ~
Robertsonlan Translocat ion Carrier
~~I
--. Unbalanced
t(14q21q)
Offspring Afte, Fertilization
Balanced
Translocation
~i
Trisomy 21
0
~
Monosomy 21
M I
Trisomy 14
Monosomy 14
Fig. 19. Diagrammatic representation of the segregation of a 14/21 Robertsonian translocation.
- Translocations usually involves chromosomes 13 and 21 • Ascertainment-prenatal/newborn studies Ascertainment by chance Serendipitous finding or through survey • Unbiased findings • High frequency ofrob(13;14) and rob(14 ;21) • Inheritance - Familial
De novo
Translocation Segregation (Figure 19) • Meiotic segregation Translocations do not disjoin as normal homologous pairs of chromosomes Forms a chain of three (trivalent) in pachytene stage of meiosis Types of segregation • Alternate segregation • Adjacent-l • No adjacent-2
53
2-22
Molecular Genetic Pathology
• No 3:1 disjunction
• For example, rob(21 ;21) (q 1O;q 10)
- Outcomes of segregations
• 100% of offspring abnormal
• Alternate segregation
- Once a rearrangement is found
• Normallbalanced segregants
• Examine family history
• Adjacent-I segregation • Unbalanced segregants o
Either monosomic or trisomic segments
o
Different than reciprocal translocation
o
Phenotypelkaryotype correlations straightforward
o
Counseling imperative but more straightforward
• Unbalanced segregants-risks - Dependent on:
• De novo translocations - Unbalanced translocation
• Obvious clinical implications •
"trisomy 21"-3% of Down syndrome cases
•
"trisomy 13"
• Spontaneous abortions • +13, +14, +15, +21, and +22 Balanced translocation de novo
• Type of translocation • Homologous vs non-homologous • Chromosomes involved
• Incidence of mental retardation • Increased in de novo rearrangements
• Sex of carrier - Unbalanced segregants risks non-homologous • Risk based on empiric data
o
Reciprocal translocations-yes
o
Robertsonian translocations-no
• Need to be concerned about uniparental disomy
• Theoretical risk of abnormal livebirth-33 % • rob(14;21) (qlO;qlO)-empiric risk o
• Study parents • Study other family members including siblings
Female carrier-IO-I5%
Male carrier-2% • rob(l3 ;14) (qlO;qIO)-empiric risk o
o
Female carrier-I %
o
Male carrier-probably
- Unbalanced segregants risks-homologous • Theoretical and empiric risks similar
• Chromosome 15 o
Prader-Willi syndrome
o
Angelman syndrome
• Prenatal diagnosis of a Robertsonian translocation - 1/10,000 amniocentesis-de novo - Determine if parent has a rearrangement - Check paternity - Do uniparental studies • If translocation involves chromosome 14 or 15
CONTIGUOUS GENE SYNDROMES
Definition • First defined by Schmickel in 1986 • Includes microdeletion syndromes • Sometimes defined as segmental aneusomy • Deletion of contiguous stretch of DNA including multiple genes on a chromosome • Includes syndromes that are caused by involvement of genes that are located physically close to one another on a specific chromosome • "Idiopathic" dysmorphic syndrome that were originally delineated clinically and subsequently found to have
54
microscopic or submicroscopic chromosome abnormalities
Microdeletions • These include syndromes that are clinically recognized • Syndromes are usually sporadic - Cytogenetic abnormalities are sometimes detected • High-resolution chromosomes needed • FISH often needed • Some patients--demonstrate submicroscopic molecular deletions
Principles of Clinical Cytogenetics
- Often specific features of syndrome demonstrate mendelian inheritance - Involves multiple unrelated loci that are contiguous • Types of classical contiguous deletions - Xp21 deletion
2-23
• Maternal disomy - 30% of patients
Angelman Syndrome • Microcephaly • Macrosomia with prominent tongue
• Duchenne muscular dystrophy
• Hypotonia
• Chronic granulomatous disease
• Ataxic gait • Excessive laughter
• McLeod phenotype • Retinitis pigmentosa • Mental retardation
• Seizures • Hypopigmentation
• Glycerol kinase deficiency
• Severe mental retardation
• Adrenal hypoplasia
• Laboratory findings - Involves a maternal deletion 15qll-q13
• Aland eye disease Xp22 .3 deletion
• 60-70% of patients
• X-linked ichthyosis
- Paternal disomy
• Kallman syndrome
• 3-5% of patients - UBE3A mutation
• Chondrodysplasia punctata • Mental retardation • Short stature • Ocular albinism - Xq21 deletion
•
10% of patients
Miller-Dieker Syndrome • Type I lissencephaly
• Choroideremia
• Dysmorphic facies
• Mental retardation
• Visible deletions - 50% of patients
• Deafness • Cleft lip and palate • Microdeletion syndromes - Prader- Willi syndrome Angelman syndrome Velocardiofacial syndrome Williams syndrome Miller-Dieker syndrome Smith-Magenis syndrome Langer-Gideon syndrome Aniridia-Wilms tumor association
• FISH or molecular testing needed to detect all cases
Velocardiofacial Syndrome • Chromosome 22q 11.21 deletions DiGeorge syndrome • Abnormalities in the development of the third and fourth brancial arches • Thymic hypoplasia • Parathyroid hypoplasia • Conotroncal cardiac defects • Facial dysmorphism - Velocardiofacial syndrome
Prader-Willi Syndrome • Neonatal hypotonia • Feeding difficulties • Genital hypoplasia • Hyperphagia/obesity (1-2 years) • Short stature, small hands and feet • Hypopigmentation • Mental retardation • Involves a paternal deletion in 15qll-ql3 - 60-70% of patients
• Palatal defects • Hypoplastic alae nasi; long nose • Learning disorders or mental retardation • Congenital heart defects • Conotruncal defects Both syndromes part of the same spectrum involving defects including : • Cardiac defects , abnormal facies, thymic hypoplasia, palatal abnormalities, hypocalcemia, and a 22q 11.21 deletion • Variable phenotypes associated with similar deletions
55
Molecular Genetic Pathology
2-24
• Ascertainment of patients • Congenital heart defects • Hypocalcemia - Deletion can be detected cytogenetically • FISH must always be used to be sure (Figure 5)
• Deletion-17p11.2 - Most of these deletions can be detected with cytogenetics; FISH can be used to confirm
William Syndrome
Familial Cases
• Developmental disorder-central nervous system and vascular connective tissue
• About 8% of cases due to familial deletions
• Dysmorphic facial features • Infantile hypercalcemia
Langer-Gideon Syndrome
• Congenital heart diseases
• Langer-Giedion syndrome - Trichorhinophalangeal syndrome
• Gregarious personality • Premature aging of skin
• Sparse scalp hair
• Mental retardation
• Bulbous/pear-shaped nose
• Deletion-7qll.23 - Involves the elastin gene
• Cone-shaped phalangeal epiphysis Multiple cartilanginous exostoses
- Not visible with high-resolution analysis
Mental retardation
- Not visible without FISH
Deletion-8q24.1
Deletion Ip syndrome Aniridia-Wilms TumorAssociation
• Malformed ears with hearing loss
• Aniridia • Wilm's tumor • Genitourinary dysplasia
• Broad root of nose
• Mental retardation
• Overlapping toes
• Deletion-lip 13
• Dorsal hirsuitism • Chronic seizures • Central nervous system abnormalities
Smith-Magenis Syndrome • Dysmorphic facial features - Brachycephaly - Flat mid-face - Prognathism • Hoarse, deep voice • Short broad hands • Delayed speech • Behavioral abnormalities - Self-destructive behavior
• Congenital heart defect • Seizures • Hypotonia • Abnormalities of the skull and brain • • • •
Deeply set eyes Malformed, malpositioned digits Growth and psychomotor retardation Deletion involves loss of 1p36.3
• Seen in 75% of Smith-Magenis syndrome patients
Rare Deletions
• More apparent in older children and adults
• Rubinstein-Taybi syndrome Dysmorphic facial features
• Onychotillomania • Pulling out of fingers and toenails
- Beaked nose
• Polyembolokolamania
Prominent columella
• Insertion of foreign objects • Head banging
Hypoplastic maxilla Down-slanted palpebral fissures Broad thumbs and first toes
• Wrist biting • Peripheral neuropathy • Sleep disorders • Mental retardation
56
• Thin lips • Fifth finger clinodactyly
Mental retardation Some involve submicroscopic deletions of l6pl3
Principles of Clinical Cytogenetics
2-25
- Beckwith-Wiedemann syndrome
• Most are due to a mutation
• Macrosomia, macroglossia
• u-Thalassemia and MR - u-Thalassemia
• Omphalocele
• (Hemoglobin H) - Facial dysmorphism
• Hypoglycemia • Transverse earlobe creases
- Mental retardation
• Hemihypertrophy
- Deletion-16pI3.3
• Advanced bone age • Increased risk of malignancy • A small percent of cases due to a paternal duplication of 11p15
• Alagille syndrome - Dysmorphic facial features - Chronic cholestasis
• About 10% of cases due to paternal disomy
- Vertebral arch defects Peripheral pulmonic stenosis/hypoplasia
- Duplication 17p11.2p12
- Autosomal dominant
• Hypotonia
- Some involve deletions-20p 11.23-20p 12.2
• Decreased reflexes
• Most are due to mutations • Greig cephalopolysyndactyly syndrome - Craniosynostosis Polysyndactyly
• Club foot • CMTlA (PMP22) • Absence of REM sleep - Cat-eye syndrome
- Mental retardation occasionally
• Coloboma of the iris
- Deletion in 7p 13
• Anal atresia • Ear abnormalities
Duplication Syndromes
• Cardiac defects
• Microduplication syndromes
• Mental retardation in some cases • Duplication of 22q
SEX CHROMOSOME ABERRATIONS
• X chromosome - -6% of total genomic DNA • 995 X linked genes • Y-chromosome - -1 % of total genomic DNA • -56 Y linked genes • Sex determining region of the Y chromosome (SRY) plays a critical role in determining gonadal sex
Sex Determination • Presence of Y chromosome - Leads to maleness • Regardless of number of X chromosomes present • Absence of Y chromosome • Results in female development • Sex differentiation - Gonads undergo sexual differentiation at 6-7 weeks
• If Y chromosome is present • Gonads-testis • Testis produces testosterone • Wolffian duct proliferation • Testis produces mullerian inhibitory substance o Regression of mullerian duct structures - Originally thought sex was determined in humans as in drosophilia • By the ratio of X chromosomes to autosomal chromosomes • 1959-determined that the Y chromosome determined sex • SRY • 199D-testis determining factor • Localized on the short arm of the Y chromosome • Close, but centromeric, to the pseudoautosomal region • SRY and testis determining factor shown to be the same • SRY sequences found in XX males
57
2-26
Molecular Genetic Pathology
Pseudo Autosomal Region
Pseudo Autosomal Region
SRY
SRY
Y chromosome
Derivative Y chromosome (without SRY)
X chromosome
Derivative X chromosome (with SRY)
Fig. 20. A repre sentation of the normal pairing of the X and Y chromosome along with a diagram showing abnormal crossing over leading to XX males and XY females .
Pseudoautosomal Region (Figure 20) • 46,XX males - In normal meiosis cross overs between the tip of the short arm of the X and Y chromosomes • Pseudoautosomal region (2.5 Mb) • SRY centromeric to pseudoautosomal region • If cross over distal (below SRY) leads to: • XX male • XY female • Androgen insensitivity syndrome - Formerly known as the testicular feminization syndrome - Frequency: 1120,000 - 46,XY karyotype - Phenotypic features • Normal female external genitalia • No uterus and blind vagina • Axillary and pubic hair-sparse • Testes present • In abdomen or inguinal canal
58
- End-organ unresponsiveness • Testes secrete androgens normally • Absence of androgen receptors in appropriate target • Defect in X-linked androgen receptor locus This is an X-linked disorder • It has associated counseling dilemmas • Individuals can develop gonadoblastoma • Phenotypic females but XY female
Sex Chromosome Abnormalities Turner Syndrome • Clinical description in 1938 • Chromosomal basis defined in 1959 • Common cause of short stature • Most common cause of primary amenorrhea • Incidence I in 1500-5000 liveborn females - I in 30-50 (3%) of all female conceptuses - I in 100 45,X conceptu ses survive to delivery
Principles of Cl inical Cytogenetics
•• •
2-27
- Vertebral abnormalities - Tendency to obesity - Hemangiomata
2/3 of patients retain maternal X Recurrence risk is very low Karyotypes seen: 45,X
Isochromosome X
12-20%
Mosaic
30-40%
45,X/46,XX
10-15 %
45,X/46,XY
2-5%
Other
• Cardiac anomalies - The single cause of increased early mortality associated with Turner syndrome
50%
• Major phenotypic findings 100%
Normal intelligence
95%
Edema, hands and feet
80%
Broad chest, hypoplastic nipples
>80%
Narrow maxilla
>80%
Unusual ears
>80%
Low posterior hair line
>80%
Micrognathia
>70%
Cubitus valgus
>70%
Nail dysplasia
>60%
Webbed neck
50%
• Internal anomalies Gonadal dysgenesis
>90%
Tibial exostoses
>60%
Cardiac anomalies
40-60%
Renal (mostly horseshoe kidney)
>60 %
Sensorineural hearing loss
>50%
• Less common include: - Scoliosis - Hip dysplasia
50%
Coarctation of aorta (includes hypoplastic arch)
20%
Aortic root dilation
9%
18-20%
• Height considerations - Tendency toward small size at birth By 2-3 years most <5th%ile for height Final height based on genetic (familial) potential as altered by genetic abnormality Incidence of short stature 100% Mean height-143 em (pre-growth hormone [GH])153 em (post-GH and anabolic steroids)
Short stature
Bicuspid aortic valve
Mitral valve prolapse
Common?
Hypoplastic left heart
Rare
• Gonadal dysgenesis - >90% have abnormal gonads Follicles develop normally by 2-3 months gestation, many still functional by birth Follicles non-functional by age 2 years • Menopause before menarche 45,X/46,XY mosaics have an approximately 30% risk of developing gonadoblastoma Intelligence and behavior - Normal intelligence • Except ring X • When XIST (X Inactive Specific Transcript) is not present on the ring - Specific cognitive defects - Spatial perception (difficulty driving, sorting, and so on.) Visuo-motor coordination - Mathematics problems Auditory verbal learning - Increased incidence of attention deficit disorder Management - Newborn period • Echocardiogram • Renal ultrasound • Rule out chromosomal mosaicism • Counsel family
•
•
• Recurrence risk very low - Growth • Frequently cannot show GH deficiency • Start GH when height drops <3%ile for age (usually age 2-5 years) • Usually require 125% of dose used for GH deficiency - Does not appear to affect aortic root dilation
59
Molecular Genetic Pathology
2-28
- Low dose anabolic steroid around start of puberty
- Delayed motor milestones, poor coordination
- Start estrogen at end of bone growth to improve secondary sex characteristics
- Speech and language abnormalities common - Behavior problems (depression, conduct disorders, and poor socialization)
• Avoid early estrogen as it suppresses growth
- Normal sexual development with no apparent increased risk of abnormal offspring
Childhood • Monitor blood pressure regularly • Echocardiogram at 3-5 years looking for aortic root dilation
XXXX syndrome - Variable phenotype-usually moderate mental retardation Other findings variable
• Annual thyroid function test • Consider cosmetic surgery for webbing, epicanthal folds
• • • • • • • •
• Ensure adequate calcium intake • Monitor hearing • Fertility As many as 8% of 45,X females may have spontaneous menses • Possibly higher in mosaics • At least 21 reported pregnancies in 13 45,X women • 8/21 (38%) fetal loss
XXXXX (penta X) syndrome
• 2/13 (15%) of liveborns malformed •
- IUGR (Intra-Uterine Growth Retardation)/poor growth/short stature
10/13 (85%) "normal"
- Severe mental retardation
• However, when mosaics are considered, numbers are less reassuring
- Facial abnormalities
• At least 62 patients with 45,X or mosaicism-l 35 pregnancies
• Hypertelorism • Upslanted palpebral fissures
• SAB or stillborn o
• Epicanthal folds
44/135 (32%)
• Malocclusion - Congenital heart disease - Multiple joint dislocations - Small hands/single palmar crease/fifth digit clinodactyly
• Anomalies (liveborns or late SABs) o 25/87 (29%) • Anomalies (liveborns only) o 23/80 (29%) • Anomalies included sex chromosome aneuploidy, trisomy 21, and single malformations • Adult issues - Hypertension - Osteoporosis - Insulin resistance/diabetes mellitus -
Obesity
- Lymphedema - Inflammatory bowel disease - Hormone replacement
XXX syndrome -
60
Incidence 1 in 1000 liveborn females No recognizable pattern of malformations Usually tall for their family (mean 172 em = 5 ft 8 in.) Head size and IQ typically low for their family
Tall stature Mid-face hypoplasia Upslanting palpebral fissures Hypertelorism Epicanthal folds Fifth digit clinodactyly Narrow shoulder girdle Amenorrhea
Klinefelter Syndrome • Clinical description by Klinefelter in 1942 • • • • •
Shown to have Barr body in 1956 47,XXY karyotype demonstrated in 1959 Incidence 1 in 500-600 male newborns No evidence of increased recurrence risk 1/2 paternal meiosis I errors (no age affect noted) - Remainder maternal M I or M II errors • Age affect seen with M I errors only
• XXYY similar, but more mental retardation • XXY-Neurocognitive profile - Full scale IQ tends to be low normal, occasional mild mental retardation Performance IQNerbal IQ difference greater than expected - Specific defect s include impairments of:
Principles of Clinical Cytogenetics
2-29
- Risk of breast cancer 66 times normal males (approaches rate for females)
• Verbal memory • Fluency • Speed of verbal processing
- Testosterone from early puberty (adult levels usually 1/2 normal)
• Overall language skills - Reading disabilities are common
• Improved body image • Improved strength and endurance
- Math skills usually normal
• Improved mood and concentration • Behavior profile - Early studies suggested that XXY boys were prone to criminal and antisocial behavior Later, prospective studies (Denver, Toronto, Denmark, Winnipeg) demonstrated that criminality is not a part of the phenotype
• Fertility - Extremely rare to be fertile - Fibrosis of seminferous tubules - Exogenous testosterone probably does not improve fertility - In the few reported cases of successful reproduction the offspring have been normal
• Behavior profile - XXY males tend to: • • • • •
Be withdrawn Have difficulty with socialization skills Have poor judgment Have difficulty with adaptation to adult life Have difficulty separating from family
• Have poor self image • These boys are felt to be at increased risk for poor outcomes in dysfunctional families • Growth - Normal birthweight and length - Tendency toward small stature (range 25-99%ile with mean at 75th %ile) Normal weight and OFC (Occipital Frontal Circumference) - Upperllower segment ratio decreased - Occasionally marfanoid habitus - Arm span may be >3 cm greater than height • Physical findings - Small testes-post puberty - Normal facies, sparse facial hair - Congenital malformations in 18% without specific pattern - Gynecomastia - Scoliosis - Low muscle tone - Tend to enter puberty normally, but testicular insufficiency develops soon after
XXXXy Syndrome • Low birthweight, poor growth, microcephaly • Hypotonia • Severe hypogenitalism • Severe mental retardation • Some phenotypic overlap with Trisomy 21, especially in newborn period
XXXY Syndrome • Very rare Klinefelter variant • Mental retardation is the rule • Other findings - Hypotonia - Facies-low nasal bridge, epicanthal folds, upslanted palpebral fissures, abnormal ears • Limited abduction at elbows • Small phallus and testes • Fifth digit clinodactyly • Flat feet
XXXXY Syndrome • Facies round, hyperteloric, upslanted palpebral fissures, epicanthal folds, low nasal bridge with upturned nasal tip • Frequent skeletal anomalies - Vertebral hips • Metacarpals, metatarsals, and phalanges Heart defects in 20% - Normal gonadotropin levels
- Phallus usually normal - Untreated hypoandrogenization leads to "Eunuchoid" habitus - Varicose veins and hypostatic leg ulceration • Management - At risk for developmental delay, learning disability - Slightly increased risk of mediastinal germ cell tumors
XYY Syndrome • • • • •
Incidence I in 1000 liveborn males No physical phenotype No increased incidence of congenital malformations IQ tends to be in low normal range Tend to be tall with normal proportions
61
2-30
Molecular Genetic Pathology
Maternal X chromosome 0 allele Paternal X chromosome
0
allele
---------
•
Fig. 21. An illustration showing a representation of random X inactivation leading to red and black patterns as seen in a calico cat. • Behavior is impulsive, with temper tantrums and poor emotional control • Usually normal fertility • Majority of offspring are normal • Few reports of XYY offspring
Lyon Hypothesis • X-inactivation Males • Hemizygous for the X chromosome • Only a single copy of the X chromosome - Female • Two X chromosomes • Mean amounts of gene products of X-linked genes; same in females as males • Why? - Mechanism of dosage compensation-Lyon hypothesis • In somatic cells X-inactivation occurs early in embryonic life • Inactivation is random • Either the paternal or maternal X chromosome Inactivation is complete • Inactivation is permanent and clonally propagated
62
- Lyon hypothesis-exceptions • Inactivation not always random • With structurally abnormal X non-randon inactivation seen • Inactivation not complete • A number of genes known to escape activity • May be up to 15% of all X-linked genes escape inactivation in some way • Inactivation reversible in development of germ cells - Lyon hypothesis-evidence • Genetic studies have helped to confirm the Lyon hypothesis • Tortoiseshell mouse • Calico cats (Figure 21) • Cytologic evidence • Barr body • Darkly staining chromatin body • Number of barr bodies = number of X chromosomes - 1
Significance • Lyon Hypothesis-significance - Explains manifestations of X-linked disorders - Explains variability of clinical manifestations in females
Principles of Clinical Cytogenetics
2-31
- Explains difficulty in biochemical carrier detection in female carriers • Mechanism of X-inactivation Involves altered chromatin structure Differential methylation of DNA epG islands in silenced genes-methylated
X-inactivation center At Xq13.2 • Cis for X-inactivation XIST • X inactive-specific transcript • Uniquely expressed from the inactive, but not the active X
SUGGESTED READING Bui TH, Blennow E, Nordenskjold M. Prenatal diagnosis: molecular genetics and cytogenetics. Best Pract Res Clin Obstet Gynaeco. 2002;16(5):629-643. Carrel L, Willard HF. X-inactivation profilereveals extensive variability in X-linked gene expression in females. Nature 2005;434:400-404. Devriendt K, VermeeschJR. Chromosomal phenotypes and submicroscopic abnormalities. Hum Genomics 2004;I(2):126-133. Gardner RJM, Sutherland GR. Chromosome Abnormalities & Genetic Counseling. 3rd ed. NewYork: Oxford University Press; 2004. Genesand disease: http://www.ncbi.nlm.nih.govlbooks/. Hall H, Hunt P, Hassold T. Meiosis and sex chromosome aneuploidy: how meiotic errorscause aneuploidy; how aneuploidy causes meiotic errors. Curr Opin Genet Dev. 2006;16(3):323-329.
Jones KL. Smith's Recognizable Patterns of Human Malformation . 6th ed. Philadelphia: Elsevier Saunders; 2006. 3-81. Keagle MK, Gersen, Steven L. Principles of ClinicalCytogenetics. 2nd ed. Humana press; 2004. Lupski JR, Stankiewicz P. Genomic disorders: molecular mechanisms for rearrangements and conveyed phenotypes. PLoS Genet. 2005; I(6):E49. Nussbaum RL, Mcinnes RR, Willard HF. Thompson & Thompson Genetics in Medicine. Revised reprint, 6th ed. Philadelphia: W.B. Saunders Company; 2004. 2-32, 135-180. Shaffer LG, Tomerup N, eds. ISCN 2005: An International Systemfor Human Cytogenetic Nomenclature. Switzerland: S. Karger AG; 2005.
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3 Diagnostic Methodology and Technology Josephine Wu, DDS, ClSp(MB), ClDir, Tao Feng, MS, MP (ASCP), Ruliang XU, MD, PhD, Fei Ye, PhD, Bruce E. Petersen, MD, Liang Cheng, MD, and David Y. Zhang, MD, PhD, MPH
CONTENTS I. Sample Collection and Processing Methods Sample Types General Considerations Whole BloodIBone Marrow Buccal Cells Cervical Cells Hair Root Paraffin-Embedded Tissue Bodily Fluids Samp le Collection and Storage Whole BloodIBone Marrow (Vacutainer Specimen Collection Tubes) Fresh Tissue Paraffin-Emb edded Tissue DNA Extractio n Met hods Manual Automated Systems RNA Extraction Methods General Considerations Ambion Versagene/Purescript (Gentra) Purescript Total RNA Purification Kits (Gentra) Ql /varnp'" RNA Blood Mini Kit Quality and Quantity Assessment Nucleic Acid StoragelHandling General Considerations DNA RNA
II . Amplification Methods 3-3 .3-3 .3-3 3-3 3-3 3-3 3-3 3-3 .3-3 3-3
3-3 3-4 3-4 3-4 3-4 3-4 3-4 3-4 3-5 3-6 3-6 3-6 3-6 3-7 3-7 3-7 3-8
General Information Signal Amplification Branched DNA Rolling Circle Amplification Ramification Amplification Invader Cleavase Technology Target-Based Amplification Polymerase Chain Reaction Variation of the PCR Ligase Chain Reaction Strand Displacement Amplification Transcription Mediated Amplification
III. Signal Detection Methods General Information DNA Binding Dyes SYBR Green Probe Based Chemistries General Information Linear Probes Structured Probes
IV. Nucleic Acid Hybridization Methods Hybrid Capture (HC) In Situ Hybridization (ISH) Southern Blot Northern Blot Allele-Specific Oligo nucleo tide (ASO) and SSO Hybridization Reverse Hybridization
3-8 3-8 3-8 3-8 3- 10 3-10 3- 11 3-12 3-13 3-16 3-19 3-2 1 3-22
3-23 3-23 3-23 3-23 3-24 3-24 3-25 3-25
3-27 3-27 3-29 3-3 1 3-32 3-32 3-33
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3-2
V. DNA Separation Methods Gel Electrophoresis Conventional Gel Electrophoresis Capillary Electrophoresis Gradient Gel Electrophoresis (GGE) and Denaturing Gradient Gel Electrophoresis (DGGE) Pulsed Field Gel Electrophoresis (PFGE) Single-Strand Conformational Polymorphism (SSCP) RFLP Analysis
VI. Sequencing of Nucleic Acids General Information SangerSequencing Dye Terminator Sequencing RNA Sequencing
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3-44 3-44
3-37
Applications Limitation of DNA Sequencing Trouble-Shooting DNA Sequencing Problems The Snapshot Method (Applied Biosystems, ABI) Pyrosequencing Technology
3-38
VII. Protein Detection Methods
3-52
3-34 3-34 3-34 .3-36
3-39 3-41
3-42 3-42 .3-42 3-43 3-44
Enzyme Immunoassay (EIA) Protein Electrophoresis Western Blot (WB) Key Technologies Used in Proteomics 20 Electrophoresis Mass Spectrometry
VIII. Suggested Reading
3-44 3-48 3-51
3-52 3-55 3-56 3-57 3-58 3-61
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SAMPLE COLLECTION AND PROCESSING METHODS Sample Types
Bodily Fluids
General Considerations
• Cerebrospinal fluid • Urine: DNA can filter through the kidneys and is present in the urine of healthy individuals, usually as small fragments. Since DNA is very unstable in urine, samples must be processed within a few hours of collection
• Ideal source of nucleic acid is fresh tissue; although paraffin embedded tissue is acceptable • If extraction is not performed immediately, flash-freezing of solid tissue or cells with liquid nitrogen preserves nucleic acids. This is particularly important with RNA, which is highly unstable and easily degraded by RNases. Fresh tissue may also be placed in commercially available reagents to preserve cellular RNA up to I week at room temperature • RNase and DNase are rapidly denatured by chaotropic agents like guanidium isothiocyanate (GITC). For effective RNA stabilization, a minimum concentration of 5 mollL is required. GITC-preserved tissue can be stored at room temperature
Whole Blood/Bone Marrow • Most often the best available DNA source requires the use of an anticoagulant to prevent clot formation
Buccal Cells • Can be either air-dried on a glass slide or collected in a saline mouthwash and pelleted for immediate analysis
• Peritoneal and pleural fluid
Sample Collection and Storage Whole Blood/Bone Marrow (Vacutainer Specimen Collection Tubes) • Specimens should be transported within 24 hours of draw and are best stored at 2-8°C for up to 72 hours. Storage at 22-25°C is not recommenced for >24 hours. Freezing blood or bone marrow specimens without prior red blood cell lysis causes contamination with heme, which can inhibit PCR amplification . Leukocyte pellets can be stored for up to I year at -20°C or for greater than 1 year at -80°C • Anticoagulants - Ethylenediamine tetra acetic acid (EDTA) (lavender topped tubes) • Preferred specimen collection type
Cervical Cells • Can be stored at room temperature for up to 2 weeks and longer when refrigerated • Fixed cytologic preparations can also be used for nucleic acid isolation
Hair Root • Useful in forensic testing , when other tissue is unavailable
Paraffin-Embedded Tissue • The most common fixative is neutral buffered formalin. When exposed to nucleic acid , formalin causes the formylation of free nucleotide amino groups, methylene bridging of bases, and cross-linking of nucleic acid with protein, resulting in increased nucleic acid fragmentation • With increasing fixation time, the amount of recoverable nucleic acid is progressively reduced . Tissue fixation in formalin for an extended period of time may reduce the yield of nucleic acid • Although RNA can be isolated, it is usually degraded ; also, formalin may inhibit subsequent reverse transcriptase-polyrnerase chain reactions (RT-PCR) • Not suitable for Southern blot techniques
- EDTA (pearl-topped tubes) • Contain the same anticoagulant as the Lavendertopped tubes in addition to a polyester material that separates most of the erythrocytes and granulocytes, and some of the lymphocytes and monocytes away from the supernatant upon centrifugation • This "Plasma Preparation Tube" is a convenient, safe, single tube system for the collection of whole blood and the separation of plasma. It can be used for certain viral load testing, i.e., HIV, HCV, and cytomegalovirus (CMV) • May contain a higher concentration of platelets than found in whole blood - Citrate (yellow topped tubes) • Acid citrate dextrose • Acceptable for molecular testing; provides a good yield of nucleic acids with>70% of the original high-molecular weight DNA - Heparin (green-topped tubes) • Least preferred specimen collection tube • Heparin concentrations as low as 0.05 U per reaction volume may cause inhibition of enzymes, i.e., DNA polymerase and Taq polymerase and prevent amplification
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• Attempts to remove heparin activity, for example , ethanol precipitation, boiling and filtration, pH modificiation with gel filtration, or titration with protamine sulfate do not appear to eliminate inhibition. Serial washing of the buffy coat with saline prior to DNA extraction may be helpful • Heparinase treatment of extracted DNA may also be helpful, but is expensive and not suitable for RNA due to its RNase activity. Alternatively, heparin-free RNA may be precipitated with lithium chloride
Fresh Tissue Should be collected in a sterile container (microcentrifuge tube) and frozen immediately in liquid nitrogen or in OCT and stored at -80°C. For travel of short distances, specimens should be placed in sterile gauze pre-wet with sterile saline to prevent drying . If longer distance transportation is required, specimens should be frozen immediately and transported in dry-ice.
Paraffin-Embedded Tissue Can be stored at room temperature. However, the longer the duration of storage, the more DNA degradation.
DNA Extraction Methods
Puregene •
150 IlL-20 mL of whole blood or bone marrow collected in common anticoagulants (EDTA, citrate, or heparin)
• The PUREGENE kit works (Gentra Systems , Inc., Minneapolis, MN) via alcohol and salt precipitation. The first step is to lyse cells with an anionic detergent in the presence of a DNA stabilizer that inhibits DNase activity, after which RNA is digested • The proteins are digested and removed along with other contaminants by salt precipitation. The DNA is then alcohol precipitated and dissolved in a DNA stabilizer • 25-60 minutes are required to process a sample • DNA yield is 35 ug/ml, of blood
Versagene (Gentra) • Employs high-capacity, glass fiber mini-columns, and proprietary non-chaotropic reagent s to produce high yields of highly concentrated, double- stranded genomic DNA, free of RNA, protein and other contaminants, and suitable for the most sensitive downstream analyses • Blood kits are available for whole blood samples ranging from 0.05 to 0.4 mL. In 60 minute s or less, VERSAGENE DNA can obtain up to 600 ug of DNA from 10 mL whole blood. Sample s from 0.05 to 0.4 mL can be completed in 30 minutes or less
Manual QIAamp®(Qiagen, Valencia, CAy DNA Blood Mini Kit
• Recovery rates are 60-90%
• The QIAamp DNA Blood Mini Kit (Qiagen ; Hilden, Germany, Valencia, California) simplifies isolation of DNA from blood and related body fluids with fast spincolumn or vacuum procedures. No phenol-chloroform extraction is required
Automated Systems
• The following samples can be processed: fresh and frozen whole blood (with common anticoagulants such as citrate , EDTA, and heparin) ; plasma; serum ; buffy coat; bone marrow; lymphocytes; platelets; body fluids • Typical yield from 200 ul, of blood is 4-12 ug with a processing time of 20-40 minute s • DNA binds specifically to the QIAamp silica-gel membrane while contaminants pass through. PCR inhibitors such as divalent cations, and proteins are completely removed in two efficient wash steps, leaving pure nucleic acid to be eluted in either water or a buffer provided with the kit • Optimized buffers lyse samples, stabilize nucleic acids, and enhance selective DNA adsorption to the QIAamp membrane. Alcohol is added and Iysates loaded onto the QIAamp spin column. Wash buffers are used to remove impurities and pure, ready-t o-use DNA is then eluted in water or low-salt buffer • The complete proces s require s 20 minutes of handling time (lysis times differ according to the sample source) • With the QIAamp DNA Blood Mini Kit, blood can be processed via a vaccum manifold instead of centrifugation, for greater speed and convenience in DNA purification
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• For blood , 50 kb fragment s are typical
• Various automated systems are available for the extraction and purification of nucleic acids (see Chapter 13)
RNA Extraction Methods GeneralConsiderations • Adequate homogenization of cells or tissues is an essential step in RNA isolation to prevent RNA loss and degradation. The method of homogenization is best tailored to the particular cell or tissue type, i.e., vortexing in a cell lysis solution for cultured cells or more rigorou s disruption technique s such as enzymatic digestion for animal tissues, plant tissues, yeast, and bacteria for maximum recovery of RNA • Endogenous RNases must be inactivated immediately upon tissue harvesting to prevent RNA degradation. This can be effectively accompli shed by - Homogenizing samples immediately after harvesting in a chaotropic-based cell lysis solution (e.g., containing guanidinium) - Flash freezing small tissue sample s (homogenized) in liquid nitrogen - Utilization of an aqueou s, non-toxic collection reagent (e.g., RNAlater® (Ambion, Foster City, California), an RNA stabilization solution) that stabilizes and protect s cellular RNA in intact, unfrozen tissue and cell
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samples when samples cannot be immediately processed. However, tissue samples must be in thin pieces (0.5 em) so that the RNAlater (Ambion, Foster City, California) can quickly permeatethe tissue before RNases destroy the RNA. Cells or tissues can be harvested into RNAlater and stored at room temperature for up to 1 week, at 4°C for up to 1 month, or at -20°C indefinitely • RNA is an unstable molecule and requires maintaining RNase-free laboratory conditions and diethylpyrocarbonate (DEPC)-treated glassware and water
Ambion Total RNA Isolation • LeukoLOCK® (Ambion, Foster City, California) Total RNA Isolation System - Method for cellular fractionation of whole blood, total RNA stabilization, and extractionof RNA from the leukocytes (includingT and B-cells, neutrophils, eosinophils, basophils, monocytes, and other less abundant cell types) Used for molecular detection of infection, inflammation, and autoimmune diseases The LeukoLOCK System employs filter-based leukocyte-depletion technology to isolate leukocytes from whole bloodand stabilize the cellson a filter. Anticoagulated bloodis passed through a LeukoLOCK filter, which captures the total leukocyte population while eliminating red bloodcells (including reticulocytes), platelets, and plasma. Afterrinsing withphosphatebuffered saline, the filter is flushed with RNAlater to stabilize the RNA in the captured leukocytes. The RNA can be isolated immediately, or stabilized cellscan be maintained for several days at room temperature, or for longer periods at -20°C or -80°C Purification of the RNA is accomplished by disrupting the capturedcells in a guanidinium thiocyanate-based solution releasing RNA while simultaneously inactivating nucleases. The cell lysate is collected and briefly treated with Proteinase K. RNA is then purified from the lysateusingAmbion's MagMAXTM magnetic bead-based technology for washing, followed by DNase treatment (with TURBODNase™) and final clean-up 10-20 ug of RNA is isolated per 9-10 mL of whole blood - RNA recovered using this method contains less than Ill0th the amount of reticulocyte-derived (X - and ~-globin mRNAs commonly present in RNA samples derived from unfractionated whole blood impurities, which can interfere with downstream expression profiling applications - mRNAs present in typical RNA samples from unfractionated whole blood • MagMAXTM AUND Viral RNA Isolation Kit, total RNA isolation
- Ambion's MagMAX system utilizes magnetic bead technology to isolate RNA from cells and viral RNA from cell-free samples, such as serum, plasma, swabs, and cell culture media. With this technology RNA is bound more efficiently than with glass fiber filter methods, resulting in higher and more consistent RNA yields. The MagMAX magnetic bead technology eliminatesfilter clogging from cellular particulates and allows the end user to concentrate RNA from large, dilute samples - As few as 10 copies of viral RNA from 100 to 400 ul, sample can be recovered - Typical viral RNA recovery exceeds 50% - Ideal when working with low viral concentrations - Extraction procedure takes approximately 30 minutes to complete
mRNA Isolation • Poly(A) RNA (mRNA) makes up between 1 and 5% of total cellular RNA • mRNA isolation procedures are used in - Detection and quantitation of extremely rare mRNAs - Synthesis of probes for array analysis - The construction of random-primed cDNA libraries, where the use of total RNA would generate rRNA templates that would significantly dilute out cDNAs of interest. Removal of ribosomal RNA (rRNA) and transfer RNA (tRNA) results in up to a 30-fold enrichmentof a specific message • Poly(A)purist® Kit (Ambion, Foster City, California) - Isolation of high-quality mRNA requires efficient removal of rRNA and specific recovery of poly(A) RNA. Yield estimates based solely on mass are not indicative of the quality or quantity of mRNA recovered since contamination from rRNA can add significant contribution to the mass - rRNA contamination is caused by non-specific adsorption to the oligo(dT) matrix and binding to/copurifying with mRNA. Ambion's poly(A) purist kit minimizes this unwanted interaction while promoting efficient oligo(dT) selection - The poly(A)purist kits are available in two formats, one that uses oligo(dT) cellulose-based selection (poly[A]purist kit and micropoly[A]purist kit), and one that utilizes oligo(d'T) magnetic bead-based purification (poly[A]purist magnetic bead based purification method [MAG] kit) • Oligo(dT) cellulose-based selection-the oligo(dT) cellulose based kits (poly[A]purist and micropoly[A]purist) utilize batch binding of RNA to premeasured aliquots of oligo(d'I') cellulose to avoid the problems of slow flow rates and clogged columns often experienced during conventional, gravity-driven chromatography. A spin cartridge is used in the wash and elution steps for speed and
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convenience. Furthermore, time savings are realized becau se a single round of selection with poly(A) purist kits is comparable with two or three rounds of selection using conventional technology • The poly(A) purist kit contain s reagent s for 6 isolations, each for up to 2 mg total mRNA, while the micropoly(A) purist kit contain s reagents for 20 isolations, each for 2-400 ug total mRNA • Magnetic bead-b ased selection employed in the poly(A) purist MAG Kit delivers the benefits of increa sed processing speed, ease of handling, and scalability. Using the same hybridization and wash system as the cellulose-based Poly(A) Purist Kits, but with magnetic particle technology, the entire selection procedure can be completed in as little as 45 minutes. In addition , the reactions are completely scalable, allowing efficient selection from samples smaller than 100 ug of total mRNA to very large (I mg) total mRNA samples • The Poly(A) Purist MAG Kit contains reagents for up to 80 isolations, each from 100 ug of total mRNA, or for 8 large preps from as much as I mg of total mRNA
Versagene/Purescript (Gentra) • This is a simple and effective technology for isolating pure RNA from blood, animal tissue , or cultured cell samples • Convenient, easy-to-use kits come complete with premixed reagents, eliminating the need to prepare reagents before beginning purification • Kits do not contain any harsh organic solvents, foulsmelling reducing agents, or reactive cyanide salts, so hazardou s waste dispo sal is not necessary • Sampl es can be processed at room temperature on the lab bench, without a fume hood
Purescript Total RNA Purification Kits (Gentra) • These kits utilize a modified salt precipitation procedure in combination with highly effective inhibitors of RNase activity • Sample s may be whole blood or bone marrow, from 200 ul, to 30 mL, collected in common anticoagulants (EOTA, citrate , or heparin ) • Sample s are processed in 60 minutes
completely removed in two efficient wash steps, leaving pure RNA to be eluted in either water or a buffer provided with the kit • Extract s total cellular RNA from fresh whole blood and other sample sources ready to use in RT-PCR and blotting procedures. The typical yield is 1-5 ug RNA per mL healthy blood or up to 100 ug RNA from tissue • Red blood cells are selectively lysed and white cells collected by centrifugation. White cells are then lysed using highly denaturing conditions, which immediately inactivate RNases. After homogenization using the QfAshredder'" spin column, the sample is applied to the QIAamp spin column. Total RNA binds to the QIAamp membrane and contaminants are washed away, leaving pure RNA to be eluted in 30-100 ul, RNase-free water (provided with the kit) for direct use in any downstream application
Quality and Quantity Assessment Analysi s of the quality, quantit y/concentration, and size of nucleic acid used is critical for the success of all aspect s of molecular testing . The following method s can be utilized to determine nucleic acid characteri stics: spectrophotometry, fluorescent dyes, and electrophoresis. • Spectrophotometry - This is the simplest and most rapid method to evaluate purity, quantity, and quality of nucleic acids - DNA and RNA demonstrate maximum absorption at approximately 260 nm. Protein absorbs at 280 nm, while background scatter absorb s at 320 nm. Protein absorption is primarily the result of the aromatic amino acids phenylalanine, tyrosine, and tryptophan - DNA and RNA quality or purity can be measured by analysis of the optical density (00) at 260 nm and 280 nm, (00 260 - 00320)/(00280 - 00320), A ratio of 1.7-2.0 is indicative of good quality nucleic acid. Less than 1.7 indicate s too much protein or the presence of other contaminants, for example , organic solvents - DNA and RNA quantit y can be measured by 00260 reading. An 00260 reading of 1.0 corre sponds to 50 ug/rnl, of double- stranded DNA, 40 ug/ml, of single stranded RNA, or 35 ug/ml, single stranded DNA. Concentrations are calculated as follows: • dsONA (ug/ml.) = (0 0 260 - 00320) x dilution factor x 50 ug/ml,
• RNA yield is 2-7 ug/rnl, of blood
• ssRNA (ug/ml.) = (0 0260 - 00 320) x dilution factor x 40 ug/rnl,
QIAamp®RNA Blood Mini Kit
• ssONA (ug/rnl.) = (0 0 260 - 00 320) x dilution factor x 35 ug/ml,
• Simplifie s isolation of RNA from blood with a fast spincolumn procedure. No phenol-chloroform extraction is required • RNA binds specifically to the QIAamp silica-gel membrane while contaminants pass through . PCR inhibitors such as divalent cations, and proteins are
70
• Fluore scent dye - Alternative method to assess purity, quantity, and quality of nucleic acids - The following fluore scent dyes bind nucleic acids: ethidium bromide (EB), acridine orange,
Diagnostic Methodology and Technology
Storage period Storage conditions
3-7
<7 days 4
I
~7 •
2-8°C
4
years
I
-20°C
• 4
Recommended for short-term storage
>7 years • 4
4
-70°C
I
• 4
Recommended for long-term storage
•
I
Recommended for indefinite storage
Fig. 1. Recommended conditions for DNA storage .
diaminobenzoic acid (DABA), and propidium iodide. The most commonly used of these is EB
• Concentrate dilute nucleic acid using ethanol precipitation
- EB can be used in quantitative assays. The intensity of fluorescence is dependent on the ratio of EB to nucleic acid
• Repeat isolation from any residual specimen, modifying sample volume to compensate for possible low cell number or poor specimen handling
- Can reliably detect as little as 5-10 ng of DNA • Electrophoresis - Small agarose gels (minigels) offer an easy method to determine size and quantity of nucleic acid Molecular-weight ladders (e.g., 100 bp marker) provide a reference standard for size determination - Nucleic acid in samples can be quantified by extrapolation from a standard curve. The standard curve is produced by performing serial dilutions of samples with known nucleic acid concentrations
- Too much nucleic acid yield • Dilute sample and remeasure OD reading within instrument range - Poor nucleic acid quality • For degradation, repeat isolation from residual specimen • For protein or other contamination, purify specimen by reisolation - Band smearing
- Band smearing indicates DNA degradation or too much DNA loaded • Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, California) - This system is an alternative to traditional gel-based analysis that integrates the quantitation of RNA samples with quality assessment in one quick and simple assay - First commercially available microfluidics instrument to provide detailed information about the condition of RNA samples - When used in coordination with the RNA 6000 LabChip®(a registered trademark of Caliper Technologies Corporation [Caliper Life Science Inc., Hopkinton, Massachusetts]), as little as I ~L of 10 ng/~L RNA is required per analysis - In addition to assessing RNA integrity, this automated system also provides a good estimate of RNA concentration and purity (i.e., rRNA contamination in mRNA preparations) in a sample - Concentration, integrity, and purity can be simultaneously analyzed in a single 5 ng sample and displayed as a gellike image, an electropherogram, or tabular formats • Troubleshooting - No or low nucleic acid yield • Ensure that ample time is allowed for resuspension/rehydration of specimen
• Due to poor DNA qualitylDNA degradation • Due to poor quality of DNA synthesis
Nucleic Acid StoragelHandling General Considerations • Nucleic acid samples are best stored as multiple aliquots in separate tubes in order to prevent degradation and damage from successive freeze-thaw events. Aliquotted tubes also minimize the potential for accidental contamination, for example, by DNase, RNase, specimen, or amplicon • To prevent amplicon contamination, exposed surfaces (bench space/work hoods, instruments, and floors) should be decontaminated with 10% bleach solution followed by 70% ethanol after use • To reduce the likelihood of exposure to ambient RNases , all laboratory surfaces, including pipetors , benchtops, glassware, and gel equipment should be decontaminated with a surface decontamination solution . RNase-free tips, tubes, and solutions should always be used and gloves should be changed frequently
DNA • Purified DNA should be stored in TE buffer at 4 DC for
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RNA • For short-term storage, purified RNA should be stored at -20°C • Purified RNA should be stored in RNase-free ultrapure water at -80°C, for long-term storage
• Although RNA resuspended in water or buffer can be stored at -80°C, RNA is most stable in an NHp Ac/ethanol precipitation mixture at -80°C
AMPLIFICATION METHODS General Information
- Both DNA and RNA can be detected
Molecular biologic amplification technologies have undergone rapid evolution since the invention of the PCR over 20 years ago. Most molecular diagnostic assays require amplification of the nucleic acid target, signal amplification, or both. Target-based amplification methods include PCR, ligase chain reaction (LCR), strand displacement amplification (SDA) , and transcription-mediated amplification (TMA). Signal amplification methods include branched DNA (bDNA), rolling circle amplification (RCA), ramification amplification (RAM), and Invader® (Third Wave Technologies Inc., Madison, Wisconsin) cleavase technology. In general, amplification methods are rapid, specific, and extremely sensitive (in many situations, as few as 10 molecules of target nucleic acid can be detected per reaction). Owing to the exquisite sensitivity of amplification processes, special care must be taken to prevent contamination of samples with nucleic acid products from previous amplification reactions and other spurious material. Laboratory staff must strictly adhere to the principle of unidirectional work flow, in which physically separated areas of the laboratory are designated for preamplification, specimen preparation, and post-amplification procedures, with one-way flow of samples, reagents, and amplification products between these areas. Other means of preventing contamination include cleaning work areas with 10% bleach at the end of each shift, and the use of uracil N-glycosylase in PCR reactions.
- Target: usually the entire genome
Signal Amplification Described below are probe-based methods of target detection, which employ enhancement of a target-specific signal without increasing the copy number of the target as such. These methods have some intrinsic advantages over PCR-based techniques, including less risk of contamination, ease of operation, isothermal nature, and better linearity.
Branched DNA • General information - First described by Mickey S. Urdea (1994), This technology was validated and developed by Chiron Diagnostics, which is now owned by Bayer Inc - The tradename is Versant 3.0 in the United States and Quantiplex 3.0 in Europe
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- It is mainly used to detect infectious agents in clinical materials including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and cytomegalovirus (CMV) Principle: - bDNA is a signal amplification nucleic acid probe assay - The technology uses dozens of probes, which mediate the attachment of signal amplification molecules to viral nucleic acid targets - One end of the bDNA molecule is designed to bind to a specific target probe, while the other end of the bDNA molecule binds to multimers linked to alkaline phosphatase (ALP). ALP then catalyzes a chemiluminescence reaction Procedure (Figure 2): - Target DNA, or RNA is isolated; double-stranded DNA is denatured into single stranded DNA. The RNA or single stranded DNA is captured to a microwell by a first set of target probes, which bind to capture probes coated on the microwell - A second set of target probes hybridizes to the DNA or RNA (the first set of target probes and the second set of target probes bind to different regions of the target DNA or RNA sequence) - The second set of target probes hybridizes to the bDNA molecules. Multiple copies of an ALP-labeled probe are then hybridized to the bDNA molecules - Detection is achieved by incubating the ALP-bound complex with a chemiluminescent substrate . Light emission is directly related to the amount of target DNA or RNA present in each sample, and results are recorded as relative light units by the analyzer. A standard curve is defined by light emission from standards . Concentrations of DNA or RNA in specimens are determined from this standard curve • Applications: - bDNA technology can be used for hepatitis C RNA viral load (VERSANT® version 3.0) detection . The assay has a broad dynamic range, accurately measures twofold drops in viral load, predicts treatment nonresponders, and provides equal genotype quantitation
Diagnostic Methodology and Technology
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OJ Fig. 3. Principle of RCA (see text for description).
Branch DNA probe
Viral target Capture probe
•
Chromogenic enzyme
Fig. 2. Principle and procedure of branched DNA signal amplification (see text for description).
- bDNA technology can also be used for HIV-I RNA viral loads (VERSANT® version 3.0 [Bayer, Pittsburgh, Pennsylvania]) detection. The assay provides a broad linear range, a high level of precision and reproducibility, and quantitates all major subtypes. It is also well suited to detect emerging subtypes - Other reported applications of the procedure include the detection and quantification of hepatitis B, CMV, and the plague agent Yersinia pestis • Advantages: - bDNA is very specific - The original amount of the target remains unmodified - bDNA allows broad detection of different genotypes in genetically diverse populations - bDNA is inherently quantitative • Limitations: - The bDNA assay has a narrower linear range for quantitation than quantitative PCR. Care must be taken when bDNA gives very low or very high results. In such cases, other methods, such as PCR-based quantitation, should be used for confirmation - bDNA requires multiple layers of probes to capture and signal the target molecule, which often produces high background - Although the bDNA technology format could be easily adapted to high-throughput screening, the assay's cost and tedious procedures limits such applications - Because ALP is used in this assay, extreme care must be taken to avoid contamination with this ubiquitous enzyme - Sensitivity of bDNA is lower as compared with other target and signal amplification methods - A tedious procedure
Rolling Circle Amplification • General information: - RCA is an isothermal signal amplification method - The amplification mode of RCA is linear and one primer is applied
- The mechanism is based on the in vivo "rolling circle" replication of bacteriophages • Principle and procedures (Figure 3): - Uniquely designed circularizable probe (C-probe or padlock probe) contains three regions : two target complementary sequences located at the 5' and 3' termini and an interposed generic linker region - Once the C-probe hybridizes to its target, the 5' and 3' ends are juxtaposed. A closed circular molecule is then generated following incubation of the C-probe-target complex with a DNA ligase - The resulting closed circular molecule is helically twisted around the target strand - The unique design of the C-probe allows its amplification by a RCA mechanism as observed in in vivo bacteriophage replication. In this scheme , a single forward primer complementary to the linker region of the C-probe and a DNA polymerase-bearing strand displacement activity are employed - The polymerase extends the bound primer along the closed C-probe for many revolutions and displaces upstream sequences, producing a long single-stranded DNA (ssDNA) of multiple repeats of the C-probe sequence that can be as long as 0.5 Mb - This type of amplification results in linear growth of the products with up to several 1000-fold amplification • Applications: - When applied to a microarray system, RCA can detect 480 fM (150 molecules) of spotted primers, corresponding to an 8000-fold increase in detection sensitivity over hybridization under the same conditions - The combination of RCA and DNA microarray technology allows for the real-time detection of multiple targets with great sensitivity and specificity - RCA can also be used to detect protein . Immuno-RCA has been developed . In this scheme, a primer is linked to an antibody and the signal is amplified by RCA • Advantages: Since this method uses polymerases (such as phi 29 polymerase) that are capable of strand displacement, the reaction takes place under isothermal conditions - Because thermal cycling is not required, this is an ideal method for in situ amplification - RCA can make target capture, amplification reaction, and detection happens on the same solid support, such as a dipstick or latex beads . This makes the technology especially suitable for use in the field
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Fig. 4. Principle of RAM (see text for description).
• Limitations: - Sensitivity is lower compared with RAM (see Ramification Amplification section), since only one primer is used - Since the procedure is technically complicated, future work will be required to evaluate its full potential
Ramification Amplification • General information: - RAM is a novel isothermal DNA amplification method that amplifies a C-probe exponentially through the mechanism of primer extension, strand displacement, and ramification Also referred to as hyperbranched RCA or cascade RCA, this method represents an extension of the RCA method described in the Rolling Circle Amplification section • Principle and procedures (Figure 4): - This method uses a specially designed circular probe (C-probe) in which the 3' and 5' ends are brought together in juxtaposition by hybridization to a target, which forms an open loop - Before amplification begins, this open loop is covalently linked by a T4 DNA ligase in a targetdependent manner, producing a closed DNA circle - This circular DNA is the template for the forward primers to attach to. Then a DNA polymerase extends the bound forward primer along the C-probe and displaces the downstream strand, generating a multimeric ssDNA - This multimeric ssDNA then serves as a template for reverse primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex - This ramification process comes to an end when all ssDNAs become dsDNAs and no new primers are available • Applications: - The practical use of RAM has been shown in several studies for detecting target nucleic acids in clinical samples, such as Chlamydia trachomatis in cervical specimens collected in PreservCyt cytologic solution
74
- The RAM assay has also been used in the identification of Escherichia coli 0157:H7 and other shiga toxinproducing E. coli in food and human samples • Advantages: - Unlike PCR, the use of a thermocycler is not necessary. RAM employs the extension and displacement nature of some polymerases, such as phi 29 polymerase, making million-fold amplification feasible in a short period of time under isothermal conditions - The primer binding sequences of the C-probe are identical, independent of the hybridization region . This feature facilitates multiplexing (simultaneous detection of multiple targets) - It is an ideal method for in situ amplification becau se RAM does not require thermal cycling - RAM can make target capture, amplification reactions , and detection happen on the same solid support , such as a dipstick or latex beads. This makes the technology especially suitable for use in the field • Limitations: - Specificity is somewhat low, and non-specific background signals can be problematic - Since the procedure is technically complicated, future work will be required to evaluate its full potential
Invader Cleavase Technology • General information: - The Invaders' assay (Third Wave Technologies, Madison, Wisconsin, WI) is a homogeneous, isothermal DNA probe-based system for highly sensitive, quantitative detection of specific nucleic acid sequences Invader reactions can be performed directly on either DNA or RNA, eliminating the need for target amplification and in the case of RNA, reverse transcription The invader system amplifies a target-specific signal but not the target itself High specificity is achieved through a combination of sequence-specific oligonucleotide (SSO) hybridization and structure-specific enzymatic cleavage
3-11
Diagnostic Methodology and Technology
Invasive structure torms trom sing le-base overlap betwe en Invade r® Oligo and WT Probe when hybrid ized to WT target DNA
A
Prim ary reaction
Mismatch between Mut Probe and W ' target DNA prevents single-base overlap ; therefore, no invasive structure is formed
B
~:I""""
WT Probe
Invade 5' (
T
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3' )
A
3'
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) 5'
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1
*
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Site of cleavage
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:3}~
3'
F2
3' '--
FRET'" cassette 1
*
~_ ~~
FRETT" cassette 2
No cteavaq
Fluorescent signal
No fluor escent signal
Fig. 5. Principle of Invader cleavase technology (see text for description) (source: www.twt.com) .
- Cleavage is carried out using one of the Cleavasef (Third Wave Technologies Inc., Madison, Wisconsin) enzymes, a family of both naturally occurring and engineered thermophilic structure-specific endonucleases - This technology can accurately detect single base changes , insertions or deletions in DNA and RNA molecules - The invader assay is also well suited for target quantitation over a broad dynamic range • Principle and procedures (Figure 5) : - The basis for the invader assay is the cleavage of a unique secondary structure formed by two partially overlapping oligonucleotides (an allele-specific
primary probe and an invader probe) that hybridize to a target sequence to create a "flap" - Cleavase VIII (flap endonuclease I from Archaeoglobus fulgidus) recognizes this threedimensional (3D) structure as a specific substrate and cleaves the 5' flap of the primary probe - The flap initiates a secondary reaction in which the released 5'-flap serves as an invader probe on a fluorescence resonance energy transfer (FRET) cassette to create another overlapping tertiary structure that is, in turn, recognized and cleaved by the cleavase enzyme - When the FRET cassette is cleaved, a fluorophore is separated from a closely adjoining quencher on the FRET cassette ; the resulting free fluorophore emits a
75
3-12
Molecular Genetic Pathology
detectable fluorescence signal proportional in intensity to the concentration of the target sequence • Applications: - The invader assay can be used to genotype DNA, and has been applied in the detection of mutations in Factor V, Factor II (Prothrombin), ApoE, and methylenetetrahydrofolate reductase (MTHFR) - The assay can be used to measure gene expression
DNA
-
- Invader assays can be designed and experimentally validated for new sequences in a matter of days or weeks depending on the target type (i.e., genomic DNA, PCR products, or total RNA) - The invader assay could also be a sensitive method for detecting certain mutations associated with drug resistance in microbial pathogens • Advantages: - The invader assay is an accurate, rapid, and costeffective tool, not only for single nucleotide polymorphisms (SNP) genotyping, but also for the characterization of gene deletion and duplication events, determination of transgenic organism zygosity, quantitation of viral targets, and DNA computing readout. Its suitability for both ultra-high-throughput and low- to medium-throughput genotyping analyse s has been well established
- It has become the most widely used PCR-independent SNP and mutation detection technology. When combined with peR, the invader DNA assay is an unparalleled system for analyzing hundreds or thousands of genotypes from a single blood sample - The Invader RNA assay has been used to quantitatively measure gene expression. Applications include detection of closely related or alternatively spliced mRNAs, target validation, and high-throughput screening • Limitations: - If DNA quality is poor and quantity is low, the assay may give invalid results
Target-Based Amplification Target-based amplification methods are designed to detect and amplify the target gene of interest. Unlike probe-based amplification, the target is amplified over and over and the final signal is dependent on the amplified target.
Polymerase Chain Reaction • General information: - The PCR was invented by Kary B. Mullis in the mid-1980's
76
Denatu ration at 94°C, 30 seconds
1
Annealing at 45-55°C, 30 seconds
....
Primer 2
Primer 1
- The assay can be used directly on genomic DNA, total RNA, or celllysates without prior target amplification, or on samples previously amplified by PCR or RT-PCR - Invader assays can be used for manual, small-scale semiautomated analysis as well as fully automated, high-throughput studies
1
---
1 1 1
Extension at 72°C, 1 minute
Thermocycling
....
.... .... .... ....
Fig. 6. Mechani sm of the PCR (see text for description).
- PCR technology has produced a revolution in molecular biology, and is now the most widely used method of nucleic acid amplification in both clinical and research settings - PCR is the prototypical methodology for target-based amplification • Principle and procedure (Figure 6): - Oligonucleotide primers are designed to be complementary to the ends of the gene sequence of interest - Following heating to denature the DNA template and cooling to promote primer annealing, the oligonucleotide primers each bind to the complementary strand of the target fragment - The primers are designed to anneal in positions such that when each is extended by a DNA polymerase, the newly synthesized strands will overlap the binding site of the opposite primer - As the steps of denaturation, annealing, and extension are repeated, the primers repeatedly bind to both the original DNA template and the newly synthesized strands and are extended to produce new copies of DNA - The end result is an exponential increase in the total number of target DNA copies • Other considerations: - Prevention of contamination • A clean lab coat should be worn during sample handling/specimen processing and frequently changed • Change gloves whenever you suspect that they are contaminated
3-13
Diagnostic Methodology and Technology
PCR products analysis area
Amplification area
Sample preparat ion area
Reagent preparation area
Airflow Buffer room
Buffer room
Buffer room
Work flow
Fig. 7. Construction and floor map of a clinical PCR laboratory illustrating the principle of unidirectional work flow. Note that the PCR products analysis area is under negative air pressure ; all other work areas are under positive pressure.
• Maintain separate areas and dedicated equipment and supplies for (see Figure 7): • Reagent preparation • Sample preparation • PCR amplification • Analysis of PCR products • Never bring amplified PCR products into the PCR setup area (unidirectional work flow) • Open and close all sample tubes carefully. Try not to splash or spray PCR samples
specificity, complementary primer sequence, GtC content and polypyrimidine (T, C) or polypurine (A, G) stretches, and 3' end sequence • Primer length: since both specificity and the temperature and time of annealing are at least partly dependent on primer length, this parameter is critical for successful PCR
° Oligonucleotides between 18 and 24 bases
°
• Keep reaction tubes and tubes containing reaction components capped as much as possible • Use a positive-displacement pipet or aerosolresistant pipet tips • Clean lab benches and equipment periodically with a 10% bleach solution followed by a 70% ethanol solution - Primer design: • Primer design is a very important parameter for PCR. The primer sequence determines several things such as the length of the product, its melting temperature (T m)' and ultimately the yield . A poorly designed primer can result in little or no product due to non-specific amplification and/or primerdimer formation, which can become competitive enough to suppress product formation • Several variables must be taken into consideration when designing primers, such as primer length, Tm'
°
are extremely sequence-specific, provided that the annealing temperature is optimal Primer length is also proportional to annealing efficiency : in general , the longer the primer, the less efficient the annealing. With fewer templates primed at each step, this can result in a significant decrease in amplified product The primers should not be too short ; however, unless the application specifically calls for it
° Designing a primer with an annealing temperature of at least SO°C is the goal. The relationship between annealing temperature and Tm is one of the "Black Boxes" of PCR. A general rule-of-thumb is to use an annealing temperature that is SOC lower than the T; ° The annealing temperature determined in this fashion will not be optimal and empirical experiments must be performed to determine the optimal temperature
77
Molecular Genetic Pathology
3-14
two primers can interfere with hybridization. If the homology should occur at the 3' end of either primer, primer dimer formation will occur, which, due to competition, will prevent the formation of the desired product
• Melting temperature (T m): o
Both of the oligonucleotide primers should be designed such that they have similar Tm
o
If primers are mismatched in terms of Tm' amplification will be less efficient or may not work at all since the primer with the higher Tm will mis-prime at lower temperatures and the primer with the lower Tm may not work at higher temperatures
o
o
The Tm of oligos are most accurately calculated using nearest neighbor thermodynamic calculations with the formula : Tm (primer) = flli [dS+ R In (cf4)] 273.15°C + 16.6 log 10 [Ks-], where H is the enthalpy and S is the entropy for helix formation, R is the molar gas constant and c is the concentration of primer. A good working approximation of this value (generally valid for oligos in the 18-24 base range) can be calculated using the formula: Tm = 2(A+T) + 4(G+C) In addition to calculating the Tm of the primers, care must be taken to ensure that the Tm of the product is low enough to ensure 100% melting at
noc
o
In general, products between 100 and 600 bp are efficiently amplified in many PCR reactions
o
The product Tm can be calculated using the = 59.9 + 0,41 [G+C (%)]formula : 675/length (under condition of 50 mM KCL)
T;
• Specificity : o
o
o
Primer specificity is at least partly dependent on primer length Primers must be chosen so that they have a unique sequence within the template DNA to be amplified A primer designed with a highly repetitive sequence will result in a smear when amplifying genomic DNA. Because Taq polymerase is active over a broad range of temperatures, primer extension will occur at the lower temperatures of annealing. If the temperature is too low, non-specific priming may occur, which can be extended by the polymerase if there is a short homology at the 3' end
• Complementary primer sequences : o
o
78
Primers need to be designed with absolutely no intra-primer homology beyond 3 bp. If a primer has such a region of self-homology, partially double-stranded structures can occur, which will interfere with annealing to the template Another concern is inter-primer homology. Partial homology in the middle regions of
• G/C content, polypyrimidine (T, C) and polypurine (A, G) stretches : o
The base composition of primers should be between 45 and 55% GC
o
The primer sequence must be chosen such that there are no poly G or poly C stretches that can promote non-specific annealing. Poly A and poly T stretches are also to be avoided as these will open up stretches of the primertemplate complex . This can lower the efficiency of amplification
o
Polypyrimidine (T, C) and polypurine (A, G) stretches should also be avoided
o
Ideally, the primer will have a near random mix of nucleotides, a 50% GC content, and be approximately 20 bases long. This will put the Tm in the range of 56-62°C
• 3' end sequence: o
It is well established that the 3' terminal position in PCR primers is essential for the control of mis-priming
It is preferable for a G or C residue to occupy the 3' terminal position of each primer. This "GC Clamp " helps to ensure correct binding at the 3' end due to the stronger hydrogen bonding of G/C residues. It also helps to improve the efficiency of the reaction o Primer3 is an example of a commonly used software for primer design - When applied in clinical settings, adequate and strict QC/QA procedures must be followed o
- Heparin inhibits PCR; thus, blood samples collected with heparin should not be used for clinical PCR diagnosis. Blood samples collected with acid citrate dextrose (ACD) or EDTA anticoagulants are acceptable for use with PCR • Advantages: - PCR is extremely sensitive and can be applied in a vast number of areas - Numerous variations on the basic PCR procedure further expand the utility of this methodology • Limitations: - Potential contamination is a major concern in PCR • The sources for contamination could be samples of high DNA concentration, DNA template controls or standards, and PCR carryover contamination
3-15
Diagnostic Methodology and Technology
• Following the guidelines discussed above will lessen the risk of contamination • Additionally, use of uracil-N-glycosylase (commercially known as Ampfirase'" [Roche Diagnostics, Indianapolis, Indiana]) specifically mitigates the potential for contamination by PCR products from prior reactions • The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine, but not DNA containing deoxythymine. Deoxyuridine is not present in naturally occuring DNA, but is always present in amplicon due to the use of deoxyruidine triphosphate as one of the dNTPs in the Master Mix reagent: therefore, only amplicon contains deoxyuridine • Deoxyuridine renders contaminating amplicon susceptible to destruction by the AmpErase enzyme prior to amplification of the target DNA. The AmpErase catalyzes the cleavage of deoxyuridine-containing DNA at the deoxyuridine residues by opening the deoxyribose chain at the CI-position • When heated in the first thermal cycling step the amplicon DNA chains break at the positions of the deoxyruidine, thereby rendering the DNA non-amplifiable • The AmpErase enzyme is inactivated by temperatures above 55°C, and therefore does not destroy target amplicon formed during amplification - Non-specific amplification is another concern in PCR. Some technologies have been invented to prevent this phenominon • Hot start PCR • Hot start PCR can improve PCR specificity by controlling mispriming events • In hot start PCR, reactions are designed such that the polymerase only becomes active at a high temperature, thus ruling out the possibility of non-specific amplification at lower temperature • Hot start PCR technique can be realized by manually adding the key components at higher temperature, which is cumbersome and timeconsuming • Various commercially available reagents can simplify the hot start procedure. For example, AmpliTaq Gold® DNA polymerase (Applied Biosystems, Foster City, CA) is a chemically modified form of AmpliTaq DNA polymerase, which is only active at high temperature. Binding to an aptamer blocks polymerase activity at low temperatures
Variations of the peR Reverse Transcription PCR (RT-PCR) • General information: - Reverse transcription coupled with the PCR (RTPCR), has proven extremely useful in the study of gene expression - RT-PCR can detect the RNA transcript of any gene, regardless of the amount of starting material or the relative abundance of the specific mRNA - In RT-PCR, an RNA template is reverse transcribed to a DNA (cDNA) using a retroviral transcriptase - The cDNA sequence of interest is then amplified exponentially using PCR. Detection of the PCR product is typically performed by agarose gel electrophoresis and EB staining or by the use of radiolabeled or fluorescent labeled nucleotides or primers in the PCR • Principle: - RT-PCR is a combination of reverse transcription and PCR - Two basic reactions are involved in RT-PCR • cDNA is synthesized from an mRNA template by avian myeloblastosis virus or moloney murine leukemia virus (MMLV or MuLV) RTase • The second cDNA strand is synthesized and subsequent PCR amplification is performed with Taq DNA polymerase as conventional PCR • Procedure (Figure 8): - Synthesis of the first DNA strand in the presence of primers, dNTPs, and an RNA-dependent DNA polymerase - RNaseH (an RNA digestion enzyme) is added to digest the RNA from the RNA-cDNA hybrid - Second strand reaction. Standard PCR is conducted using DNA oligo primers specific for the sequence of interest • Applications: - RT-PCR is the most sensitive technique for mRNA detection and quantitation - The procedure can be either qualitative or quantitative
- It is also used to create cDNA libraries • Advantages: - Sensitivity: RT-PCR is the most sensitive technique for mRNA detection and quantitation currently available . Theoretically, a single copy of mRNA can be detected by this technique. In practice, tens to hundreds of copies are required for reliable quantitation - Sample integrity requirements: since most RT-PCR methods amplify only a few hundred bases rather than the complete mRNA sequence, the slight degradation of sample can be tolerated
79
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Molecular Genetic Pathology
Synthesis of first DNA strand from mRNA (1) Random primer
5"- - - - - - - - - - - - - - - - - - .. N6
" N6
.. N6
.. N6
AAAAAA
3'
mRNA First strand DNA
3' 5'
mRNA First strand DNA
(2) Oligo(dT) primer
5'
AAAAA _ - - - - - - - - - - - - - - - - - - TTTTT (3) Sequence specific primer AAAAAA
3'
5'
3'
mRNA First strand DNA
I Fig. 8. Principle and procedure of RT-PCR (see text for description).
- Quantitation: • Like other methods of mRNA analysis , RT-PCR can be used for relative or absolute quantitation • Relative quantitation compares transcript abundance across multiple samples, using a co-amplified internal control, which ideally has invariant expression within those samples, for sample normalization, i.e., the bcr-abl assay • Absolute quantitation using competitive RT-PCR measures the absolute amount of a specific mRNA sequence in a sample . Dilutions of a synthetic RNA molecule (identical in sequence, but slightly shorter than the endogenous target) are added to sample RNA replicates and are co-amplified with the endogenous target. The PCR product from the endogenous transcript is then compared with the concentration curve created by the synthetic "competitor RNA" • It is also possible to do real-time RT-PCR quantitation by measuring an internal control in the samples • Limitations: - Sample purity requirements: because of its sensitivity, the technique of RT-PCR requires that sample s be free of genomic DNA or other DNA contaminants. Special care must be taken during RNA isolation to ensure that the sample RNA is DNA-free - Optimization of RT-PCR assays can be technically challenging. The design of suitable primers and
80
controls often requires substantial pre-experimental planning - In relative RT-PCR, the choice of internal standard is critical. An ideal internal standard is one with invariant expression during the cell cycle, between cell types, and in response to the experimental treatment under analysis. p-actin, GAPDH and GUS are commonly used housekeeping genes used as internal standards. - In relative RT-PCR, the products must be analyzed while the PCR is still in exponential phase for both the target and the reference amplicon. Thus, pilot experiments are required both to validate the internal control and to determine cycling parameters for the exponential amplification phase of all targets to be studied - Competitive RT-PCR makes use of an exogenous RNA transcript (competitor) that must be accurately quantitated and added to replicate samples in amounts that span the range of the target mRNA levels. Experimentation is needed to determine the amount of competitor required and to ensure that the target and competitor sequences are amplified with equivalent efficiencies, yet are discernible by gel electrophoresis or by fluorescent probes
Real-Time
res
• General information: - Real-time PCR systems are based on the detection and quantitation of a fluorescence signal
3-17
Diagnostic Methodology and Technology
The fluorescence signal increa ses in direct proportion to the amount of PCR product formed . Fluorescence is measured repeatedly with each PCR cycle. Quantitation of product is therefore based on measurements performed during the amplification process, as opposed to the end point detection of conventional PCR - A fixed fluorescence threshold is set significantly above the baseline fluorescence level and can be altered by the operator. The parameter CT (threshold cycle) is defined as the cycle number at which the fluorescence emission exceeds the fixed threshold - The first cycle at which the level of fluorescen ce exceeds the threshold correlates to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed . Samples with a lO-fold target difference demonstrate a 3.3 cycle difference Real-time PCR quantitation eliminates post-PCR processing of PCR products. This helps to increase throughput and reduces the chances of carryover contamination - In comparison with conventional PCR, real-time PCR offers a much wider dynamic range (7-8 log). Thu s, accurate quantitation can be achieved over a very large range of initial target concentration - Real-time PCR can also be used to detect mutations and SNPs, with the help of melting curve analysi s • Principle and procedure: - Real-time PCR is based on the same fundamental principles as conventional PCR; however, the incorporation of fluorescent markers in the reaction mixture permit s real-time monitoring of amplification - There are two types of probes used in real-time PCR: • DNA-binding dyes, such as SYBR® Green (Molecular Probes , Invitrogen, Carlsbad , CA) • Sequence-specific probes, including TaqMan® (Applied Biosystems, Foster City, CA), hybridization probes , molecular beacons , and scorpions'" (DxS Ltd., Manchester, United Kingdom) (see Signal Detection Methods section) • Applications: - Quantitation of gene expression - Array verification - Drug therapy efficacy/drug monitoring - Real-time immuno-PCR (IPCR) - Viral quantitation - Pathogen detection, including CMV detection, rapid diagnosis of meningococcal infection, penicillin susceptibility of Streptococcus pneumoniae, Mycobacterium tuberculosis and its resistant strains, and waterborne microbial pathogens in the environment - DNA damage (microsatellite instability) measurement
-
Radiation exposure assessment In vivo imaging of cellular processes Mitochondrial DNA studies Methylation detection Detection of inactivation of X chromosome Determination of identity at highly polymorphic human leukocyte antigen (HLA) loci
- Monitoring post-transplant solid organ graft outcome - Monitoring chimerism after haematopoietic stem cell transplantation - Monitoring minimal residual disease after haematopoietic stem cell transplantation - Genotyping by fluorescence melting-curve analysis (FMCA) or high-resolution melting analysis (HRMA) - Detection of trisomies and single-gene copy numbers - Microdeletion genotypes - Haplotyping - Quantitative microsatellite analysis - DNA pooling and quantitative allelic discrimination - Prenatal diagnosis/sex determination using single cell isolated from maternal blood or fetal DNA in maternal circulation - Prenatal diagnosis of hemoglobinopathies - Intraoperative cancer diagnostics - Linear-after-the-exponential (LATE)-PCR: a new method for real-time quantitative analysis of target numbers in small samples, which is adaptable to highthroughput applications in clinical diagno stics, biodefense, forensics, and DNA sequencing • Advantage s: - Homogeneous assay: i.e., amplification and detection steps occur simultaneously and in the same reaction vessel. Since the PCR products do not need to be manipulated after amplification in order to perform a separate detection step, this limits the potential for contamination Elimination of post-PCR processing saves time and labor costs, facilitating high-throughput Measurement of PCR product during exponential phase of amplification (rather than the plateau phase) permits precise quantitation, as during the exponenti al phase, none of the reaction components are limiting Large dynamic range (7-8 log) allows for very high sensitivity It can detect as little as a two-fold target change High degree of reproducibility Multiplexing capability (internal control , single or multiple targets, may be amplified and detected simultaneously in one reaction tube using multiple fluorescent probes of different colors) Can use integrated quantification standard (obviates need for external standard curve)
81
Molecular Genetic Pathology
3-18
-
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Fig. 9. Principle and procedure of Nested PCR. (see text for description).
• Limitations: - Special instruments are required to perform real-time PCR - Real-time PCR is susceptible to PCR inhibition by compounds present in certain biologic samples . To circumvent this problem, alternative DNA polymerases (e.g., Tfl, Pwo, Tth, and so on) that are resistant to particular inhibitors can be used - Increased risk of false-negative values without use of an internal standard
Nested peR • General information: - Nested PCR is a modification of conventional PCR - Two internal primers are used to amplify the PCR products of two external primers - Nested PCR is intended to increase fold of amplification and amplification specificity • Principle: Nested PCR involves two sets of primers and two successive PCR procedures. The second primer set functions in the amplification of a secondary target within the first run product • Procedure (Figure 9): - The target DNA undergoes the first run of PCR with the first set of primers - The amplicons from the first reaction undergo a second run with the second set of primers • Advantages: - Increased sensitivity over conventional PCR - Increased specificity due to enrichment for the target sequence with the first round of amplification
82
• Limitations: - Because the first-round PCR products need to be transferred prior to the second round, nested PCR is highly susceptible to contamination
Amplification Refractory Mutation System (ARMS) • General information: - ARMS is a well-established PCR-based method for the detection of SNPs and other genetic variations. ARMS is a variation of allele specific PCR • Principle: - In traditional allele specific PCR, two parallel reactions are carried out in separate tubes; one primer is common to both tubes; however, the other primer (the terminal 3'-nucleotide of which overlaps the mutation site) differs between the two tubes. The "control" tube primer set includes a wild-type-specific primer, and the other tube includes a mutation-specific primer - Since the primer sets are allele-specific, the reaction containing the wild-type primer set is refractory to PCR amplification of mutant template DNA. Conversely, the reaction containing the mutant primer set is refractory to PCR amplification wild-type DNA - An important phenomenon to note is that, in some instances, a single 3'-mismatched base does allow amplification to proceed . To ameliorate this problem, an additional deliberate mismatch near the 3' end (in most situations, two bases way from the 3' terminus) of the mutation-specific and wild-type-specific primers is introduced In ARMS, both allele specific primers (wild type and mutant) are included in one tube along with a set of outer primers that span the SNP site
3-19
Diagnostic Methodology and Technology
- The outer primers will amplify sequences 1) between themselves, 2) between the wild type primer and 3) between the mutant primer. Thus, three differently sized amplification products (i.e., the longer outer products or the shorter wild type and mutant products) are formed and can be separated via gel electrophoresis • Applications: - Genotyping: • • • • • •
Genetic testing Pharmacogenetics Personalized medicine Predispositon testing Research genotyping Advantages in this application: • Very reliable mutation detection method allowing detection of all SNPs and indels • Converts non-specific PCR detection methods such as intercalation, amplifluor, and lux into genotyping methods
Q-genotyping • Sample pooling • Viral genotyping • Tumor analysis • Cancer screening and early detection • Cancer monitoring • Advantages in this application: • Allows quantitative genotyping when combined with real-time PCR • Excellent SNP discrimination ability allows detection of genetic variations when only a small proportion of the sample carrie s the mutation Haplotyping • • • •
Haplotyping HLA testing Promoter analysis Advantages in this application: • Combination of two ARMS primers allows direct analysis of the phases of associated SNPs
• Identifies whether SNPs are on the same chromosome Multiplexing • Multiplex genetic testing • High-throughput genotyping • Advantages in this application: • Several ARMS primers can be combined into one reaction • Compatible with micro-array detection • Limitations: - Assay optimization and validation may be difficult to perform
Ligase Chain Reaction • General information - It was first described by Francis Barany in 1991 - LCR achieves amplification in a similar fashion to PCR except that DNA ligase is used - Unlike other probe-based amplification technologies, LCR requires temperature cycling - LCR uses two sets of primers that hybridize to the target DNA strand at adjacent locations • Principle: - LCR utilizes two pairs of oligonucleotides as primers ; one pair is complementary to each strand of the target sequence - Each pair of primers is designed to cover the entire sequence to be amplified, without leaving space between them - The 3'-hydroxyl end of the first primer is adjacent to the 5'-phosphate end of the second primer of one pair when they anneal to the template. Therefore, the sequence to be amplified is defined by the continuous span of the primers brought together on the template - The nick between the primers is covalently sealed by DNA ligase. Provided that the nucleotides nucleotides at the junction are correctly base-paired to the target, a fragment is generated equating to the total sequences of the primers of each pair - The products of one round of ligation serve as templates for subsequent cycles. Consequently, an exponential amplification of the desired fragment defined by the primers is achieved by using a thermostable DNA ligase and performing sequential cycles of denaturation and ligation • Procedure (Figure 10): - At low temperature, the two pairs of adjoining probe s each anneal to their respective target strand - The 3' end of one probe is covalently linked to the 5' end of the adjoining probe through the action of a thermostable DNA ligase at a temperature of
n oc
- The temperature is then further raised, such that the ligated probes dissociate from the target DNA strand and are available to serve as templates for another set of probes to hybridize and ligate - Multiple cycles of annealing, ligation, and denaturation are performed resulting in exponential target amplification • Applications: - Since a single nucleotide substitution in the template sequence can prevent the ligation reaction , LCR can be used for genotyping point mutations and SNPs - LCR can also be used for the detection of many microorganisms, such as HIV, HBV, M. tuberculosis, C. trachomatis, and Neisseria gonorrhoeae in clinical specimens
83
3-20
Molecular Genetic Pathology
~~I 1.Anneal oligonucleotides
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2. Ligate with thermophilic ligase
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iii
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Fig. 10. Principle and procedure of LCR (see text for description) .
- The LCR assay kits (LCx) for C. trachomatis and N. gonorrhoeae are marketed by Abbott Diagnostics (Abbott Park, IL). An HIV-I LCx commercial test is available outside the United States • Advantages: - LCR is extremely specific because it amplifies only the fragment comprised by the primer sequences, with no intervening nucleotides • Limitations : - Its limitations come from the specificity of the LCR, which is restricted to the region of the ligation junction. Mutations outside that region are not detected - Due to more specific ligation events (0.1-1 .0%), false positive results can occur.
Strand Displacement Amplification • General information - SDA is a mostly isothermal nucleic acid amplification method -
109 copies of target DNA sequence can be generated in one reaction
- Requires use of special polymerases (e.g., Bst DNA polymerase or Phi 29 polymerase) and special thiolated dCTP • Principle and procedure (Figure llA,B): - SDA is a 2-step procedure : target creation and exponential target amplification • Target creation: two sets of primers are applied in this step: Bland S1; B2 and S2. S1 and S2 are
84
special primers that contain a restriction enzyme recognition sequence (enzyme BsoBI) 5' to the target-binding region. S1 primed product is displaced and serves as a template for another set of primers, B2 and S2. BsoBI cleaves between the first and second nucleotides at the 5' end of S1 and S2 but cannot cleave between nucleotides joined by a phosphorothioate linkage • Exponential target amplification: BsoBI enzyme is used in this step to nick the S 1- and S2-primed amplicons. After nicking, the DNA polymerase binds to this nick and begins the synthesis of a new strand while simultaneously displacing the downstream strand. This cycle of alternate nicking and displacement repeats. The displaced strands are capable of binding to opposite strand primers, which produces exponential amplification at 52.5 °C • Applications: - The broadest application of SDA is in the diagnosis of infectious diseases The BD ProbeTec ET system is widely used with screening assays for C. trachomatis (CT) and N. gonorrhoeae (GC) in urogenital specimens - SDA is readily coupled with FRET probes to permit homogenous real-time detection of amplified products • Advantages : - A rapid DNA amplification method that can produce target in excess of 109 copies in less than 15 minutes - Easily adapted to microarray-based applications
Diagnostic Methodology and Technology
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Fig. 11. (A) Target generation in SDA: the S 1 primer (which contains a restriction enzyme recognition site, indicated by doubleline) binds to the target DNA sequence. The B I "bumper" primer binds upstream to the S1 primer (1). Extension from the B I primer displaces the extension product of the S I primer (2). The S2 primer (which also contains a restriction enzyme recognition site) then binds to the SI extension product. The B2 "bumper" primer then binds upstream of the S2 primer (3). The S2 extension product is in turn displaced by extension from the B2 primer (4). Binding of an S1 primer to the S2 extension product (5), with subsequent extension, produces a double-stranded molecule with a restriction site at each end (6). Since extension incorporates phosphothiorate-modified nucleotides, newly synthesized portions of the strands are resistant to cleavage, and restriction enzymes can only cleave restriction sites that were present in the original S1 or S2 primers. Therefore, the action of the restriction enzyme produces single-strand nicks (designated by arrows) in the double-stranded molecule. Extension from the nick sites displaces strands into solution, which contain partial restriction sites (7); it is these strands that enter the exponential amplification stage of the reaction (see B). Meanwhile, the double-stranded molecule can regenerate itself, in order to undergo additional cycles of strand displacement. (B) Exponential amplification of the target sequence : single strands produced by the target-generation reaction (in A) hybridize with SI and S2 primers (1). Both the primer and its corresponding target strand then undergo extension from their 3' ends. The resulting double-stranded molecule (2) is nicked by the restriction enzyme. 3' extension from the nicked site releases a single strand into solution (3). Binding of an S1 or S2 primer to the displaced strand continues the cycle.
85
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Molecular Genetic Pathology
• Limitations: - Not completely isothermal; an initial heat denaturation step is required - Complicated reaction mechanisms and primer designs
Transcription Mediated Amplification • General information: - TMA is an isothermal nucleic acid amplification method - TMA uses RNA transcription (RNA polymerase) and DNA synthesis (RTase) to produce RNA amplicons from a target nucleic acid
It can be used to target both RNA and DNA - TMA produces 100-1000 copies per cycle in contrast to PCR that produces only two copies per cycle. This results in a 10 billion-fold increase of copies within about 15-30 minutes - Commercial TMA kits are available with the GenProbe" Amplified" (Gen-Probe Inc., San Diego, California) products-APTIMA® Combo 2™ (Aptima, Woburn, Massachusetts) for C. trachomatis and N. gonnorhoeae, Amp CT Assay for C. trachomatis and MTD Test for M. tuberculosis - Contamination is not a major issue because of the labile nature of RNA - TMA has been combined with the Gen-Probe" hybridization protection assay (HPA) detection technique in a single tube format • Principle: - TMA is an RNA transcription amplification system using two enzymes: RNA polymerase and RTase, and two primers: a promoter primer and a regular primer. RNA polymerase mediates transcription, resulting in 100-1000 copies of RNA amplicon per DNA template. Reverse transcriptase creates a DNA copy of the target RNA. The promotor primer contains a promoter sequence recognized by RNA polymerase. The other primer works for RTase - First, the promoter-primer binds to the target rRNA, and RTase creates a DNA copy of the target rRNA. The RNA in the resulting RNA:DNA hybrid is cleaved by the RTase - The regular primer hybridizes to the DNA copy, and RTase produces the second DNA strand . RNA polymerase recognizes the promoter sequence in the DNA template and initiates transcription - An exponential amplification is achieved by repeated cycles of reverse transcription and DNA synthesis • Procedure (Figure 12): - Promoter-primer binds to rRNA target - Reverse Transcriptase (RTase) creates a DNA copy of the rRNA target - RNA:DNA duplex is formed
86
- RNAseH activity of RTase degrades the RNA strand - Primer 2 binds to the DNA, and RTase creates a new DNA copy - Double-stranded DNA template with a promoter sequence is formed - RNA polymerase (RNA Pol) initiates transcription of RNA from DNA template
- 100-1000 copies of RNA amplicon are produced - Primer 2 binds to each RNA amplicon and RT creates a DNA copy - RNA:DNA duplex is formed - RNAse H activity of RT degrades the RNA strand - Promoter-primer binds to the newly synthesized DNA. RT creates a double-stranded DNA and the autocatalytic cycle continues, resulting in exponential amplification • Applications: - The amplified M. tuberculosis direct test (Gen-Probe) for detection of M. tuberculosis in clinical samples and provides accurate same day test results - Other assays for C. trachomatis, HIV, chronic myelogenous leukemia (CML) and M. avium complex are also available or in development • Advantages: Improved reliability by targeting abundant ribosomal RNA. Since rRNA is present in thousands of copies per cell, the likelihood of initiating amplification is greater than when single copy DNA targets are used. This advantage is very important when organisms are present in low numbers, which is when target amplification methods are most useful - Single temperature exponential amplification. The procedure is simple to perform , does not require costly thermocycler equipment, and provides rapid amplification of target sequence present in the sample - Primary RNA amplicon. The RNA product of the amplification system is more labile outside the reaction tube than DNA product made by other amplification systems . The risk of laboratory contamination and false-positive results is thus substantially reduced - Single tube solution format with no wash steps. Reagents are only added to the amplification tube and never removed or transferred . This again minimizes the chance of cross-contamination and false-positive results. The single tube, no-wash format also allows for the development of relatively simple instrumentation to automate the amplification and detection steps - Simplicity. With few reagent additions, and hybridization protection assay detection, the format is user-friendly and familiar to laboratories already using the Gen-Probe DNA probe assays • Limitations: Complicated reaction mechanisms and experimental designs - Inefficient in amplification of long target sequences
3-23
Diagnostic Methodology and Technology
(1)
rRNA target
(2)
(3)
...........
1 I::I'= ..::J: i:I II: ! iC1I:I1I:I:l: 1I:l ! I::II:III:! 'C'I:I'I: ' :I: 'C':I'I: I ' :I: "I:lI::II:I 'C1I:I!I: I :l: ' ' 'I: I' : l
OJ
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,
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-
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~ I I I I " I ! II I I III IIIII !iii ! ii i ! lii l l l l ! 1
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t
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t (9)
+-
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I I! 11111 III I! 11111 11 111 I II! 11111 I I ! I ! ! !
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Fig. 12. Mechanism of transcription-mediated amplification (TMA) (see text for description).
SIGNAL DETECTION METHODS General Information Before the application of fluorescence detection methods , gel electrophoresis was the standard method for detection of amplified nucleic acid products (see below) . Currently, most real-time amplification detection is via fluorescence. Realtime fluorescence detection of amplifi cation products is rapid, reducing assay time. An additional benefit is that the product can be detected within the reaction tube, markedly reducing the potential for carryover contamination.
DNA-Binding Dyes SYBR Green • DNA binding dyes such as SYBR @green are cost effective and easy to use, especially for researchers who
are new to using real-time PCR. SYBR@green is a double-stranded DNA-binding dye. When free in solution, SYBR@Green I displays relatively low fluorescence, but when bound to double -stranded DNA, its fluorescence increases by over lOOO-fold. The greater the quantity of double- stranded DNA present, the more binding sites are available for the dye; thus the intensity of the fluorescent signal is proportional to the DNA concentration (Figure 13) • SYBR@Green is the simplest, easiest to use, and most economical chemistry for real-time PCR. Since SYBR@ Green binds double-stranded DNA in an indiscriminate manner there is no requirement for target-specific probes . Use of SYBR@Green is compatible with existing PCR reagent s and protocols
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Molecular Genetic Pathology
Unbound SYBR green
Bound SYBR green
Fig. 13. SYBR®Green binding to DNA.
• SYBR ®Green is often used for initial expression validation screening of micro array sample s as well as for other gene expression applications not requiring exceptional sensitivity and specificity. Optimization of primers to use with SYBR ®green is straightforward and provides a high level of experimental design success • At high concentrations, SYBR®green inhibits enzymes, including RTase and Taq DNA polymerase. Therefore, optimal concentration of SYBR®green must be experimentally determined in order to balance amplification efficiency and sensitivity with fluorescence intensity • Several kits are commercially available for optimization of PCR reactions using SYBR green dye . Examples of these kits include: Power SYBR ®Green RT-PCR Reagents Kit from Applied Biosciences (Wilmington, North Carolina, USA); and iQ SYBR Green Supermix" from Bio-Rad (Bio-Rad Laboratories, Hercules, CA, USA) • The limitations of SYBR ® Green methodologies derive primarily from the dye's ability to bind to any doublestranded DNA in the reaction mixture, including primer-dimers and other non-specific reaction products. Therefore, the dye cannot distinguish between specific and non-specific products accumulated during PCR . This problem can be mitigated by inclusion of a non-template control in each run. In addition, though non-specific signal cannot always be prevented, its presence can be easily and reliably detected by performing melting curve analysis on the PCR products
Probe-Based Chemistries GeneralInformation As compared with non-specific chemistries such as SYBR® Green, a higher level of detection specificity is provided by the use of fluorescent sequence-specific probes to detect PCR products. For the most part, these detection mechanisms rely on the principle of FRET, in which a
88
fluorescent dye in the excited state, can transfer energy to either another (acceptor) fluorophore, with subsequent emission of a fluorescent signal from the receptor molecule, or to a quencher dye, which dissipates the energy without emission of a detectable fluorescence signal. In order for this energy transfer to take place , the donor and acceptor molecules must be situated in close physical proximity. Probes are designed such that in the absence of a specific target sequence in the reaction mixture, the desired fluorescence signal is not produced . However, when the probe (or probes) hybridizes to the target sequence, the level of fluorescence detected is directly related to the amount of amplified target in each PCR cycle. In addition to enhanced specificity, another significant advantage of using sequencespecific probes is that multiple probes can be labeled with different reporter dyes and combined to allow detection of more than one target in a single reaction. There are two general types of sequence-specific fluorescent probes: linear probes, which include TaqMan probes (hydrolysis probes) and hybridization probes; and structured probes, which include molecular beacons and scorpions.
LinearProbes • General information: Linear probes (TaqMan® probes and hybridization probes) are the most widely used detection methods in realtime PCR . - Taqlvlan'" probes: • The TaqMan® probe is a linear oligonucleotide with one end labeled with a fluorescent reporter dye and the other end bonded to a quencher. More specifically, a fluorescent dye, typically 5'fluorescein phosphoramidite (FAM) (reporter), is attached to the 5' end of the probe, and a quencher, historically tetramethyl-6carboxyrhodamine (TAMRA), is attached at 3' end. Increasingly, more effective quenchers such as the Black Hole Quenchers are replacing the TAMRA because they provide lower background fluorescence • In the absence of gene targets , the reporter and quencher dyes are maintained in close proximity, such that FRET takes place and no fluorescence is detected at the reporter dye's emission wavelength. TaqMan probes use a FRET quenching mechani sm where quenching can occur over a fairly long distance (100 A or more, depending upon the f1uorophore and quencher used). Thus, as long as the quencher is on the same oligonucleotide as the f1uorophore, quenching will occur • The probe is designed to anneal to one strand of the target sequence just slightly downstream of one of the primers . During PCR, when the polymerase replicates a template on which a
3-25
Diagnostic Methodology and Technology
Quencher
Fluorophore
0 \ '-_ _/
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........................- --
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.
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Fig. 14. TaqMan probe chemistry mechanism.
TaqMan probe is bound, the 5'-exonuclease activity of the polymerase cleaves the probe into separate single bases, releases free reporter dye into solution, and the fluorescence is detected (Figure 14) o
It is very important to adjust the thermal profile to facilitate both the hybridization of probe and primers, and the cleavage of the probe. To meet these requirements, probes will generally have a two-step thermal profile with a denaturing step (usually at 95°C) and a combination annealing/extension step at 60 oe , 7-lOoe below the Tm of the probe. If the temperature in the reaction is too high when Taq DNA polymerase extends through the primer (such as at a standard extension temperature of n°C) the probe will be stranddisplaced rather than cleaved and no increase in fluorescence will be seen
• In addition to DNA quantification in biologic samples, TaqMan can also be used for SNP detection or mutation analysis. For these applications, a separate probe is designed to be complementary to each allele, and each probe is labeled with a different fluorophore (e.g., with FAM and 5'-Hexachloro-Fluorescein Phosphoramidite [HEX]). In these assays , it is often a challenge to optimize conditions as to prevent the probes from cross-reacting with the wrong allele . Most often, enhanced specificity for SNP and allele discrimination analysis is achieved by using either one of the structured probe
chemistries (see Structured Probes section) or by using a new type of TaqMan probe known as an MOB (minor groove- binder) probe. • MOB probes are similar to the standard TaqMan probes, but they include the addition of a minor groove-binding moiety on the 3' end that acts to stabilize annealing to the template . The stabilizing effect that the MOB group has on the Tm of the probe allows for the use of a much shorter probe (down to around 13 bp). The shorter probe sequence is more susceptible to the destabilizing effects of single bp mismatches, which makes these probes better than standard TaqMan®probes for applications that require discrimination of targets with high-sequence homology - Hybridization probes : • As opposed to hydrolysis probe methodologies, where only one probe is applied, hybridization probe methodologies utilize two oligonucleotide probes. One probe is labeled with a donor fluorophore at the 3' end, and the other is labeled at the 5' end with an acceptor fluorophore. The probe s have sequen ce specificities such that when both probes bind to the target sequence, the donor and acceptor fluorophores are brought into close proximity, allowing FRET to occur. When the donor fluorophore absorbs energy from an excitatory signal, the energy is then transferred to the acceptor fluorophore , which in turn emit s light at a specific wavelength that can be measured
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Molecular Genetic Pathology
9••
Dark quencher on separate oligo
I
+
Molecular beacon
PCR prime r
I Target
Hybrid
Fig. 15. Molecular beacon chemistry mechanism (http://www. stratagene.com/tradeshows/lntroduction_to_Quantitative_PCR_ web.pdf).
I
I
Fluorophore
Blocker Probe
Fig. 16. Elements of a scorpion primer (courtesy of DxS, used with permission).
Structured Probes • General information: Structured probes contain stem-loop structure regions that confer enhanced target specificity when compared with traditional linear probes. This characteristic enables a high level of discrimination between similar sequences. Therefore, structured probes are well suited for SNP and allele discrimination applications. Molecular beacons and scorpions are common types of structured probes. • Molecular beacons: - Molecular beacons form a stem-loop structure, where the central-loop-sequence is complementary to the target of interest and the stem arms are complementary to each other. One end (typically 5') of the stem is labeled with a fluorescence reporter and the other end is bound to a quencher - Instead of using FRET, molecular beacons use ground-state or static quenching, which requires the fluorophore and quencher to be in very close proximity for quenching to occur. Historically DABCYL, or Methyl Red, has been used for this application - In the absence of target, the close proximity of the reporter and quencher prevents the probe from fluorescing. However, when a molecular beacon hybridizes to a target, the probe becomes linear, the fluorescent dye and quencher are separated, and a fluorescence signal is omitted. Unlike TaqMan probes, molecular beacons utilize shape change to emit fluorescence instead of the exonulease activity of polymerase. Thus, the probe remains intact during the amplification reaction (Figure 15) In the absence of binding to the target sequence, the molecular beacon's thermodynamic properties favor the formation of a hairpin structure over mismatched binding. This property gives molecular beacons the increased mismatch discrimination that makes them well suited for applications such as SNP detection and allele discrimination
90
- Careful design of the molecular beacon stem is critical. If the stem structure is too stable, target hybridization can be inhibited. Conversely, if the molecular beacon probe does not fold in the expected stem loop conformation, it will not quench properly. Melting curve analysis can be used to determine whether or not molecular beacons have performed according to expectation • Scorpions: - Scorpions are bifunctional molecules containing a PCR primer covalently linked to a probe (Figure 16). The fluorophore in the probe interacts with a quencher, which reduces fluorescence. During a PCR reaction the fluorophore and quencher are separated, which leads to an increase in light output from the reaction tube (Figure 17) - There are two formats of Scorpions. One is the bimolecular linear scorpion format. The alternative is the uni-molecular stem-loop format. The fluorescence of scorpion primer-probes is normally quenched. Upon primer-mediated DNA synthesis of the gene targets, the scorpion probes hybridize to the newly formed complementary sequences, separating the fluorescent reporters from the quenchers thus producing fluorescence - Sensitivity-the limit of detection is a few molecules even in the presence of very high levels of background DNA - Specificity-both the scorpion primer region and probe region can be made sequence specific . This gives unparalleled discrimination allowing the detection of single nucleotide changes even in admixtures where the alternative sequence is in vast excess - Speed-signal generation is exceptionally fast. This means that the technology can support very rapid PCR, allowing detection in less than 10 minutes - Detecting difficult sequences-the intramolecular signal generation mechanism means that scorpions are particularly good at detecting targets in regions of high G+C content or secondary structure
3-27
Diagnostic Methodology and Technology
Step 1 - The scorpion primer is extended on target DNA
Step 2 - The extended primer is heat denatured and the quencher disassociates
Step 3 - As it cools the extended scorpion undergoes an internal rearrangement and begins to fluoresce in a target specific manner. Unextended primer is quenched
Fig. 17. Scorpion primer fluorescence mechanism (courtesy of DxS, used with permission).
NUCLEIC ACID HYBRIDIZATION METHODS
Hybrid Capture (HC) • General information - An in vitro, solution hybridization, signalamplification test for detecting DNA or RNA targets, mainly used in molecular microbiology - Probe: target-specific, single-stranded RNA probes (riboprobes) - Target: usually the entire genome • Principle and procedure (Figure 18): - Target DNA is isolated
- Double-stranded DNA is denatured (producing single-stranded DNA) - Sample DNA in solution is hybridized with an RNA probe or probe cocktail to form specific DNA-RNA hybrids - Hybrids are immobilized by antibodies bound to the tubes or wells of a microtiter plate that specifically recognize RNA-DNA hybrids - Multiple detector antibodies bind with each immobilized target-probe hybrid (first signal amplifier). The detector antibody is a second RNA-DNA-specific
91
Molecular Genetic Pathology
3-28
2
3
4
5
Fig. 18. Principle and procedure of HC hybridization (see text for description). Adapted from http://www.diagnostictechnology. com .au/products/digene/ overview.html .
antibody, with each antibody molecule conjugated to multiple molecules of ALP (second signal amplifier) - After removal of excess antibodies and unhybridized probes , each immobilized ALP enzyme reacts with numerous molecules of dioxetane substrate to produce a chemiluminescent product (third signal amplifier) . The resulting light signal is detected by the photomultiplier tube of a luminometer - The intensity of emitted light, expressed as relative light units, is proportional to the amount of target DNA present in the specimen, providing a semiquantitative measurement • Applications: - The major clinical use is to detect human papilloma viruses (HPV) in cervical specimens to determine appropriate follow-up, primarily in older patients and in patients with atypical squamous cells of undetermined significance (ASCUS) • First introduced by Digene in 1995 to detect 14 HPV-types including high-risk types (HPV 16, 18, 31, 33, 35, 45, 51, 52, and 56) and low-risk types (HPV 6, II , 42, 43, and 44) • Increases the sensitivity of the conventional Pap test • Provides a meaningful negative predictive value for assessing cervical dysplasia • Second generation of HC assay for HPV DNA detection developed recently • Approved by the Food and Drug Administration (FDA) for the detection of high-risk HPV types • Replaces tubes with a microtiter plate. Currently available in a 96-well microplate format • Often only the high-risk cocktail is used, in order to reduce the time and cost of the test • Can detect viral loads as low as 1 pg of viral DNA per milliliter • Has a higher diagnostic sensitivity (exceeding 90 percent) than cervical cytology
- It is also used to detect other infectious agents in clinical materials including: • N. gonorrhoeae and C. trachomatis in a single specimen (one specimen, two separate probes)
92
• Herpes simplex viruses (HSV) in vesicle material , and CMV in blood and other fluids • Other viral gene detection such as HBV • Advantages - Non-radioactive and relatively rapid method - No target amplification, no cross-contamination like PCR - No unwanted side reactions such as reannealing - RNA-DNA hybrids more stable than DNA-DNA hybrids - Semiquantitative measurement - Easy to perform in clinical settings, and suitable for automation (HC II) - HPV DNA testing : • Higher sensitivity than culture or immunologic methods and higher specificity than PCR method • Almost 100% negative predictive value for the detection of high grade squamous cervical intraepitheliallesions (HSILs) • Identification of women with concurrent cervical disease as well as those at risk of developing disease in the future • Effective way to perform quality assurance of cytology • Currently the only FDA approved test for high-risk HPV • Limitations - Lower sensitivity than PCR or other target amplification methods For HPV HC II testing • It only detects the most common high-risk HPV types • It cannot determine the specific subtype of virus, thus having multiple positive tests does not necessarily indicate persistent infection with the same type of high-risk HPV because serial infection with different HPV-types is a common phenomenon • No benefits to women who are younger than 30, those who are immunosuppressed (including AIDS), and those who have had a total hysterectomy (with removal of the cervix) for benign gynecologic disease • Not a quantitative assay • Cross-hybridization can occur between HPV-types 6 and 42 (low risk) and the high-risk probes , producing false-po sitives for high-risk HPV • False-positives can also occur due to crossreactivity of HPV probes with bacterial plasmid pBR322 present in some genital samples • Very low levels of infection or sampling error can produce false-negatives • Specimen contamination with antifungal cream, contraceptive jelly, or douche can produce false-negatives • Not recommended for evaluation of suspected sexual abuse
Diagnostic Methodology and Technology
3-29
Biotin
:!!;!!~;;;;;; ~!!l;;~
Labeled probe!~ Target DNA-.
XXXXXXXXXXXYXY Fig. 19. Schematic presentation of in situ hybridization (FISH as an example) . Biotin-labeled probe first hybridizes with the target DNA sequence, and fluorescence-labeled avidin subsequently binds to biotin on the probes. A fluorescence-labeled and biotinylated anti-avidin antibody can be used to further amplify signals .
In Situ Hybridization (ISH) • General information: - A hybridization technique introduced independently by Gall and pardue, Buongiomo-Nardelli and Amaldi, and John et al. in 1969 - It uses a DNA or RNA probe to detect the target DNA or RNA in the cell cytoplasm or nucleus (i.e., in situ) - A signal amplification method, no sequence amplification involved - Probes: • A short DNA or RNA sequence complimentary to the target DNA or mRNA sequence • Usually between 20 and 50 bp long (oligonucleotide) or hundreds of nucleotides (DNA probes), but can be as long as thousands of nucleotides - Specimens suitable for in situ hybridization test: • Cells in suspension or fixed on glass slides • Frozen-sectioned tissue • Paraffin embedded tissue sections • Principle - Probe (DNA or RNA) undergoes hydrogen bonding (annealing) to complimentary target sequences in fixed or fresh tissue, thus revealing the location and quantity of DNA or RNA - Factors that affect hybridization • Temperature. The binding of probe to the target DNA or RNA depends upon the Tm or melting point, the temperature at which 50% of double strands are denatured. The optimal temperature for hybridization is just below the Tm • Others include pH, monovalent cation concentration, and presence of organic solvents in the hybridization solution - Hybridization conditions • High stringency conditions allow highly specific hybridization of the probe with the identical or very
similar homology to the target sequence to increase the specificity • Low stringency conditions allow less specific binding of probe with lower homology to the target sequence to increase the sensitivity • Procedure (Figure 19) - Prepare probe . There are four common types of probes. The advantages and disadvantages of those probes are listed in Table 1 • Oligonucleotide probe: • Synthesized by an automated DNA synthesizer • Size: around 40-50 bp • Single-stranded DNA probe: • Produced by PCR or reverse transcription of RNA • Size: 200-500 bp Double-stranded DNA probe : • Produced by bacteria or PCR • Size: in the range of hundreds of base pairs • RNA probe (cRNA probe or riboprobe): • Prepared by an RNA polymerase-catalyzed transcription of mRNA in the 3' to 5' prime direction or in vitro transcription of Iineralized plasmid DNA with RNA polymerase (T3, T7 , or SP6) • Size: in the range of hundreds of nucleotides - Probe labeling : • Radiolabeling with 3H, 32p, 35S, or less commonly 14C, and 1251: quick, easy and sensitive but needs long exposure • Fluorescence-labeling with fluorescein isothiocyanate (FITC), Texas red, or Rhodamine among others. The fluorescence is detected with fluorescence microscopy • Non-radioactive, non-fluorescence-labeling using biotin, digoxin, digoxigenin (DIG) , and so on. This type of probe labeling needs an intermediate step, such as avidin or antibodies
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Molecular Genetic Pathology
Table 1. Comparison of Probes Used for In Situ Hybridization Probe type
Advantages
Disadvantages
DNA (double strand)
Easy to use Subcloning unnecessary Choice of labeling methods High-specific activity Possibility of signal amplification (networking)
Reannealing during hybridization (decreased probe availability) Probe denaturation required, increasing probelength and decreasing tissue penetration Hybrids less stable than RNA probes
DNA (single strand)
No probe denaturation needed No reannealing during hybridization (single strand)
Technically complex Subcloning required Hybrids less stable than RNA probes
RNA
Stable hybrids (RNA-RNA) High-specific activity No probe denaturation needed No reannealing Unhybridized probe enzymatically destroyed, sparing hybrid
Subcloning needed Less tissue penetration
Oligonucleotide
No cloning or molecular biology expertise required Stable Good tissue penetration (small size) Constructed according to recipe from amino acid data No self-hybridization
Limited labeling methods Lower-specific activity, so less sensitive Dependent on published sequences Less stable hybrids Access to DNA synthesizer needed
Reproduced with permission from Feldman et al., In: Principles of Neurop sychopharmacology. Sunderland, MA : Sinauer Associates, lnc ., 1997,31-35
-
Prehybridization (RNase-free if RNA-DNA or RNAIRNA hybridization). The tissue or cells are fixed with paraformaldehyde, treated with acid (HCI), proteinase K, or detergents to increase signal, and HzO z or acetylated to chemically modify proteins and reduce non-specific binding
-
Hybridization (RNase-free if RNAIDNA or RNAIRNA hybridization): labeled probe binds to the target DNAIRNA sequence in a hybridization solution under a cover slip
-
Post-hybridization: rinse with washing solution saline sodium citrate (SSC) to remove excess or non-specific bound probes or eliminate single-stranded RNA by adding RNase
-
Detection • Radioactive detection: bound radiolabeled probes are directly detected using either photographic film or photographic emulsion • Fluorescent in situ hybridization (FISH): bound fluorescent labels are detected either directly (d irectly labeled probe) or indirectly (biotin-avidinlabeled probe or DIG-anti-DIG antibody-labeled probe) by using a fluorescent microscope or plate
94
reader. More than one different probe can be visualized at one time in the same location • Chromogenic in situ hybridization. Bound probes are detected by chromogenic reaction. This usually involves three steps: • Probes labeled with either biotin or DIG bind to the target sequences • Avidin or anti-DIG antibody labeled with peroxidase or alkaline phosphatase binds to the probe-target complex
-
• Enzymatic reaction catalyzes the substrates to display the color in situ Setting up controls: • Control for DNAIRNA quality in tissue section or cells to avoid false-negative results • Poly(dT) probe to check the quality of mRNA by detecting mRNA poly A tail s • Probes against constitutively expressed hou se keeping genes such as actin or ~-tubulin • Positive control (test efficacy) : • Use of a probe to hybridize with the target sequence in tissue section or cells known to have the target sequence
Diagnostic Methodology and Technology
• Northern blot analysis of tissue or cells to show that the labeled probe binds to an mRNA target of the correct molecular size, or immunohistochemical detection of the gene product to correlate the co-expression of the protein and mRNA not commonly used • Negative control (test specificity): • Digest the target mRNA with RNase prior to hybridization with the oligonucleotidelDNA probe. RNase treatment will abolish the mRNA in the tissue or cells and no hybridization shall occur. It will also reduce the background • Hybridize labeled sense probe in parallel with labeled antisense probe with the target sequence. Detection of signal only by the antisense probe indicates specific binding • Pretreat tissue sections or cells with excess nonlabeled probe (usually 10 times more) before hybridization with labeled probe (competition) - Interpretation. Interpreting the results should follow general guidelines as follows: • A positive result should be reported only if the negative control is negative • A negative result should be reported only if the positive control is positive • If there is discrepancy between the control and test results, the result is "inconclusive" or should be repeated • If there are mixed populations of cells (normal and abnormal cells) , hybridization results should be correlated with morphology • It is always advised to read multiple foci of tissue sections after in situ hybridization. Heterogeneity of gene expression in different parts of a lesion may lead to misinterpretation • Applications: - Localization of DNA sequences or genes on chromosomes (normal, mutations, translocations, deletions, and so on) - Useful in genetic testing, diagnosis, monitoring early relapse, and assessing efficacy of therapeutic regimens - Study of the microscopic distribution and cellular localization of DNA and RNA sequences in a heterogeneous cell population - Quantification of DNA or RNA in situ - Study of gene expression (mRNA) - Detection of viral nucleic acids • Advantages: - Relatively rapid and precise localization of DNA or RNA of interest in anatomic location or specific types of cells - Great sensitivity to detect a target present in only a small fraction of the cells in the tissue or mixture - Higher specificity than immunohistochemistry
3-31
- Suitability to study the gene expression by detection of mRNA in situ - Both qualitative and quantitative - Automation and high-throughput analysis possible • Limitations: - The sequence of DNA or RNA of interest must be known - Probes must be designed for specific applications - The sensitivity is relatively lower than that of peR. A low level of gene expression in a small number of cells can go undetected - Significant experience is required to perform the test and interpret the results - Special precautions are required for handling radioactive probes - Relatively expensive equipment for FISH
Southern Blot • General information: - A hybridization technique developed by Edwin Southern in 1975. - Used to locate a particular sequence of DNA within a complex mixture or genome and measure its relative quantity - Probe: a short segment of ssDNA or oligonucleotide - The minimal detectable amount of DNA by Southern blot in general is approximately 0.1pg under optimal conditions. However, it is dependent on the size and specificity of the probe • Principle: DNA transferred onto a membrane after agarose gel electrophoresis is hybridized with a DNA probe usually labeled with 32p, or a biotin/streptavidin-enzyme complex. The hybridization is based upon the complimentarity between the target DNA fragments and probe. The degree of hybridization of a complimentary probe is proportional to the amount of the specific target sequence in the sample . The signal of autoradiography or amount of enzymatic reaction measured by color or chemoluminescence is used to measure the degree of hybridization. The presence of a specific pattern by the above methods is used to detect or rule out a mutation in the target gene or sequence. • Procedure (Figure 20) - Isolate and digest genomic DNA or large DNA fragments with restriction enzymes Separate denatured DNA fragments by gel electrophoresis, usually agarose gel, based upon the sizes of DNA fragments Transfer separated DNA fragments onto a charged membrane, usually nitrocellulose or nylon Hybridize labeled probe with the target DNA on the membrane
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Molecular Genetic Pathology
~
Cleavage with restriction enzyme
)
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Membrane ..........
~ Gel electropho resis
•
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Transfer of DNA to solid membrane
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-----
--
Hybr idization with labeled probe
--
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Detection
Fig. 20. Schematic presentation of Southern blot using a radiolabeled probe (see text for description of steps).
- Visualize hybridization using autoradiography (radioactive probe), colorimetric, or bioluminescent methods (enzyme or biotin-avidin-enzyme complexlabeled probes) • Applications: - To detect a particular gene and the number of gene copies in the genome (genetic testing, oncogene/tumor suppressor gene detection, and so on) - To analyze restriction fragment length polymorphisms (RFLP) and variable number tandem repeat (forensic DNA testing, transplantation, genetic epidemiology, and so on) - To identify the degree of similarity between a gene and probe sequence (molecular detection of intraspecies variations, gene mutation) - To detect gene rearrangements (T-cell receptor gene rearrangement) - Used in gene cloning, chromosome walking , genome mapping, and so on • Advantages: - Qualitative and quantitative method - High analytic sensitivity and specificity - Detection of major deletions and rearrangements in large DNA fragments Analysis of multiple samples at the same time • Limitations: - Time-consuming and laborious - Requires a relatively large amount (microgram scale) of high quality DNA, usually from fresh or frozen tissue May not detect a clonal population if it represents < 10% of the total cells in specimen Cannot detect a single or a small number of nucleotide differences in the total genome or in large fragments of DNA
Northern Blot • General information: A hybridization technique developed by Alwine and coworkers in 1977
96
- Used to identify RNA (mRNA) and its relative quantity on a membrane using a DNA or RNA probe • Principle and procedure (similar to those of Southern blotting except the following): - The target sequence is RNA, not DNA Northern blot uses formaldehyde in the electrophoresis gel as a denaturant. Sodium hydroxide, as used in the Southern blot procedure, would degrade RNA The procedure requires an RNase-free environment. Maintaining the purity and integrity of RNA is essential for performing efficient Northern blots - Molecular weight is measured in nucleotides (nt) or kilonucleotides (knt), and occasionally in bases (b) or kilobases (kb) - More than one mRNA may be probed after stripping off the bound probes • Applications: Mainly for research purposes, seldom used in clinical diagnosis - Detection and quantification of gene expression (mRNAs) - Comparison of mRNA abundance among different samples based on the intensity of the signal on a single membrane - Determination of transcript size and detection of alternatively spliced transcripts • Advantages: - Qualitative and quantitative method - Relatively sensitive - Exceptionally versatile . Virtually any type of probe can be used - Multiple samples can be analyzed at the same time - Relatively fast • Limitations: - RNase-free environment required - Easy degradation of RNA, often fresh or frozen tissue is necessary - Less sensitive than nuclease protection assays and RTPCR
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Diagnostic Methodology and Technology
- Difficult to use multiple probe s to detect multiple mRNA transcripts
ASO/SSO hybridization
Conventional DNA hybridization
Allele-Specific Oligonucleotide (ASO) and SSO Hybridization • General information: Allele specific oligonucleotide (ASO) hybridization was introduced to identify specific alleles by Conner et al. in 1983. ASO may be mistakenly referred to as SSO hybridization, which is similar to ASO, but used to detect specific sequences instead of alleles - ASO and SSO probes are short and specific for particular DNA sequences, typically 15-20 nt long - ASO and SSO probes are usually designed as such that a single nucleotide difference between alleles or specific sequences occurs in a central segment ?f the . oligonucleotide sequence, so that the single n~c~eotld~ . mismatch maximally enhances thermodynarruc Instability • Principle: Hybridization between the probe and the tm:get seq~ence is stable only if there is perfect base complimentarity and unstable even if there is a single mismatch between the probe and the target sequence (Figure 21). • Procedure: It is often used in combination with dot blot assay (dot blot-ASO assay), Southern blot (see Southern blot), or PCR. Below is the procedure of ASO/SSO-dot blot assay. - Spot an aqueou s solution of denatured target DNA onto a charged membrane, allowing it to dry - Denature the target DNA sequences by exposure to alkali if spot target DNA is non-denatured - Expose the immobilized denatured target D~~A to . single-stranded labeled ASO or SSO probe In solution - Visualize hybridization by autoradiography, chemiluminescence, or chromogenic reaction • Applications: Genetic testing to discriminate alleles with a single nucleotide difference (can identify diseased patients and carriers) - Pharmacogenetic testing , such as detection of drug resistance alleles due to SNPs - Forensic DNA testing based upon multiple SNP - Genetic epidemiology (population genetic s and evolution) • Advantages - Suitable for both screening and diagno stic purposes - Highly specific - Easy, rapid (dot blot-ASO), efficient , and relatively inexpensive - Adaptable to automation and high-throughput detection (SNP Scanning, microarray, and so on) • Limitations - The sequences of the target alleles or loci must be known
Perfect match -
stable
Single NT mismatch - unstable under high stringency cond ition
Perfect match -
stable
Single NT mismatch - stable ever under high stringency condit ion
Fig. 21. Comparison of hybridization using a conventional DNA probe and an allele-specific/sequence-specific oligonucleotide probe.
- Only a specific nucleotide difference or mutation is detected. However, many diseases are caused by multiple mutations - Its sensitivity is relatively lower than that of PCR
Reverse Hybridization • General information: - A hybridi zation technique using the opposite order of the conventional method , i.e.,the target DNA/RNA binds probes, which are fixed on a membrane or solid phase matrix - Probes usually are oligonucleotides or single strand DNA (or denatured double-stranded DNA) Common types of reverse hybridization techniques • Reverse dot blot • Reverse Northern blot • DNA/oligonucleotide microarray • Line probe assays (Lipa) • Principle and procedure (Figure 22): Similar to other hybridization methods , based upon the complimentarity between probe sequence and target DN.A or RNA sequence . The major differences from conventional hybridi zation include: Labeling the target (DNA or RNA) rather than probes . The target sequences may be directly labeled or amplified by PCR before labeling Dotting , blotting, or linking unlabeled probe s onto the charged membrane or solid phase matrix Hybridizing labeled target sequences to immobilized probes. Target DNA sequences must be denatured before hybridization - Using multiple probe s to simultaneously examine target sequences allows efficient high-throughput detection and automation
97
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Molecular Genetic Pathology
151... ........ • o'!!· ·.()SUbstra;~" Bl....• '" O'~~trePtavid !!··.() Product ~... in-enZyme
(!
«
(!
Biotin labeled target
<:!2
Immobilized gene probe Solid phase
Fig. 22. Schematic presentation of reverse hybridization assay using a biotin-avidin-enzyme system.
• Applications: - Large-scale genetic testing and viral genotyping - Other uses similar to corresponding hybridi zation methods • Advantage s: quick and suitable for automat ion and highthroughput analysis • Limitations: - Similar to corre sponding hybridization method s - Difficult to correctly identify and distingui sh between two closely spaced mutation s - More costly than the conventional hybridization methods
DNA SEPARATION METHODS Gel Electrophoresis Conventional Gel Electrophoresis Agarose GeL ELectrophoresis • General information - One of the most common and easiest electrophoretic methods to separate DNA or RNA by size - Agarose acts as a molecular sieve, through which nucleic acids are driven by an electric field - The pore size is determined by the agarose concentration. The pore size decreases as the agarose concentration increa ses - Most agarose gels are made between 0.7 and 2%. The resolution ranges from 0.2 to 10 kb depending upon the gel concentration • Principle: - DNA and RNA molecules are negatively charged at neutral pH due to the presence of phosphate groups. Consequently, these molecules will migrate toward the positive pole when an electrical potential is applied - The migration rate of DNA in agarose gel electrophoresis depend s upon four main factors • The agarose concentration (inversely related to the logarithm of the electrophoretic mobility). Lower concentration gels provide better resolution for larger DNA fragment s, and vice versa
98
Among three conformations (closed circular or typically supercoiled DNA, nicked circular DNA, and linear DNA), the supercoiled form migrate s the fastest, and the linear form migrates the slowest • The applied voltage. For a given DNA fragment , the higher the voltage, the faster the migration. In general , the voltage applied on an agaro se gel is 5 Vfcm (length of gel) - The DNA is visualized by staining with EB a dye that strongly binds to DNA by intercalating between bases and emits visible orange light when absorbing invisible ultraviolet (UV) light - The molecular size of an unknown band of linear DNA is estimated by comparing its traveling distance to molecular weight standards. Supercoiled DNA must be linearized before it can be evaluated for molecular weight • Procedure (Figure 23): - Prepare the agarose gel by mixing agarose powder in buffer solution, then heating and pouring the liquified gel into the electrophoresis apparatus and allowing it to solidify - Load DNA ladder into the gel. The DNA ladder (molecular weight markers) is pretreated with loading buffer containing denaturing solution and a marker dye - Mix DNA sample s with loading buffer and load them into the gel - Start electrophoresis by applying electric field
• The molecular sizes of the DNA fragments . The migration rate of linear duplex DNA is inversely proportional to the logarithm of the DNA fragment size. Smaller fragment s migrate faster than larger fragments
- Separate nucleic acid fragment s in the gel on the basis of size - Stop electrophoresis when the marker dye approaches the end of the gel
• The conformation of the DNA. The migration rate within in the agarose gel of individual DNA fragments varies with different conformations.
- Stain DNA bands in the gel with EB. Optionally, EB can also be added into electrophoresis buffer to stain the DNA during electrophoresis
3-35
Diagnostic Methodology and Technology
A
2
Sample wells
c
3
4
5
6
D
•---
- --
'---------"'-'"
,,'------------'"
Fig. 23. Schematic presentation of agarose gel electrophoresis. Agarose gel is prepared (A). DNA markers and samples are loaded into wells (B, C). Fragments of DNA (or RNA) are separated by electophoresis (D).
- Visualize the EB-stained DNA under UV light
(Figure 24) - Cut the band(s) of interest in the gel and retrieve or purify the DNA if needed • Applications: - One of the most common analytic methods to determine the presence, the size, and the amount of DNA fragments - Preparative method for the isolation or purification of a particular DNA species - The method that can also be used for RNA separation • Advantages: - Versatile (wide range of separation) - Easy to use, fast separation, relatively inexpensive
Fig. 24. An example of agarose gel electrophoresis of DNA fragments . Lanes 1 and 6: molecular weight markers; lanes 2 and 3: two patient samples; lane 4: positive control; lane 5: negative control. (Adapted from Richter et ai, Emerging Infec Dis. 2002 ;8(7):729-731)
• Limitations: - Only useful in separation of relatively large fragments of DNAJRNA due to large pore size of gels - Lower resolving power than polyacrylamide gel electrophoresis (PAGE) (see below) - Low sensitivity
Polyacrylamide Gel Electrophoresis • General information - An electrophoretic technique introduced by Raymond and Weintraub in 1959 - Uses polyacrylamide gel as a molecular sieve to separate DNA when an electric field is applied • Principle: - Similar to agarose gel electrophoresis. Unique features include: • Polyacrylamide gels are formed from the polymerization of two compounds, acrylamide and a cross-linking agent, N,N I-methylene-bisacrylamide (or Bis) • The size of the pore of polyacrylamide is determined by the gel concentration and usually ranges from 3-30% of acrylamide and Bis. The separation of molecules within a gel is determined by the relative sizes of the pores formed within the gel • The resolution of PAGE ranges from 10 to 2000 bp and is dependent upon the gel concentration
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• Procedure - Prepare polyacrylamide gel. Acrylamide and Bis solution is mixed in the proper proportion. Ammonium persulfate along with either ~-dimethyl aminopropionitrile (DMAP) or N,N,N',N',tetramethylethylenediamine (TEMED) is added to the mixture for polymerization - Pour the gel into the plate - Load the sample and DNA ladder with marker dye (bromophenol blue) - Separate DNA fragments by electrophoresis - Stop electrophoresis when marker dye approaches the bottom of the gel - Stain the gel with EB Illuminate the gel with UV light - Alternately, if 35S or 33p nuclides were incorporated into the DNA during synthesis , autoradiograhy can be performed after the gel is dried • Applications - Primarily used for DNA sequencing - Analysis of small fragments of DNA (quantification, size determination) - Small-scale preparation • Advantages : - Ability to separate smaller DNA fragments than agarose gel, attributed to finer porosity - Very high resolution ; separation of strands with single base pair difference, under optimal conditions - Stronger than agarose gel - Dried gel can be used for permanent archival record • Limitations : - Small range of separation - More difficult to use and more expensive than agarose gels - Acrylamide is neurotoxic ; appropriate precautions must be taken
Capillary Electrophoresis • General information Capillary electrophoresis is a special type of electrophoretic technique introduced by Hjerten in 1983. CE is a family of separation technologies with wideranging applications in chemistry and the biologic sciences. Separation is achieved by the flow of analytes through a narrow capillary, propelled directly or indirectly by a strong electric field. • Principles - Capillaries are very narrow (normally ranging from 25-100 urn in internal diameter and 0.5-1.0 m in length) and are usually comprised of fused silica - Capillaries may be filled with either buffer solution or replaceable liquid polymers
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Molecular Genetic Pathology
- Depending on the precise technique employed, separation occurs on the basis of charge-to-mass ratio, size, or other physical properties of the analyte - In fused silica capillaries, cations from the buffer solution are attracted to negatively charged silanoate groups that line the inner capillary wall, producing a layer of positively charged mobile ions. These ions are drawn toward the negative pole (cathode) along with associated water molecules, producing a bulk flow of buffer, which carries all analytes (regardless of charge) towards the cathode, a phenomenon known as electroosmotic flow, or EOF Capillary gel electrophoresis (CGE) is a variant of CE, which has broad applicability in the analysis of nucleic acids, and is particularly useful in DNA sequencing - In CGE, the capillary is filled with solution-phase polymers which act as a molecular sieve, resolving DNA fragments on the basis of their sizes - The gel matrix used in CGE includes cross-linked polyacrylamide and non-cross-linked matrix linear polymers such as polyacrylamide, polyvinyl alcohol, dextran, and agarose In CGE, it is necessary to minimize EOF so that the polymer is not drawn out of the capillary during the run; this is achieved by coating the capillary walls - In the absence of EOF, nucleic acids, which are negatively charged, migrate towards the anode. Smaller fragments , which are less encumbered by the gel matrix, move more quickly than larger fragments - Detection is usually fluorescence-based (e.g., by incorporation of dye-labeled terminators in sequencing reactions) - The liquid gel matrix is replaced after each run, minimizing the potential for carry-over contamination • Procedure (CGE) (Figure 25) DNA fragments are prepared (isolation, synthesis, purification, and/or enzyme digestion) The DNA sample is introduced into the capillary at the cathodic (negative) end either by application of electrical current or mechanical pressure - DNA fragments are resolved as they migrate through the matrix - Resolved DNA fragment s are detected near the anode (positive pole) of the capillary. Detection methods include fluorescence , absorbance, electrochemical, and mass spectrometry (MS) - Data is analyzed and electropherogram is produced • Applications - Separation of ds DNA: the most frequent application of CEo It can separate dsDNA up to 40 kbp in a homogenous electric field using a mixture of polyethyleneoxide - Separation of DNA sequencing fragments : it can separate DNA fragments up to 1000 bp under certain conditions
3-37
Diagnostic Methodology and Technology
1~
~
Computer Cat hode
Anode Cap illary tubing
+ Laser beam
•............. Migration
ISamp le I
Fig. 25. Schematic presentation of CE as used in DNA sequencing (see text for description of steps). A light (usually laser) is used to elicit a fluorescence signal that is captured by a detector.
- Genotyping, allele analysi s, or RFLP detection - Combination with other DNA separation methods • Advantages - Highly efficient and quick separation of a large number of DNA fragments at the same time - High resolution - Small capillary diamet er permits rapid dissipation of heat - A small sample volume (pico to nanoliter) sufficient - Automated and high-throughput applications are possible - Quantitative and qualitati ve detection - Replaceable gel or matrix - Minimal quantities of reagent s required - Low cost of individual runs, short analysis time, and simplicity • Limitations - Cannot perform separations at preparative scales - High concentration of DNA in a small volume required - Relative low sensitivity compared with highperforman ce liquid chromatography (HPLC) - Initial cost of instrumentation
DNA migrates, fragment s with minor, even single nucleotide change s can be separated - In practice, the den aturants used are heat (a con stant temperature of 60°C) and a fixed ratio of formamide (ranging from 0 to 40%) and urea (ranging from 0-7 M) - PCR-coupled with DGGE permits the separation of almost all DNA variants. A GC-rich sequence is incorporated into one of the primer s used for enzymatic amplification, producing PCR products with altered melting points • Principle (Figure 26) - The migration rate of the small DNA fragments (100-700 bp) through a low-to-high conventional or denaturant gradient acry1amide gel initially is determined by the molecular size - With heightening denaturing conditions, individual DNA fragment s reach a point where the duplex DNA fragment is melted - The partial melting severely retard s the migration of the DNA fragment s in the gel, and a mobilit y shift is observed - As the denaturing conditions become more extreme , the partially melted fragment completely dissociates into single strands The denatured DNA is essentially stopped (arrested) at the melting point in the gel, and form s a sharp band - Minimal differences between DNA fragment s (as little as a single base pair change ) can result in a significant mobility shift - The final mobilit y of DNA in DGGE depends upon the molecular size and compo sition of each fragment • Procedure - Prepare gradient gel with the aid of a gradient maker at room temperature Place the solidified gel into a tank containing gel buffer at a temperature of 60°C, with recirculation pump turned on - Load the sample s Run electrophoresis at an appropriate voltage (usually 65-75 V)
Gradient Gel Electrophoresis (GGE) and Denaturing Gradient Gel Electrophoresis (DGGE)
- Stop electrophoresis and stain the gel with EB - Examine the gel by UV illumination
• General information - A variant of gel electrophoretic technique introduced by Fischer and Lerman in 1983
- Transfer the DNA onto nylon blots if necessary
It uses a gradient gel with pore size decrea sing from the top to the bottom to increase the resolution. The gradient gel is almost invariably a polyacrylamide gel The concentration of gel typically varies from 5% at the top to 25% at the bottom
Denaturation is accomplished by heat, or heat in conjunction with a chemical or pH gradient. As the
• Applicat ions - To type alleles of a polymorphism (e.g., alleles of the ABO blood group system) - To detect non-RFLP - To screen exons of a mutated gene in human genetic s and oncolog y - To be used in molecular microbiology such as identification of microorganisms and epidemiologic studies
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Molecular Genetic Pathology
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DGGE
A Non·mutant DNA (N)
Mutant DNA (M)
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- High reproducibility in experienced hands - Rapid and relatively inexpensive and '00000"
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Homoduplex DNA
Heleroduplex DNA
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• Advantages - High sensitivity. DGGE can detect virtually all mutations in a given piece of DNA. It displays the highest detection rate among mutation scanning methods
M N+M
B
- Possibility of optimizing the analysis by computer simulation - A non-radioactive protocol • Limitations: - The denaturant gradient slope and running times vary for every DNA region to be analyzed, greatly affecting the routine application of the method - It requires great technical expertise in the preparation of denaturants and pouring of acrylamide gels to yield reproducible results - The long running required to resolve hetero- and homoduplexes, often produce curtains and smears instead of sharp zones Without DNA sequencing, heteroduplexes and homoduplexes that co-migrate in the gel can confound DGGE interpretations
Pulsed Field Gel Electrophoresis (PFGE) • General information - An electrophoresis technique introduced by Schwartz and Cantor in 1984 - Alternating electrical fields are used to separate long DNA strands based on size in a low-density agarose gel matrix - Raises the upper size limit of DNA separation in agarose from 20 kb to > 10Mb (10,000 kb)
Fig. 26. Mechanism of DGGE and an example of its application (analysis of sequence variation in the second internal transcribed spacer (ITS-2) of ribosomal DNA of strongyloid nematodes). Schematic representation of heteroduplex formation and principle ofDGGE (A). Mutant homoduplexes (lane M) melt at a lower denaturant concentration than non-mutant homoduplexes (lane N) and are consequently retarded at a higher position in the gel. Heteroduplexes (lane N+M) melt at even lower denaturant concentrations. DGGE analysis of sequence variation within ITS-2 PCR products (350 bp in size including the GC-clamp) amplified from single adults of the nematode Haemonchus contortus originating from a natural population. (B) Each bright (homoduplex) band in DGGE represents an enriched clonal sequence type of ITS-2 as determined by direct sequencing of excised bands. Bands in the
102
• Principle - The theory behind PFGE is controversial. In general, DNA molecules greater than 30-50 kb migrate with the same mobility regardless of size during continuous field electrophoresis; thus they are seen in the gel as a single large diffuse band. Multiple fragments of DNA in such a band can be separated from each other if an electric field is applied to change direction of electrophoresis. With each reorientation of the electric field relative to the gel, smaller-sized DNA will begin to move in a new direction more quickly than the larger DNA, thereby leaving the larger DNA fragments behind. This provides a separation of long strands of DNA based upon size
upper third of the gel represent heteroduplex molecules produced during PCR. (Reprinted from Gasser and Zhu, Parasitol Today 1999;215(11):462-465 . (© 1999 with permission from Elsevier)
3-39
Diagnostic Methodology and Technology
FIGE
rn
- Cloning large DNA using yeast artificial chromosomes (YAC's) and PI cloning vectors
A-
Detecting in vivo chromosome breakage and degradation - Epidemiologic studies, e.g., to establish the degree of relatedness among different strains of the same specie s +
RGE
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Fig. 27. Electrode configurations of commonly used pulsed field gel electrophoresis units .
• Advantages - Ability to separate and characterize large DNA fragment s, for example, chromosomes of microorganisms - Ability to examine the elongated and oriented configuration of large DNA molecules in agarose gels at finite field strengths - High sensitivity and reproducibility. It is one of the most reliable techniques for determining strain genetic similarity in many bacteria • Limitations - Intact DNA is required . Special care must be taken not to shear or damage the DNA - Faulty results may arise due to
- PFGE instrumentation can be generally categorized into four different types (Figure 27): • Field inversion gel electrophoresis (FIGE) works by periodically inverting the polarity of the electrodes during electrophoresis • The other type of instrumentation functions to reorient the DNA at smaller oblique angle, to move the DNA fragment s forward in a zigzag pattern down the gel. This type of instrumentation includes transverse-alternating field gel electrophoresis (TAFE), contour-clamped homogeneous electric field (CHEF) and rotating gel electrophoresis (RGE) - PFGE special equipment consists of a gel box, a chiller and pump, a switch unit, a programmable highvoltage power supply, and computer software • Procedure - Prepare intact or unsheared DNA by embedding intact cells in agaro se plugs and digesting away the proteins in the plugs using enzymes to avoid shearing of large DNA fragments - Digest intact DNA in the plugs with a rare-cutting restriction endonuclease - Separate DNA fragments by gel electrophoresis using PFGE special equipment - Detect and interpret the banding patterns • Applications (Figure 28) - Identifying RFLP's and fingerprinting - Determining the number and size of chromosomes (electrophoretic karyotype) from fungi and parasites such as Leishmania, Plasmodium, and Trypanosoma - Gene mapping and construction of physical maps of the chromosomes of human and prokaryotic organisms
• Easy contamination of the agarose plugs with DNases by accidental introduction of non-specific DNA-degrading enzymes • Incomplete (partial) digestion of DNA by the restriction enzyme that generates unusually large fragments Methodology is complicated, time consuming - High cost
Single-Strand Conformational Polymorphism (SSCP) • General information - An electrophoretic technique introduced by Sunnucks et al. in 1989 and used to separates ssDNA or RNA based on mutation-related conformational differences • Principle (Figure 29): - Single-stranded DNA molecules assume unique conformations that depend on their nucleotide sequences under non-denaturing conditions and reduced temperature. Subtle differences in sequence, often a single base pair variation , may cause a different secondary structure by altering intrastrand base pairing, resulting in a measurable difference in electrophoretic mobility. Small or tightly packed molecules migrate more quickly through the gel than large or loosely packed molecules Most SSCP protocols are designed to analyze the polymorphism at a single locus using a specific pair of PCR primers bracketing the target region . The target ssDNA is amplified by asymmetric PCR, in which one primer is present in exces s over the other. After the low-concentration primer supply is exhausted, continued PCR only allows amplification of the target ssDNA
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Molecular Genetic Pathology
2
3
4
5
6
7
8
9
10
11
12
13
14
15
---------------
-
-
-
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Fig. 28. PFGE of group B Streptococcus (GBS) strains isolated from college women with urinary tract infections and their most recent sex partner. Lane 1 is a DNA ladder. Lanes 2-3 and 14-15 represent rectal and vaginal GBS isolates from two different women. Lanes 4-6 are two urine isolates and one rectal isolate from one woman and lanes 7 and 8 are the urine and rectal isolate from her sex partner, respectively. Lanes 9-11 represent a female vaginal isolate and her sex partner's urine and rectal isolate. (Adapted from Manning, SD, Frontiers Biosci. 2003;(Review)8: sl-18)
• Procedure - Prepare DNA fragments by digesting genomic DNA with restriction endonucleases and/or PCR amplification of the target ssDNA fragment or peR amplification of the dsDNA followed by denaturation into single strands - Denature DNA samples in an alkaline (basic) solution - Separate DNA fragments by neutral PAGE - Stain the gel or transfer DNA onto a nylon membrane followed by hybridization with probes - Compare the mobility of control with unknown DNA sample fragments - Confirm identity of mutations by DNA sequencing
- Limited sensitivity. Under optimal conditions, approximately 80-90% of the potential base exchanges are detectable by SSCP. Changing the pH and adding glycerol may increase sensitivity - No information provided for the position of the change - A constant temperature is required during the electrophoresis for best results, because ssDNA mobilities are temperature-dependent • Applications: - Detection of DNA polymorphisms and mutations at multiple sites in DNA fragments - Serving as genetic markers because they are allelic variants similar to RFLPs
• Advantages: - Simple procedure - Inexpensive equipment - No need for precise knowledge of the sequence polymorphism
RFLP Analysis
• Limitations: Limited size range of DNA fragments (150-300 bp) for optimal separation results . Adding certain reagents such as glycerol to the gel may increase the size limit. RNA-SSCP electrophoresis may allow for separation of larger-sized fragments
• General information - RFLP analysis is a methodology that combines restriction enzymatic digestion and conventional gel electrophoresis to separate and anlyze the DNA fragments. The resolved DNA fragments can either be visualized with EB or by Southern blot
104
-
Use in molecular genetics with modifications, such as heteroduplex analysis, ribonuclease protection assay, SNP techniques
3-41
Diagnostic Methodology and Technology
A
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Fig. 29. SSCP principle. Wild-type (A) and polymorphism or mutant (B) double-stranded DNA molecules have equal lengths , but their corresponding single-stranded fragments show different conformations. The banding results after gel electrophoresis of both double-stranded and ssDNA fragments is shown (C) . The migration patterns of the doublestranded fragments are indistinguishable. However, three single-stranded fragments display different migration patterns, or mobilities , according to their conformations.
- Differences in the size and/or number of restriction fragments result from sequence changes (base substitutions, additions, deletions, and so on) that involve restriction enzyme recognition site(s) - The term "RFLP" also refers to these sequence changes (polymorphisms) themselves - Special types of RFLPs include • Minisatellites, detecting hypervariable RFLP loci (variable number tandem repeat loci) • Restriction landmark genomic scanning , 2D RFLP analysis • Principle : Each restriction enzyme recognizes a specific sequence (4--6 bp, or occasionally 8 bp in length). Any change of this
sequence (e.g., by mutation, insertion, or deletion) results in loss of the splice site due to lack of recognition by the enzyme. Digestion of a DNA sample by restriction enzymes produces a collection of DNA fragments of precisely defined length (restriction fragments), which can be resolved by gel
1.35 kb
_
1.15kb
-
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-
-
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Fig. 30. Schematic presentation of RFLP (an example of detecting a missing site (for MstII) in hemoglobin S). Hemoglobin S produces a longer DNA fragment (1.35 kb) than hemoglobin A (U5 kb) after MstII restriction enzyme digestion due to mutation at an MstII cleavage site (upper panels). Gel electrophoresis can separate the two types of hemoglobin genes based upon the different sizes of their corresponding restriction fragments (lower panel).
electrophoresis. RFLPs can represent normal variation or be associated with disease. • Procedure (Figure 30) - Isolate and purify genomic DNA with or without PCR amplification - Digest DNA with restriction enzyme(s) - Separate DNA fragments according to their sizes by gel electrophoresis - Detect DNA bands by staining the gel or blotting DNA onto a membrane followed by hybridization using a labeled probe (Southern blot) • Applications - Genetic testing or screening of human DNA for the presence of potentially deleterious mutations - Forensic DNA testing or fingerprinting - Genetic epidemiology or population studies - Molecular microbiology to identify related species • Advantages - No prior sequence information required - Inexpensive • Limitations - RFLP sites recognized by enzymes must be present in association with known mutations - The informativeness is limited. Polymorph isms are only detected if they affect restriction sites - It requires a large amount of DNA with high integrity - Sensitivity is variable - Difficult to standardize - Time-consuming and laborious
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Molecular Genetic Pathology
SEQUENCING OF NUCLEIC ACIDS
General Information • DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. Currently, almost all DNA sequencing is performed using the chain termination method developed by Frederick Sanger. This technique uses sequence-specific termination of DNA synthesis reactions using modified nucleotide substrates • Another method was developed by A Maxam and W Gilbert, hence called Maxam and Gilbert sequencing. This method utilizes a base-specific chemical modification followed by cleavage of modified DNA. This method is no longer used for routine DNA sequencing because the reagents are toxic and the procedure is not amenable to automation. However, it is still valuable in special applications such as assaying protein and DNA interactions known as "footprinting" • Other technologies, such as Pyrosequencing, SNaPshot, and Invader, are also being used for sequencing and SNP detection
Sanger Sequencing • Principle : In chain terminator sequencing (Sanger sequencing) extension is initiated at a specific site on the template DNA by using a short oligonucleotide primer complementary to the template at that region. The oligonucleotide primer is extended using a DNA polymerase. Included with the primer and DNA polymerase are the four deoxynucleotides (dATP, dCTP, dGTP, dTTP) along with a low concentration of a chain terminating nucleotide (most commonly a di-deoxynucleotide, ddNTP). Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular nucleotide is used. Four separate DNA polymerase reactions, each using a different one of the four di-deoxynucleotides are carried out. The products of these reactions are then sizeseparated by electrophoresis in separate lanes of a slab polyacrylamide gel, or more commonly now, by CEo • Procedures - The DNA sequencing reactions are somewhat analogous to PCR (see Amplification Methods section) • The reaction mix includes the template DNA, free nucleotides, an enzyme (usually a variant of Taq polymerase) and a "primer"-a short piece of ssDNA about 20-30 nt long that can hybridize to one strand of the template DNA • For detection purposes, the primers are labeled with 32S radioisotope • DNA needs to be purified to remove unincorporated nucleotides and primers, which can interfere with the sequencing reaction and results (see below) The Sanger technique utilizes 2', 3'-di-deoxynucleotide triphospates (ddNTPs) , molecules that differ from
106
deoxynucleotides by having a hydrogen atom attached to the 3' carbon rather than an OH group (Figure 31) • These molecules terminate DNA chain elongation because they cannot form a phosphodiester bond with the next deoxynucleotide - The sequencing reaction is conventionally performed in four separate sequencing reactions choosing a different ddNTP of interest for each reaction • For example, a mixture of a particular ddNTP (such as ddCTP) with its normal dNTP (dCTP in this case), and the other three dNTPs (dATP, dGTP, and dTTP). The concentration of ddCTP should be 1% of the concentration of dCTP. The logic behind this ratio is that after DNA polymerase is added, the polymerization will take place and will terminate whenever a ddCTP is incorporated into the growing strand
• If the ddCTP is only 1% of the total concentration of dCTP, a whole series of labeled strands will result (Figure 32). Note that the lengths of these strands are dependent on the location of the base relative to the 5' end. These reactions can be done as PCR to improve the sensitivity and specificity - When these reactions are completed, PAGE is performed. The products of each reaction are loaded into separate lanes for a total of four lanes (Figure 33) • The DNA is transferred to a nitrocellulose filter. Autoradiography is performed so that only bands containing DNA with radioactive label will appear - In PAGE, the shortest fragments will migrate the farthest. Therefore, the bottom-most band indicates the particular di-deoxynucleotide that was added first to the labeled primer. In Figure 33, for example, the product that migrated the farthest was from the ddTTP reaction mixture . Thus , ddTTP must have been added first to the primer, and its complementary base, thymine, must have been the base present on the 3' end of the sequenced strand. One can continue reading in this fashion . Note in Figure 33 that if one reads the bases from the bottom up, one is reading the 5' to 3' sequence of the strand complementary to the sequenced strand. The sequenced strand can be read 5' to 3' by reading top to bottom the bases complementary to those on the gel
Dye Terminator Sequencing • An alternative to labeling of the primer is to label the terminators instead, commonly called "dye terminator sequencing". The major advantage of this approach is that the complete sequencing set can be performed in a single reaction, rather than the four needed with the labeled primer approach. This is accomplished by labeling each of the dideoxynucleotide chain-terminators with a differently colored fluorescent dye
3-43
Diagnostic Methodology and Technology
Structures of nucleic acids
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Fig. 31. Comparison of normal nucleotide structure with the structure of a chain terminating dideoxynucleotide. Asterisk indicates the absence of a hydroxyl group at the 3' position of the dideoxynucleotide that prevents formation of DNA. Red arrow highlights hydroxyl group position.
• Products of the sequencing reaction are separated by capillary or slab gel electrophoresis. Laser detection is used to identify the bases at each position. The sequence is "read" from the bottom up, using a key where "A" is green, "C" is blue, "G" is yellow, and "T" is red. Using software provided by the manufacturers of sequencing machines , the signal/noise ratios of the dyes is determined for each position so that the proper base can be "called". The order of the bases is displayed in a format known as a "chromatogram," "electropherograrn," or "trace" file (Figure 34) • This method is easier and quicker than the dye primer approach, but may produce more uneven data peaks (different heights), due to a template-dependent difference in the incorporation of the large dye chain terminators. This problem has been significantly reduced with the introduction of new enzymes and dyes that minimize incorporation variability. Nonetheless, the context of a given nucleotide has a significant influence on peak height (Figure 35) . This feature may be useful in the interpretation of a trace, since artifactual peaks have no effect on the heights of adjacent peaks
• Common artifacts in dye-terminator sequencing traces include "dye blobs" (Figure 36) and large peaks that occur if bubbles are present in the capillary (Figure 37) • This method is now used for the vast majority of sequencing reactions as it is both simpler and cheaper. The major reason for this is that the primers do not have to be separately labeled (which can be a significant expense for a single-use custom primer), although this is less of a concern with frequently used "universal" primers
RNA Sequencing • As RNA is generated by transcription from DNA, the information is already present in the cell's DNA. Therefore, RNA sequence can be deduced from the coding DNA sequence • However, it is sometimes desirable to sequence RNA molecules. In particular, in eukaryotes RNA molecules are not necessarily co-linear with their DNA template , as introns are excised • To sequence RNA, the usual method is first to reverse transcribe the sample to generate DNA fragments . The
107
3-44
Molecular Genetic Pathology
---
A ATTCGACGGCAGTATGCCTAGC ATTCGACGGCAGTATGCC ATTCGACGGCAGTATG~
ATTCGACGGC ATTCGAC ATTC
Fig. 32. Schematic diagram of one lane of a sequencing gel containing products of a sequencing reaction using ddCTP. The position of the chain-terminating dideoxynucleotide in each strand is underlined.
DNA can then be sequenced as described above (Sanger or dye terminator sequencing)
C
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- -- - - --- - --- - - Fig. 33. Conventional sequencing gel. Sequence output: 5' TACGTACACGTGACACGTACTTAC 3' .
Applications • Gene sequencing • Detection of SNP
Limitations of DNA Sequencing • Usually can only read out 500-700 nucleotides accurately. Therefore, one needs to break the DNA into several fragments for sequencing if the sequence is too long • Limited sensitivity for SNP detection, if the SNP is present in <25% of the sample DNA • Relatively low-throughput and expensive • Misreading by DNA polymerase may occur. Therefore, reading from both sides is necessary
Trouble-Shooting DNA Sequencing Problems • Automated DNA sequencing is one of the most common and robust techniques performed in molecular biologic laboratories . Unfortunately, however, when a problem arises, trouble-shooting can be difficult. Some common problems are listed as follows - The trace chromatogram has noisy or "messy" sequence peaks with low quality scores. Can have peaks that look real and have high-quality scores, especially when the trace has been base called using the KB base caller - No or little signal in the raw data channels except for leftover dye at the beginning of the trace (Figure 38) - Signal strength in the raw channel usually below 100 - The trace sequence does not match either the expected sequence or other sequences in GenBank
108
• Causes of failed DNA sequencing reactions - Poor quality DNA. Very common when sequencing plasmid miniprep templates - Loss of DNA during clean up. This can be a particular problem when using ethanol precipitation clean up protocols - Too much template DNA. Excess template can kill the sequencing reaction - Wrong primer used (more common than you might think); or absence of expected primer binding site (Figure 39) - Bad water. The water used contains a sequencing inhibitor - Degraded primer or problems in primer synthesis. Oligonucleotide synthesis is chemically complex and primer synthesis failure is fairly common (Figure 40) - Dead sequencing chemistry. This can occur if the reagents are stored under the wrong conditions or are frozen and thawed too many times, causing degradation of either the Taq DNA polymerase or dyelabeled nucleotides - Blocked capillary. Every trace using a particular capillary fails. Can be identified by tracking trace quality on a trace-by-trace basis • Mixed signal (overlapping peaks in the same location) - Under certain circumstances the sequencing trace may have more than one peak at the same location, or when the target DNA itself contains more than one primer binding site. Errors in base calling can occur when
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Fig. 34. An example of a dye-terminator read. This 3.2 kb pGEM3zf-plasmid template was sequenced using the ml3 forward -21 primer on an ABI 3730xl sequencer. The sequence could be read unambiguously starting about 20 nt from the primer with 100% accuracy out to around 1000 bp. The dye terminators were completely removed by absorption to silica-coated paramagnetic particles. (© 2007 David F. Bishop, used with permission.)
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3-46
Molecu lar Genetic Pathology
Electropherogram peak heights are context-sensitive C
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Fig. 35. Due to the different kinetic s of fluore scent dye-terminator incorporation, electropherogram peak height s are not uniform , and vary by context due to the enzymatic propertie s of DNA polymera ses. While in general, the peak height variability is not too great, the most significantly attenuated signals are found for Gs following after As. In electroph erograms A-C the G after the first A is weak, as are the two G's after A's in electropherogram D. If there are significant background problem s, the weak G after A peaks may be ambiguous, especiall y if the sequence is unknown. Sequencing in both orientation s obviate s this problem, as the corre sponding Cs after Ts are strong. Electropherograms A to C also demonstrate the specific effects of sequence context on peak height. Sequence B is that for an individual heteroz ygous for a T to C polymorphi sm. Note when comparing the homozygous C sequence in C to the homozygous T in A, the peak height s for at least 6 nucleotide s surrounding the altered base change in height with the arrows approximating the magnitude of change. Such changes can help one discriminate artifactual peaks from real ones as the artifacts do not result in other peak changes. (© 2007 David F. Bishop, used with permission .)
secondary peaks exceed about 20% of the height of the primary peak • Causes of mixed temp late sequencing traces • Two or more templates were present in the reaction (Fig ure 41). This is the most common cause of mixed template o A "double pick" of two colonies. This can occur when colonies are too close together on the colony plate • Two primers were present in the sequencing reaction s. Thi s can occur when using premixed PCR reagents for sequencing where the primer stock is actually a mix of universal forward and reverse oligonucleotide primers • The PCR fragment was not purified of leftover primers before sequencing • Two priming sites are present in the DNA template (Fig ure 42) • Poor-quality PCR template containing multiple DNA fragments was used
110
• Too Iowa primer annealing temperature was used in the sequencing reaction • Different sequencing reactions were accidentally mixed at the clean up stage. This can also sometime occur if the same tip is used without rinsing • Polymerase slippage on template mononucleotide regions - The trace data becomes mixed after a long mononucleotide (single base) run in the DNA temp late (Fig ure 43) - Can also occur on long dinucleotide repeats in a manner analogous to stutter bands that occur in microsatellite PCR amplification • Cause s of sequence slippage • Long runs of a single nucleotide causes the DNA polymera se to "slip" on the template. Thi s occurs by either the template or extension product looping out and rehybridizing, and results in the generation of seque ncing products
3-47
Diagnostic Methodology and Technology
Dye blobs due to carryover and degradation of dye terminators
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Fig. 36. If the fluorescent Dye terminators used in the cycle sequencing reaction are not completely removed, they will show up as multiple broad peaks in the first 50 bases of the sequence, as seen in A. The dye blob peaks are recognized by their broad elution profile relative to the sharper peaks of each fluorescent sequence fragment. For example , there is a broad T blob centered at 42 nt in electropherogram A that spans from around nt 40-46. Even though the base-calling software is confused, it is possible to read the correct sequence by ignoring the dye blob. Even after dye terminators are completely removed, there is a slow hydrolysis of dye moieties with storage of the reaction products . This frequently shows up as a broad T peak around nt 100. Electropherograms B through D show varying degrees of this degradation product, but the sequence can be easily read manually. (© 2007 by David F. Bishop, used with permission.)
of varying size. These sequencing products then produce a mixed signal in the trace downstream of the single nucleotide run • Insertions or deletions in one allele. This can occur when two alleles are sequenced together (peR templates) and one of the two mononucleotide runs has an InslDel polymorphism
• Premature termination of the sequencing reaction - Strong stops resulting from inverted repeats producing stem-loop secondary structures
(Figure 44) - Strong stops resulting from interstrand hybridization
(Figure 45)
111
3-48
Molecular Genetic Pathology
Peaks of all bases in same base position C
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Fig. 37. Large peaks corresponding to all four nucleotides in the same base position are most likely caused by a bubble migrating though the capillary (A, B). The optical refraction caused by the bubble disperses the wavelengths and focuses them causing an intensity increase. This is most frequently observed after installation of a new capillary. (© 2007 by David F. Bishop, used with permission.)
Mixed sequence caused by low template conce ntration
Fig. 38. The presence of 2-4 nucleotide peaks in each base position along with very weak detector signals indicates low or no template in the sequencing reaction. In the example, the signal intensity was about 1I500th the typical intensities. Frequently, dye blobs will be seen, as even extremely low levels of contaminating dye terminators will show up at these low sequence signal strengths. To the dye blob peaks centered at 5, 15, 32, and 55 nt in trace A. This mixed sequence continues throughout the sequence read as seen for subsequent region s in traces Band C. (© 2007 by David F. Bishop , used with permission.)
Smear sequenc ing due to the absence of the primer binding site
Fig. 39. Even weaker signal strengths are observed when the expected primer binding site is absent from the sequencing reaction. The resulting pattern feature s a predominant higher intensity area in the first 50 bases due to sequencing of small amounts of primer dimer s, with the remaining weak sequence due to sequencing of the smear of diminishing amounts of higher weight fragment s that the polymerase makes from the primers alone in absence of an effective template. (© 2007© by David F. Bishop, used with permission.)
112
3-49
Diagnostic Methodology and Technology
Adjacent peak duplication at lower intensity caused by N-1 primer contamination
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Mixed sequences due to two templates
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Fig. 41. Co-purification of two plasmids containing different insert sequences results in sequences where the shared vector sequence is unique, followed by a mixed sequence obtained from the simultaneous sequencing of both inserts in the same reaction. This is shown in the example with the arrow marking the beginning of the two different insert sequences. A multiple cloning sequence ending in Eco RI can be seen above the unique sequence electropherogram. (© 2007 by David F. Bishop, used with permission.)
113
3-50
Molecular Genetic Pathology
Mixed sequences due to duplicated T7 sites
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Fig. 42. Sequences of a plasmid containing the minichromosome maintenance deficient 8 (MCM8) cDNA were obtained using T7 as a primer. The sequences looked like they were contaminated with a second plasmid due to the presence of two different nucleotides at each base position, but interestingly, after a couple of hundred bases, the sequence became readable (electropherograms B-D). It was noted that one of the contaminating sequences petered out after a TO repeat region, allowing the underlying sequence of the forward strand of the MIN8 cDNA to predominate. It was then noted that the 3' end of the MIN8 cDNA contained a CA repeat of the same length as the TO repeat (Panel A). Thus, it became clear that the plasmid had T7 priming sites at both ends of the cDNA sequence, resulting in both sequences being read simultaneously. Synthesis of gene-specific unique primers permitted clean sequencing of the insert. (© 2007 by David F. Bishop, used with permission)
Mixed sequ ences due to polymerase slippage
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Fig. 43. DNA polymerase has difficulty faithfully replicating homopolymers and occasionally skips a base, resulting in a fraction of the sequencing reaction having one or more bases deleted in the homopolymer region. Since the subsequent sequences are out of phase by one or more bases, shadow bands of increasingly lower intensity will precede the correct sequence. One can read through such regions by using homopolymer primers anchored at the 3' end by the next base in the sequence, for example, 5' AAAAAAAAAAAAC 3' for this sequence. (© 2007 by David F. Bishop, used with permission)
114
3-51
Diagnostic Methodology and Technology
Strong stops caused by inverted repeats
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Fig. 44. Sequencing of the human transferrin receptor (TFR) cDNA resulted in a strong stop a few bases before a highly stable inverted repeat, stem-loop secondary structure. In A, the top line is that of the published TFR sequence , while the bottom line is the sequence obtained by Cycle Sequencing and CEoThe middle line is the sequence obtained after heat denaturing the cycle sequencing reaction products just prior to loading for CEoThe corresponding electropherograms for the heated and unheated reaction products are shown in Band C, respectively. The structure predicted in D resulted from the presence of an inverted repeat sequence. While heat denaturation often succeeds in melting secondary structures, and is typically more effective than additions such as DMSO, or the use of modified nucleotides, more extended perfect inverted repeats, such as those found in shRNA (short hairpin RNA) and AAV (adeno-associated virus) retroviral constructs are resistant to these treatments. In these cases, the method of choice is to use or introduce a restriction site in the loop and after cutting, sequence the loop region from both sides . For shRNA, a detailed treatment of this approach has been published (McIntyre and Fanning, BMC Biotechnol. 2006;6 : I). (© 2007 by David F. Bishop , used with permission)
The Snapshot Method (Applied Biosystems, ABI) • This method is a modified form of Sanger sequencing used to detect SNPs • The SNaPshot primer targets a sequence immediately upstream of the SNP site and is extended by a single base in the presence of all four fluorescently labeled dideoxynucleotides (ddNTPs) • Each fluorescent ddNTP emits a different wavelength, which is translated into a specific color for each base. The size of the product is the size of the initial probe plus one fluorescent base • The reactions are run on an ABI 3700 and genotypes are determined by the color and location of the peak that is generated from the emitted fluorescence • Data are then analyzed with the ABI Gene Scan software package using size standards for verification of the peaks . Primer design and DNA template purification can significantly affect genotyping accuracy • Failure to remove unincorporated ddNTPs can yield extraneous fluorescence
• This can prevent a sample from being genotyped , or cause it to be genotyped incorrectly. Genemapper software is available to automatically determine sample genotypes
Pyrosequencing Technology • Principle: Pyrosequencing (Pyrosequencing Inc., Westborough, MA) is a recently developed DNA sequencing method based on detecting the formation of pyrophosphate, the by-product of DNA polymerization. In a number of enzymatic steps, pyrophosphate is converted to ATP, which fuels a luciferase reaction and converts luciferin to oxyluciferin (Figure 46). Light is generated with each addition of a nucleotide in the growing DNA chain. The intensity of light generated is proportional to the amount of nucleotide incorporated. Scores are determined by computer-automated comparison of predicted SNP patterns with raw data. Samples generally do not require manual interpretation, which provides reliability and accuracy in scoring. • Procedure - Step I : a sequencing primer is hybridized to a singlestranded, PCR-amplifed DNA template, and
115
3-52
Molecular Genetic Pathology
Strong stops caused by direct repeats
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Fig . 45 . Three independent sequencing reactions of a region of the human tumor necros is factor receptor (TNFR) cDNA resulted in truncated sequence in a repeat region; the sequences are shown in A and the electropherograms in B-D. A dot blot analysis of the TNFR sequence revealed an extensive region of direct repeats between nucleotides 900 and 1200 of the sequence in E. Heat denaturation of the cycle sequencing products is particularly effective, since the secondary structure is caused by inter-strand hybridization. (© 2007 by David F. Bishop , used with permission)
Fig. 46. Mechanism of light generation in pyrosequencing reactions (see text for explanation).
116
incubated with DNA polymerase, ATP sulfurylase, luciferase, and apyrase enzymes, as well as the substrates adenosine 5' phosphosulfate (APS) and luciferin - Step 2: one of four deoxyribonucleotide triphosphates (dNTPs) is added to the reaction . DNA polymerase catalyzes the incorporation of the deoxyribonucleotide triphosphate into the DNA strand only if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide - Step 3: ATP sulfurylase quantitatively converts PPi to ATP in the presence of APS. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge-coupled device (CCD) camera and seen as a peak in a Pyrogram''". The height of each peak (light signal) is proportional to the number of nucleotides incorporated - Step 4: apyrase, a nucleotide degrading enzyme, continuously degrades ATP and unincorporated dNTPs.
3-53
Diagnostic Methodology and Technology
This switches off light production and regenerates the reaction solution. The next dNTP is then added - Step 5: addition of dNTPs is performed one at a time. It should be noted that deoxyadenosine alfa-thio triphosphate (dATPaS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP) since it is efficiently used by the DNA polymerase, but not recognized by the luciferase
sequence is determined from the signal peaks in the Pyrogram • Advantages of pyrosequencing: - Pyrosequencing not only generates sequence information, but also it is quantitative, ideal for measuring the relative amounts of alleles. This property allows the quantification of DNA methylation, heterozygosity, ploidy levels, multi-copy genes, pooled DNA samples, hematopoeitic chimerism, and mixed genotypes in heterogeneous samples (e.g., tumor and normal cells)
- As the process continues, the complementary DNA strand is built up and the nucleotide
PROTEIN DETECTION METHODS
Enzyme Immunoassay (EIA)
A
(1)
• General information:
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- EIA is the newer name of enzyme-linked immunosorbent assay (ELISA), which was developed in 1971 as a way of detecting proteins in serum
y
- EIA is an immunoassay technique combining an in vitro antigen-antibody reaction with a subsequent enzymatic reaction - EIA is a highly sensitive and specific assay and compares favorably with other methods used to detect substances in the body, such as radio immunoassay
(2)
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Competitive EIA assay
B
- EIA tests are usually performed in microwell plates, and use an enzyme linked to an antibody or antigen as a marker for detection of a specific protein , especially an antigen or antibody • Principle (Figure 47):
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Sandwich EIA assay
- EIA techniques have many variations. Currently the three most important are: • Competitive immunoassay: relies on the principle of competition between antigen in a test specimen and antigen-enzyme conjugate for binding with a constant amount of antibody. Usually, secondary antibody (such as goat anti-rabbit IgG or goat anti-mouse IgG) is pre-coated on microtiter plates, and is used to bind with an antigen-specific mono- or poly-antibody. In the meantime, a fixed amount of enzyme-labeled antigen competes with antigen in the test specimen for a fixed number of binding sites of the antigen-specific mono- or polyantibody. Thus, the amount of enzyme-labeled antigen immunologically bound to the well progressively decreases as the concentration of antigen in the specimen increases • Sandwich immunoassay: is based on the capture of antigen by one antibody and the detection by another antibody. In general , a monoclonal antibody, which is pre-coated on microtiter plates,
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Fig. 47. Three types ofElA assay: (A) competitive EIA assay : (1) coat plate with secondary antibody ; (2) add primary antibody; (3) add antigen (sample); (4) add antigen conjugate; (5) add substrate to produce color. (B) Sandwich ElA assay: (1) coat plate with primary antibody no. 1; (2) add antigen (sample); (3) add primary antibody no. 2; (4) add secondary antibody conjugate; (5) add substrate to produce color. (C) Indirect EIA assay: (1) coat plate with antigen; (2) add primary antibody; (3) add secondary antibody conjugate; (4) add substrate to produce color.
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Molecular Genetic Pathology
is used to capture antigen in a test specimen. Antigen-specific polyclonal antibody, which is conjugated to an enzyme, then binds with the immobilized antigen • Indirect immunoassay: is used to detect specific antibody, and is somewhat different from the first two immunoassays, which are for detection of antigen. Antibody in a test specimen is captured by antigen pre-coated on microtiter plates. Detection occurs by means of a secondary antibody conjugated to an enzyme - The enzyme label: most of the immunoassays employ horse-radish peroxidase (HRP), ALP, or ~-galactosidase. Substrates used with HRP include 2,2'-azo-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), and 3,3'5 ,5'tetramethylbenzidine base (TMB), which yield green, orange, and blue colors, respectively. TMB is gradually replacing mutagenic substrates such as OPD, leading to increased sensitivity and safety - Color development: with the addition of antibody or antigen conjugated to enzyme (such as HRP) and followed by the addition of substrate (such as TMB), the amount of antigen or antibody is detected through measurement of the color intensity with a spectrophotometer. This results in a direct or inverse relationship between optical density (OD) and concentration: the higher the OD the more antigen or antibody (for sandwich or indirect EIA type); or the higher the OD the less antigen (for competitive EIA) • Procedure : - An EIA is a five-step procedure: • Coat the microtiter plate wells with antigen or antibody • Block all unbound sites to prevent false-positive result • Add antibody or antigen to the wells • Add primary or secondary antibody conjugated to an enzyme • Introduce substrate - General Procedure for the Competitive EIA Method : • To coat the plate with secondary antibody, add diluted secondary antibody to each well. The appropriate dilution should be determined using a checkerboard titration prior to testing samples. A microtiter plate will bind approximately 100 ng/well (300 ng/cm-). The amount of antibody used will depend on the individual assay, but if maximal binding is required , use at least I ug/well . Allow to incubate for 4 hours at room temperature or 4°C overnight • Wash the coated plate by filling wells with PBS. Flick the plate over a suitable container, and rinse with PBS two more times
118
• To block residual binding capacity of the plate, fill each well to the top with blocking buffer (3% BSAlPBS with 0.02% sodium azide) and incubate for at least 2 hours at room temperature . Rinse plate three times with PBS as in step b. After the last rinse, remove residual liquid by wrapping each plate in a large paper tissue and gently flicking it face down onto several paper towels laid out on a benchtop • Add antigen-specific antibody to the coated wells. The antibody should be diluted in blocking buffer (3% BSAlPBS with 0.05% Tween-20) • Add standard or sample (antigen) to plate. Standard or antigen sample should be diluted in blocking buffer • Add antigen-conjugate solution to the coated wells. All dilutions should be done in the blocking buffer. Incubate for at least 2 hours at room temperature in a humid atmosphere • Wash the plate four times with PBS as in step b • Add substrate (such as TMB) and measure OD: TMB is added to each well and incubated for 30 minutes at room temperature, resulting in the development of blue color. The color development is stopped with the addition of IN H2S04 or 3N HCI, and the OD is measured spectrophotometrically at 450 nm • A standard curve is obtained by plotting the concentration of standards versus OD. Competitive EIA yields an inverse curve, where higher values of standards or antigen in the samples yield a lower amount of color change • The concentration of the antigen specimen can be calculated from the standard curve - General procedure for the sandwich EIA method : • Coat plate with antigen-specific I st monoclonal antibody • Wash the wells three times with PBS • Block residual binding capacity of plate • Add standard or sample (antigen) to plate: Incubate for at least 2 hours at room temperature in a humid atmosphere • Wash the plate four times with PBS • Add antigen-specific 2nd polyclonal antibody to plate: the amount to be added can be determined in preliminary experiments. For accurate quantitation, the 2nd antibody should be used in excess. Incubate for at least 2 hours at room temperature in a humid atmosphere • Wash with several changes of PBS • Add HRP-conjugated secondary antibody (such as goat anti-rabbit antibody) to plate: incubate for I hour at room temperature in a humid atmosphere • Wash with several changes of PBS
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Diagnostic Methodology and Technology
• Add substrate (such as TMB) and measure OD • Make a standard curve • Perform calculation - General Procedure for the Indirect EIA method: • Coat plate with antigen • Wash the wells three times with PBS • Block residual binding capacity of plate • Add standard or sample (antibody) to plate • Wash the plate four times with PBS • Add HRP-conjugated secondary antibody to plate • Wash with several changes of PBS • Add substrate (such as TMB) and measure OD • Make a standard curve • Perform calculation • Applications: - EIA can be performed to evaluate the presence of antibody in a sample, which is recognized by an antigen. Therefore, it is a useful tool for determining serum antibody concentrations, such as with the HIV test or West Nile Virus - EIA also can be used to detect and quantify the concentrations of antigens that are recognized by antibodies, such as disease-related substances - The most commonly used EIA assay format is the sandwich assay. It can be used to measure antigen s that are bound between two antibodies. Competitive assay is often used when the antigen is small and has only one epitope, or antibody binding site • Advantages : - EIA is a sensitive and specific assay for the detection and quantitation of antigens or antibodies. It combines the specificity of antibodies with the sensitivity of simple enzyme assays - EIA is also a rapid and relatively easy assay when compared with conventional GCIMS and HPLC. Wherea s a conventional method may require I hour to analyze one sample, EIA can analyze about 30 samples per hour. The Immunoassay instrumentation kit is portable and can be used for testing right at the sampling site - EIA can be performed without the use of radioactive material s, and is also considerably less expensive than radioimmunoassay - ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. In quantitative ELISA, the optical density of the sample is interpolated into a standard curve, which is typically based on a serial dilution of the target • Limitations: EIA requires skilled laboratory technicians and specialized laboratory equipment
- Cross-reactivity may occur with the secondary antibody, resulting in non-specific signal - Because of the design of the immunoassay, sample contaminants that might interfere with the antigenantibody reaction can produce positive reading s when samples are indeed negative (false-positive results) - Lower molecular weight molecules often lack specific antigenic sites, and sometimes there are cross-linking problems
Protein Electrophoresis • General Information: - Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a very common method for separating proteins using a polyacrylamide gel as the support medium and SDS as a denaturing agent - SDS-PAGE is used to separate proteins based on molecular weight - Gel density can be controlled by varying the monomer concentration. Gels can be of constant density or they can be variable (gradient gels) • Principle: SDS has two important features: • It is an anionic detergent that binds quantitatively to proteins , giving them uniform negative charge, which means they will all migrate towards the positive pole when placed in an electric field. The number of SDS molecules that bind to a protein is proportional to the number of amino acids that make up the protein. Each SDS molecule contributes two negative charges , overwhelming any charge the protein may have • SDS also disrupts the forces that contribute to protein folding (tertiary structure), ensuring that all proteins are denatured to the same linear configuration (see Figure 48) - Since all proteins become linearized and uniformly negatively charged, separation occurs solely on the basis of size. Smaller molecules are able to navigate the gel faster than larger ones, thus they migrate more rapidly - The polyacrylamide gel is a cross-linked matrix that functions as a sieve that differentially retards the motion of molecules as they move through the electric field - The preparation of the gel require s casting two different layers of acrylamide between glass plates. The lower layer (separating or resolving gel) is responsible for actually separating polypeptides by size. The upper layer (stacking gel) includes the sample wells; it is designed to sweep up proteins in a sample between two moving boundaries so that they are compressed (stacked) into micrometer thin layers when they reach the separating gel - Staining of proteins in gels may be done using the standard coomassie brilliant blue, amido black, or
119
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Molecular Genetic Pathology
- The size of proteins corresponding to each band is determined based on comparison with molecular weight markers, which are run along with the sample proteins on the gel
Before SDS
• Applications : - Estimating relative molecular mass of proteins - Determining protein purity in a sample - Identifying the composition of protein complexes - Preparation for blotting
After SDS
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Fig. 48. Cartoon depicting changes in a protein when incubated with the denaturing detergent SDS. The top portion of the figure shows a protein with negative and positive charges due to charged R-groups. The lower diagram shows how SDS coats protein molecules with a negatively charged layer, which overwhelms any positive charges intrinsic to the protein. SDS also disrupts hydrophobic interactions. As a result, proteins are denatured (reduced to their primary structures), and thus linearized.
• Advantages: 1D gel electrophoresis is a relatively easy technique and is very reliable - Can be performed using large gels or smaller "micro" gels, giving a choice as to how much sample and reagent(s) will be utilized • Limitations: - Occurrence of false-positives and -negatives due to co-migrating contaminants - Abnormal migration of proteins due to presence of large numbers of charged amino acids
Western Blotting (WB) silver stain reagents of various kinds. Coomassie brilliant blue G-250 is probably the most widely used due to its convenience . It binds non-specifically to virtually all proteins and can visualize bands containing as little as 0.3 flg protein - Protein molecular weight standards are used to measure the relative sizes of the unknown proteins • Procedures: - A sample of proteins is first denatured . An appropriate amount of electrophoresis sample buffer (IX = 125 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 0.003% bromophenol blue, and 1% ~-mercaptoethano1) is then added to all proteins. The mixture is then heated to 95°C for 3-5 minutes - The SDS-PAGE gel is prepared in two steps. First the resolving gel is prepared and then the stacking gel, which sits atop the resolving gel - The gel is placed into the gel apparatus, in an appropriate buffer, and protein samples are loaded onto the stack ing gel. Load 5-100 ug total protein in a volume that is appropriate for the well size. Be sure to use protein markers, which produce bands of known size - When power is supplied, the proteins migrate from the cathode (upper chamber of gel apparatus) to the anode (lower chamber) - Electrophoresis is stopped when the bromophenol blue dye front reaches the bottom of the gel. The protein bands are then stained (e.g., with coomassie brilliant blue G-250)
120
• General information: - WB was first introduced in the late 1970's. Since then, it has become rapidly accepted and widely applied - WB is used to detect a specific target protein in a sample containing a complex mixture of proteins by using a polyclonal or monoclonal antibody specific to that protein - WB allows investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples • Principle : - SDS, a reducing agent such as dithiothreitol (DTT) or 2-mercaptoethanol, and heat are responsible for the actual denaturation of proteins • SDS breaks up the 2- and 3D structure of proteins by adding negative charge to the amino acids • Disulfide bonding is covalent and is not disrupted by SDS. DTT or 2-mercaptoethanol is a strong reducing agent. Its specific role in protein denaturation is to remove the last remnant s of tertiary and quaternary structure by reducing disulfide bonds • Many proteins have significant hydrophobic properties and may be tightly associated with other molecules . Heating the proteins to at least 60°C separates the molecules, allowing SDS to bind in the hydrophobic regions to complete the process of denaturation - The denatured proteins are separated based on weight and electrical properties by gel electrophoresis, usually
3-57
Diagnostic Methodology and Technology
SDS Polyacryl amide gel electrophoresis
Protein blot on nitrocellulose
-
Label with specific antibody
Detect antibody
-
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Fig. 49. Schematic presentation of how WB is performed (see text for description of steps).
-
-
-
-
SDS-polyacrylamide (SDS-PAGE) (see Protein Electrophoresis [SDS-PAGE]) The proteins are then electrophoretically transfered to a membrane of charged nylon, nitrocellulose, or polyvinylidene fluoride The membrane must be incubated with generic proteins (such as milk protein s) to block remaining hydrophobic binding sites on the membrane. This reduces background and prevents binding of the primary antibody to the membrane itself A primary antibody with specificity for the protein of interest is introduced, and antibody-protein complexes are formed. The appropriate working concentration of the primary antibody is dependent upon its binding characteristics The method of detection is dependent upon the label that has been conjugated to the primary (or secondary) antibod y. The most common antibody label used in WB is an enzyme such as ALP or HRP, which can be detected visually through the conversion of a colorimetric substrate (chromagen) to a colored precipitate at the site of antibody binding. Alternatively, chemiluminescent substrates may be employed which emit light upon conversion by the enzyme . The light emitted at the site of substrate
conversion can be captured on X-ray film. Chemiluminescent substrates are much more sensitive than colorimetric substrates • Procedure (Figure 49): - Preparation of celllysates: • Process cells by trypsinization and spin or tissue by mincing and digestion • Lyse the pellet or tissue with lysis buffer on ice for 10 minutes • Sonication for 10-30 seconds • Spin at 14,000 rpm in an Eppendorf® (Hamburg , Germany) microfuge for 20 minutes at 4°C • Transfer the supernatant to a new tube and discard the pellet • Determine the protein concentration using Bradford assay or BCA method SDS-PAGE is performed (as earlier) Membrane transfer: • Prechill transfer buffer at -20°C • Cut a piece of a suitable membrane (e.g., nitrocellulose, polyvinylidene fluoride , and so on)
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Molecular Genetic Pathology
3-58
• Prewet the membrane, sponges, and filter papers in transfer buffer • Assemble a "sandwich" following the sequences: sponge- filter paper-gel-membrane-filter paper-sponge • Transfer proteins from the gel to the membrane using electrophoresis for I hour at 100 V at 4°C. Bigger protein s might take longer to transfer. The negative pole must be on the side of the gel and the positive pole must be on the side of the membrane to drive the negatively charged proteins over to the positively charged membrane. One must ensure that there are no air bubbles between the membrane and the gel or the proteins will not transfer • When finished, the membrane is imprinted with the same protein bands as the gel - Blocking: • Immerse the membrane in blocking buffer • Block for 30 minute s at 37°C, 1 hour at room temperature, or overnight at 4°C - Incubation with primary antibody: • Decant the blocking buffer and add a primary antibody, diluted in blocking buffer as suggested in its product description sheet • Incubate with gentle shaking for 30 minutes at 37°C , 1 hour at room temperature, or overnight at 4°C • Decant the antibody, and wash for 30 minutes with agitation in wash buffer (TBS or PBS with 0.1 % Tween-20), changing the wash buffer every 5 minutes - Incubation with secondary antibody : • Decant the wash buffer and add secondary antibody conjugated to HRP, diluted in blocking buffer, for 1 hour at room temperature • Decant the antibody conjugate, and wash for 30 minutes with agitation in wash buffer (TBS or PBS with 0.1 % Tween-20 ), changing the wash buffer every 5 minute s - Enzymatic chemiluminescence substrate incubation and visualization: • Decant the wash buffer and place the membrane in a plastic bag or clean tray containing the development working solution (0.125 mlzcm'') for 1-5 minute s following the manufacturer's instructions for specific enzymatic chemiluminescence reagents and procedures. Agitate the bag or tray to cover the surface of the membrane • Remove the membrane from the bag or tray and wrap it using plastic paper • Expo se to X-ray film and enhancing cassette for the appropriate time period. For best results, use a range of exposures (10 seconds, 1 minute,
122
5 minutes, and 20 minutes ) to visualize the chemiluminescence signal corresponding to the specific antibody-antigen complex • Bands appear wherever there is a protein-primary antibody-secondary antibody-enzyme complex. These bands correspond to the location of the target protein • Applications: - WB provides information about presence and concentration of an antigen in a sample - WB also reveals data about the nature of the antigen detected, such as its molecular weight, tertiary structure, and in some cases, its biologic activity - In clinical settings, WB is routinely used to confirm serious diagnose s suggested by ELISA such as HIV seroconversion • Advantages: - WB is a sensitive and specific technique - WB is especially helpful when dealing with antigen s that are insoluble , difficult to label, or easily degraded , and thus not amenable to procedures such as immunoprecipitation - WB, unlike EIA, can be used to detect multiple protein antigen s • Limitations: - WB is a relatively imperfect quantitati ve technique. For example, variations in epitopes can affect the intensity of staining or derivatization, and chemiluminescence exposure times are known to vary from blot to blot - WB sometimes produce s serious errors such as background staining or extra bands in the blot
Key Technologies Used in Proteomics Proteomics is the large-scale study of proteins, particularly their structures and function s. This term was coined by Marc Wilkins and colleagu es in the early 1990s and made an analogy with "genomics", which describes the entire collection of genes in an organism. Proteomics is much more complicated than genomic s. Most importantly, while the genome is a rather constant entity, the proteome differs from cell to cell and is constantly changing through its biochemical interactions with the genome and the environment. One organi sm has radically different protein expression pattern s in different parts of its body, in different stages of its life cycle, and in different environmental conditions. Since proteins playa central role in the life of an organism, proteomics is instrumental in the discovery of biomarkers, such as markers that indicate the presence of a particular disease. Proteomics studies usually require three stages of sample preparation (Figure 50). Several key technologies are involved, including 2D electrophoresis and MS. Protein s are first separated using 2D electrophoresis, and then individual protein spots of interest are cut from the gel and digested into
3-59
Diagnostic Methodology and Technology
Unidentified protein extracted from gel
Split into fragments of 5-10 amino acids .~.Il.l!J IJr:b i: 1_ j/;! :~t~)~l
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Fig. 50. General flow scheme for proteomic analysis .
smaller polypeptide fragments (5 to 10 amino acids in length) by enzymes. The polypeptide fragments are then analyzed by MS. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS is used primarily to measure the masses of peptides. Electrospray ionization (ESI) tandem MS is used to obtain peptide structure and sequence data.
2D Electrophoresis • General Information : - 2D electrophoresis is a method of protein separation, by which proteins in a mixture are separated according to their isoelectric point (pI) in the horizontal direction (isoelectric focusing, or IEF) and molecular weight in the vertical direction (SDS-PAGE) 2D eletrophoresis is the most effective means of resolving complex protein mixtures, and was first introduced in the early 1970s 2D eletrophoresis is used for the isolation/separation/ purification of proteins for further characterization with MS and identification of specific proteins. Thus, this separation method has become synonymous with proteomics 2D electrophoresis can effectively separate multiple isoforms of a protein
• Principle: - Sample preparation and solubilization are crucial factors for the overall performance of 2D electrophoresis. Protein complexes and aggregates should be completely disrupted in order to prevent the appearance of artifactual spots due to incomplete protein solubilization. Proteins can be completely solubilized, typically by a reagent such as urea 2D electrophoresis separates proteins based on size, as in regular electrophoresis, but also based on charge, or isoelectric point (pI) - The first step is isoelectric focusing (IEF). The mixed protein sample is run on an immobilized pH gradient, the range of the gradient used depends on the expected proteins in the sample. The sample is added to the gradient and an electric current is applied. Proteins will be positively charged at pH's below their pI and negatively charged at pH's above their pl. When a given protein is at the point in the gradient where the surrounding pH is equal to its pI, the protein will have a net charge of zero and it will stop moving - The second step is SDS-PAGE. Once enough time has passed for the proteins to settle in the gradient, the
123
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Molecular Genetic Pathology
with forceps, rinsed with water, and positioned on a focusing tray as specified by manufacturer (e.g., Biorad, Amersham, Pharmacia Biotech) • Running conditions: depending on different IEF equipment, follow manufacturer's protocol for IEF run. For example, based on Pharmacia-Hoeffer Biotechnology AB, the voltage is linearly increased from 300-3500 V during 3 hours, followed by 3 additional hours at 3500 V, whereupon the voltage is increased to 5000 V. Focusing is carried out for a total of 100 kVh in an overnight run. After running, strips can be frozen (-20°C) for several weeks (remove oil), or used immediately for the second dimension
Size (mW)
1
Isoelectric point (pI) -
-
-.
Fig. 51. 2D protein electrophoresis (polyacrylamide gel). Proteins are separated based on two different physical properties: isoelectric focusing is followed by standard separation based on size .
current is removed and the gradient is laid horizontally along an SDS-PAGE gel. An electric current is then applied and the proteins move horizontally out of the IEF gradient and into the SDS-PAGE gel where they are separated based on molecular weight - Once the proteins have been separated, they can be visualized by conventional staining techniques, including the standard coomassie brilliant blue, amido black, and silver stains. Silver staining is one of the most sensitive protein detection methods, with sensitivity greater than lOO-fold that of coomassie brilliant blue staining. A stained 2D gel is shown in Figure51 • Procedures:
• Prepare resolving gels (SDS-PAGE) as described in Western blotting section • IPG strip transfer: after equilibration, the IPG strips are picked up using two pairs of clean forceps and are carefully placed atop the SDS-PAGE (resolving) gels facing the front of the gel cassettes, by carefully sliding them down the gel cassette via the plastic laminated side of the IPG strips. For consi stency, the IPG strips are positioned with the basic side closer to the anode (Red/+) and the acidic side closer to the cathode (Blackl-)
Sample preparation: proteins for 2D electrophoresis are first extracted from tissue or cells using an appropriate amount of urea lysis buffer (8 M urea , 4% CHAPS, 65 mM DTE, 40 mM Tris, and a trace of bromophenol blue) depending on the size of tissue or the number of cells and strip size
• The tops of the gel cassettes are sealed with 0.5-1 % agaro se in electropheresis buffer. Ensure that there is no formation of bubbles between the IPG strips and the SDS-PAGE gel. The combination of the IPG strips and agarose avoids the need for a stacking gel
Immobilized pH gradient (IPG) as first dimens ion:
• Running conditions: run the gels at 40 mNgel at 8-12°C until the trackingdye reaches the bottom of the gels
• IPG gel strip rehydration: 100 Ilg-15 mg of protein sample is pipeted into rehydration tray; IPG gel strips are positioned such that the gel of the strips is in contact with the protein samples (upside down). The gels and samples are covered with several milliliters low viscosity paraffin oil to prevent evaporation. The strips are then left at room temperature for rehydration. A minimum of 10 hours is required for rehydration , and overnight is recommended • Sample application: the rehydrated IPG gels carrying the proteins are removed from the rehydration tray
124
• IPG gel strips equilibration: After the first dimension of electrophoresis is carried out, the strips are equilibrated in order to resolubilize the proteins and to reduce disulfide bond s. The strips are transferred into an equilibration tray and equilibrated in equilibration buffer I (50 mM TrisHCI pH 6.8, 6 M urea, 30% glycerol, 2% SDS , and 2% DTE) for 10 minutes, and then equilibrated in equilibration buffer II (50 mM Tris-HCl pH 6.8, 6 M urea, 30% glycerol, 2% SDS , 2.5% iodoacetamide, and a trace of bromophenol blue) for another 10 minutes - SDS-PAGE as second dimension:
- Protein detection: At the end of the second dimen sion run, the proteins can be detected by conventional staining techniques, such as coomassie brilliant blue , amido black , and silver stains . Silver staining is popular owing to its high sensitivity. The procedure for silver staining is described below (the gel should be gently agitated throughout the various steps): • After the SDS-PAGE is carried out, the gels are removed from the glass plates and washed in deionized water for 5 minutes
Diagnostic Methodology and Technology
• Soak in ethanol: acetic acid: water (40:10:50) for I hour • Soak in ethanol: acetic acid: water (5:5:90) for 2 hours or overnight • Wash in deionized water for 5 minutes • Soak in a solution containing glutaraldehyde (1%) and sodium acetate (0.5 M) for 30 minutes • Wash three times in deionized water for 10 minutes each • Soak twice in a 2,7 Naphthalene-disulfonic acid solution (0.05%) for 30 minutes each • Rinse four times in deionized water for 15 minutes each • Stain in a freshly made ammoniacal silver nitrate solution for 30 minutes • After staining, the gels are washed four times in deionized water for 5 minutes each • The images are developed in a solution containing citric acid (0.01%) and formaldehyde (0.1%) for 5-10 minutes • When a slight background stain appears, development is stopped with a solution containing Tris (5%) and acetic acid (2%) - Image analysis: • Stained gels are scanned with a scanning device, and the proteins of interest are marked for excision • Applications: - Resolution and analysis of highly complex protein mixtures - Separation of the isoforms of a protein - Proteomic analysis - If sufficient sample is present on the gel (>300 I1g of total protein) then proteins can be excised from the gel, subjected to in-gel proteolysis and analyzed by MS • Advantages: - The main advantage of using 2D electrophoresis is the large mass range and the amount of proteins that can be analyzed at anyone time. 2D electrophoresis is particularly effective in the analysis of proteins within the mass range of 20-250 kDa and pI of 3-8, and can separate 2000-3000 proteins in one gel - 2D electrophoresis is the single best method for resolving highly complex protein mixtures • Limitations: - It is difficult to isolate proteins with isoelectric points outside of the range of 3.0-8.0. This is due to problems associated with creating stable pH gradients outside that range - 2D electrophoresis is a low-throughput, timeconsuming process (3-4 days per run) that involves many steps and requires a high level of laboratory skill to obtain good results
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- 2D electrophoresis has diminished utility in the analysis of extremely acidic, basic, or hydrophobic proteins such as membrane-bound proteins, and also in the analysis of smaller proteins and peptides (<15 kDa)
Mass Spectrometry MS is an analytical tool which can be used to determine chemical structure on the basis of the mass/charge (mlz) ratio of molecular ions derived from a fragmented parent molecule. Mass spectrometer instruments have three fundamental components (Figure 52). The ftrst componentis the ion source, which converts sample molecules into fragmented molecular ions. The second component is the mass analyzer, which resolves these ions based on their mlz ratios. The third component is the detector, which detects the ions resolved by the mass analyzer. In brief, the samplehas to be introduced into the ionization source of the instrument. Once inside the ion source, the samplemolecules are ionized. The resulting ions are extracted into the analyzer region of the mass spectrometer where they are separatedaccordingto mlz ratio. The separated ions are detected and information pertaining to the mlz ratio and relativeabundance of each ion is stored for presentation as a mass spectrum. Some instruments are capable of analyzing intact molecules with little fragmentation. Two different types of instrument are used for most proteomics MS work: MALDI-TOF instruments, which measure peptide masses, and ESI-tandem MS instruments, which are used to obtain structure and sequence data. The two types operate in entirely different ways and generate different, but complementary information. Indeed, the best-equipped proteomics laboratories have both types of instruments available.
Matrix-Assisted Laser Desorption Ionization- Time of Flight • General Information: - The first term "MALDI" (matrix-assisted laser desorption ionization) refers to the ion source, and describes a method of ionization, whereas the term "TOF" (time of flight) refers to the mass analyzer - MALDI-TOF mass spectrometry is a method used for measuring the mass of a sample. For large samples such as biomolecules (proteins, peptides, oligosaccharides, and oligonucleotides), molecular masses can be measured to within an accuracy of 0.0I% of the total molecular mass of the sample. For small organic molecules, the molecular masses can be measured to within an accuracy of 5 ppm or less, which is often sufficient to conftrm the molecular formula of a compound
• Principle: - In-gel digestion of proteins separated by 2D electrophoresis: • Before peptide masses can be obtained using MALDI-TOF, the proteins must be cleaved into peptides. This is because there are errors in the
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The components of a mass spectrometer
...
...
... I
t Vacuum
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Fig. 52. The components of a MS.
00
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measurement of intact proteins. The greater the mass of the protein, the greater the absolute magnitude of the error • Proteins can be digested into smaller polypeptide fragments (5-10 amino acids) with a suitable enzyme. Trypsin is useful for MS studies because each proteolytic fragment contains a basic arginine (R) or lysine (K) amino acid residue, and thus is eminently suitable for positive ionization MS analysis - MALDI principle (Figure 53): • The sample to be analyzed is dispersed in a large excess of a chemical matrix, which typically is composed of a small molecular weight organic molecule, which functions as a chromophore that strongly absorbs applied laser energy. Common matrix compounds include sinapinnic acid (SA) for protein samples and a-cyano-4-hydroxycinnamic acid (ACH) for peptide samples
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• The admixture of the sample and matrix is then spotted onto a small plate or slide and left to evaporate in air. The evaporation of residual water or other solvent from the sample alIows the formation of a crystal lattice into which the peptide sample is integrated • The target (plate or slide) is then placed into the MALDI source. The source is equipped with a laser, which fires a beam of light at the target. The matrix chemicals absorb photons from the beam and become electronicalIy excited. This excess energy is then transferred to the peptides or proteins in the sample, thereby ionizing them. The matrix also functions to help overcome molecular photodissociation of the sample ions induced by direct laser irradiation, thereby preventing unwanted fragmentation • The ionization process can produce either positive or negative ions, depending on the nature of the sample. Positive ionization is used in general for protein and peptide analyses. In positive ionization mode the protonated molecular ions (M+H)+ are usually the dominant species. Negative ionization can be used for the analysis of oligosaccharides and oligonucleotides. In negative ionization mode the deprotonated molecular ions (M-Ht are usually the most abundant species • The ions formed in the MALDI source are then extracted and directed into the TOF mass analyzer - TOF principle (Figure 54): • The TOF analyzer separates ions according to their mlz ratios by measuring the time it takes for ions to travel through a field-free region known as the flight or drift tube, before striking a
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Diagnostic Methodology and Technology
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Fig. 54. Simplified schematic of MALDI-TOF mass spectrometry (A) linear mode and (B) reflecting mode.
detector. The heavier ions are slower than the lighter ones. In this way, each molecule yields a distinct signal • TOF analyzers can operate in either of two ways. In "linear mode," formed ions are extracted from the MALDI source, and then directly sent down the flight tube (TOF analyzer) to the detector (Figure 48A). The resolution of instruments running in linear mode with continuous extraction of ions is, however, relatively poor; ("resolution" refers to the extent to which the instrument can distinguish between ions of slightly different mlz values). In "reflecting mode," a reflectron (a device, which uses static electric field s to alter the paths of ions) located at the end of the flight tube, is used to compensate for differences in flight times of ion s having the same m/z value. This results in ions of the same mlz value reaching the detector at the same time. The reflectron dramatically improves resolution in TOF analyzers • Procedures: - Spot excision: • Excise spots of interest from 2D gel by using a clean pipet tip, cutting as close to the edge of the spot as possible, or by using a spot cutter • Transfer the gel spots into 0.5-mL siliconized tubes or a 96-well plate, one spot for one tube or one well • Rinse the spots using water; then remove water from tubes or well s - In-gel digestion of proteins: • Add 50 ul, of 50 % acetonitrile/l 00 mM ammonium bicarbonate (ABC) buffer to each tube or well, and incubate for 5 minutes • Remove supernatant using pipet • Repeat the above two steps
• Add 50 ul, of Acetonitrile to each tube or well • Remove the acetonitrile • Dry in speed vacuum • Swell gel pieces in 10-20 ul, 50 mM ABC for 4 minute s at 37°C • Add equal volume of trypsin solution to soaked gel pieces • Digest at least 1 hour at 37°C -
Extraction of tryptic peptides from gel : • Add 50 ul, of 60% acetonitrile/5% trifluoroacetic acid (TFA) to the gel pieces • Sonicate for 15 minutes in ice bath • Centrifuge to bring down liquid for 30 seconds • Transfer supernatant to a 0.5-mL siliconized tube • Repeat the above four steps , pool the supernatants • Dry pool supernatants in speed vacuum
- Sample preparation for MALDI analysi s: • The above supernatants may be concentrated by speed-vacuum to dryness, and then brought up in no more than 5-10 ul, of 0.1% TFN60% acetonitrile. A sample concentration of 1 mg/mL is ideal. Usually from I to 10 pm of sample is required for analysis • Take an aliquot (1-2 ilL) of this peptide solution and mix with an equal volume of a solution containing a vast excess of a matrix. The two most commonly used matrices are ACH and SA. ACH is usually dissolved at 30 mg/mL in 0.1 % TFN60% acetonitrile. SA can be prepared at 50 mg/mL in 0.1 % TFN60 % acetonitrile - MALDI-TOF analysis: • The mixture is applied to a stainless steel sample target, and allowed to dry. It is essential that all spots on the target be completely dry before the
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Source
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target is inserted into the sample chamber. Moisture on the target leads to rapid degradation of the electrodes • Insert target into MALDI-TOF MS instrument • With the target under high vacuum, the laser is fired . The energy arriving at the sample/matrix surface is optimized • Data is accumulated until an mlz spectrum of reasonable intensity has been amassed • The mlz scale of the MS is calibrated with a known sample that can either be analyzed independently (external calibration) or premixed with the sample and matrix (internal calibration) • Applications: MALDI- TOF MS is used for molecular mass measurements of various analytes such as peptide s, proteins, oligosaccharides, and oligonucleotides
- It is an important analytical tool in proteomics It is also used in the analysis of the products of peptide synthesis - It is used as a method of N-terminal and C-terminal protein/peptide sequencing • Advantages: - MALDI -TOF MS is a very sensitive technique, allowing detection at the low fmole level - Produces highly accurate data with high resolution - MALDI-TOF instruments are among the easiest of MS instruments to operate - MALDI-TOF MS is well suited to high-throughput proteomics work • Limitations: - MALDI-TOF instrument is best suited to measuring peptide masses. This type of information, although useful for protein identification, is nevertheless limited - The success of MALDI-TOF analyses is highly dependent on the quality of the sample. Contamination of the peptide digest sample with significant levels of detergents, buffer salts, metals, or organic modifiers may greatly inhibit peptide ionization in the MALDI source
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Tandem MS (MS/MS) • General Information: - The tandem mass spectrometer is an instrument consisting of two mass spectrometers in series connected by a chamber known as a collision cell. The sample to be examined is essentially sorted and weighed in the first mass spectrometer, then broken into pieces in the collision cell, and the piece or pieces subsequently sorted and weighed in the second mass spectrometer (Figure 55) - The term "tandem mass spectrometry" is often abbreviated as "tandem MS" or "MSIMS" - Tandem MS can be used for structural and sequencing studies - Tandem mass spectrometry was first introduced in the 1970's and was quickly accepted in the analytical community • Principles: - The underlying principle of the MSIMS method is the use of filiation relations : The first mass spectrometer (MS I) is used to select, from the primary ions, those of a particular mlz value, which then pass into the fragmentation region . The ion selected by the MS I is the parent ion or a precursor ion and can be a molecular ion or an ion resulting from primary fragmentation. Dissociation occurs in the fragmentation region . The daughter ions are analyzed in the second mass spectrometer (MS2) . In fact, MS I can be viewed as an ion source for MS2: • In MS 1, ionization is produced by electron impact. One ion is then selected (by a quadrupole, magnetic sector, or ion cyclotron resonance mechanism). The selected ion proceeds through the outlet to the fragmentation region • In the fragmentation region, there is a neutral gas (e.g., He, Ar, N2) under high pressure . The ions selected interact with these molecules of gas. During the collision , the kinetic energy of ions is transformed into internal energy, which leads to fragmentation into daughter ions • In MS2, the daughter ions are detected and the final spectrum shows the peaks of the selected ion and all its daughters
Diagnostic Methodology and Technology
- Instrumentation for MSIMS: a tandem mass spectrometer can be thought of as two mass spectrometers in series connected by a chamber that can break an ion into pieces • Types of sources: the abbreviations ESI (electrospray), fast atom bombardment, or MALDI before the term "tandem MS" indicate the manner in which the analyte is introduced into the tandem mass spectrometer • Dissolvation-nebulization sources (ESI, "electrospray"): this type of sourceis used to produce ions from molecules in solution. The solution is placed into a metallic capillary. An electric field is applied between the capillary's point and an electrode; multi-charged droplets are produced and accelerated toward the electrode • Ionization-desorption sources: based on secondary emission. The bombardment of a solid or liquid sample with a primary beam (ion, atom, or photon) induces the secondary emission of particles: electrons, neutral particles, and ions. Only these particles are analyzed by mass spectroscopy. The name of the source depends on the nature of the incident beam: ions in secondary ion mass spectrometry, atoms in fast atom bombardment, photons in laser desorption, andMALDI • Analyzers: a tandem mass spectrometer is a mass spectrometer that has more than one analyzer, in practice usually two. The two analyzers are separated by a collision cell into which an inert gas (e.g., argon or xenon) is admitted to collide with the selected sample ions bringing about their fragmentation. The analyzers can be of the same or different types, the most common combinations being: • quadrupole-quadrupole • magnetic sector-quadrupole • magnetic sector-magnetic sector • quadrupole-time-of-flight • Collision cell: collisional activation can be divided into two categories, involving high or low energy, to which different types of collision cells are appropriate. In sectorinstruments, where high energy collisions are most common, the cell is usually a tight chamberof 1-3 em length with entrance and exit slits, which transmit the ion beam. Good pumping is essential to maintain a low pressure outside the cell. In some instruments, the collision cell is electrically insulated from the mass spectrometer and can be heldat a high potential to retard the ion beam and reaccelerated it on exit.This allows control of the collision energy and also reduces the kinetic energy spread of daughter ions formed • Types of detectors: the detector's purpose is to translate ion arrival into electrical signals measured
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by an electronic system. There are two different classical types of detector • Electron multiplying: the principal sorts of electron multiplying detectors are the "channeltron" and the "micro channel plate". In these devices ions impact upon a surface composedof half conductors. The impact releases electrons, which are accelerated to another surface where additional electrons are released, and so on, thereby amplifying the signal. The resulting current pulse is proportional to the original signal intensity • Photodetectors: electrons are created in the same manner as above but then interact with a phosphorescent surface, which generates photons. The photons are recovered; their number is proportional to the signal intensity • Procedures: - Spot excision: same as "MALDI-TOF Mass Spectrometry procedures" - In-gel digestion of proteins: same as "MALDI-TOF Mass Spectrometry procedures" - Extraction of tryptic peptides from gel: same as "MALDI-TOF Mass Spectrometry procedures" - Sample preparation for tandem MS analysis: for maximum sensitivity, thoroughly desalt the tryptic digest before analysis using HPLC, which gives considerable advantage in sensitivity • HPLC separations are performed with a CapLC (Waters) using trapping guard and analytical columns in series • Withthe eluentof the guardcolumn directed to waste, sample (20 mL) is loaded at a flow rate of 30 mL/minute with pumpC (aqueous 0.1 % formic acid), washed for 5 min at 30 mUminute, and then the 10 port valve is actuated so the acetonitrile-water-formic acid mixture from pumpsB and A is directed to the trapping column to elute the peptides in-line with the analytical column, which is pre-equilibrated with 5% solvent B from the last run and the flow reduced to 200 nLlminute. The flow (1 mUminute) from pump A and B is reduced to 200 nLlminute by splitting the flow with a Valco Teeand 5 m of fused silica(75-rom innerdiameter) on the waste arm - Tandem MS analysis: • The eluent from the analytical column is ionized by electrospray ionization (ESI) with tandem MS analysis • Carryout the analysis usingautomated data analysis. The SEQUEST computer program is used to match the sequence information in the spectra to a database of known protein sequences. SEQUEST is a powerful suite of programs that can take the information in the peptide MSIMS spectra and correlate it to a database of known DNAor protein sequences
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• Applications: An important application of tandem mass spectrometry is protein identification by peptide sequencing Oligonucleotide sequencingcan also be achieved by tandem mass spectrometry Tandemmass spectrometry has been used for structure elucidation of unknowns and for analysis of complex mixtures • Advantages: Tandemmass spectrometry does not require extensive sample purification
It can produce protein sequence data that cannot be obtained using other mass spectrometry methods
Produces data much faster than other mass spectrometry methods due to its use of non-sequential sample analysis Tandem mass spectrometry is a very sensitive technique • Limitations: With this methodology it is difficult to elucidate the identity of N-terminal andC-terminal residues in a peptide The instrument is expensive and complex. Performing analyses demands a very high level of expertise
SUGGESTED READING Additional Methodologies for Nucleic Acid Detection Alwine JC, Kemp DJ, Stark GR. Methodfor detection of specific RNAsin agarosegels by transferto diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci USA 1977;74(12):5350-5354. Buongiorno-Nardelli M, Amaldi F. Autoradiographic detectionof molecularhybrids between RNA and DNA in tissue sections. Nature 1970;225(5236):946-948. Conner BJ, Reyes, AA, Morin C, et al. Detectionof sickle cell beta Sglobin allele by hybridization with syntheticoligonucleotides. Proc Natl Acad Sci USA 1983;80:278-282.
Darby lA, Bisucci T, Desmouliere A, et aI. In situ hybridization using cRNA probes: isotopicand nonisotopic detectionmethods. Methods Mol Bioi. 2006;326:17-31. Gall JG, Pardue ML. Formation and detectionof RNA-DNA hybrid molecules in cytologicalpreparations. Proc Natl Acad Sci USA 1969;63(2):378-383. Hicks DG, Longoria G, Pettay J, et al, In situ hybridization in the pathologylaboratory: general principles,automation, and emerging research applications for tissue-based studies of gene expression. J Mol Histol.2004;35 :595--601.
Hubbard RA. Human papillomavirus testing methods. Arch Pathol Lab Med.2003;127:940-945.
Iftner T, Villa LL. Human papillomavirus technologies. J Natl Cancer Inst Monogr . 2003;31:80-88. John HA, Birnstiel ML, Jones KW, RNA-DNA hybrids at the cytological level. Nature 1969;223:582-587. Porchet N, Aubert JP. Northern blot analysisof large mRNAs. Methods Mol Bioi. 2000;125:305-312.
Princivalle AP, Parker RM, Dover TJ, et al, mRNA: detectionby in situ and northernhybridization. Methods Mol BioI. 2005;306:51-91. Ragoussis J, Elvidge G. Affymetrix GeneChipsystem: movingfrom research to the clinic. Expert Rev Mol Diagn. 2006;6:145-152. Southern EM. Blottingat 25. Trends Biochem Sci. 2000 ;25:585-588. Southern EM. Detectionof specific sequencesamong DNAfragments separatedby gel electrophoresis. J Mol Bioi. 1975;98(3):503-517.
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Stoneking M, Hedgecock D, Higuchi RG, et aI. Population variation of human mtDNA control regionsequencesdetected by enzymatic amplification and sequence-specific oligonucleotide probes. Am J Hum Genet . 1991;48:370-382.
Amplification Methods Andras SC, Power JB, Cocking EC, et al, Strategies for signal amplification in nucleicacid detection. Mol Biotechnol. 2001;19:29-44. Barany F. Geneticdisease detectionand DNAamplification using cloned thermostable ligase. Proc. Natl. Acad of Sci USA 1991 ;88(I):189-193. Heid CA, Stevens J, Livak KJ, et al. Real time quantitative PCR. Genome Res. 1996;6(I0):986-994. Hellyer TJ, Nadeau JG. Strand displacement amplification: a versatiletool for moleculardiagnostics. Expert Rev Mal Diagn. 2004;4(2):251-261 . Mickey SU, 1994. Mullis KB, Faloona FA.Specific synthesis of DNAin vitrovia a polymerasecatalyzed chainreaction. Methods Enzymol. 1987;155:335-350. Schneeberger C, Speiser P, Kury F, et aI. Quantitative detection of reverse transcriptase-PCR productsby means of a novel and sensitive DNA stain. PCR Methods Appl. 1995;4(4):234-238. Urdea MS. BranchedDNA signal amplification. Nature Biotechnol. 1994;12:926-928. Zhang D, Feng T, YeF, et aI. Recent advances in probe amplification technologies, In: TangYW. StrattonCW,eds. Advanced Techniques in Diagnostic Microbiology. NewYork, NY: Springer; 2006:210-227.
DNA Separation Methods Arshad MF, Dunn FJ, Vega R, et aI. Progressin developing improved programsfor pulsed field agarosegel electrophoresis of DNA. Electrophoresis 1993;14:344-348. Cariello NF, Skopek TR. Mutational analysis using denaturinggradient gel electrophoresis and PCR. Mutat Res. 1993;288:103-112. Fischer SG, Lerman S. DNAfragments differing by single base-pairsubstitutions are separatedin denaturinggradient gels: correspondence with meltingtheory. Proc Natl Acad Sci. USA 1983;80(6): 1579-1583.
Diagnostic Methodology and Technology
Fodde R, Losekoot M. Mutation detection by denaturing gradientgel electrophoresis (DGGE). Hum Mutat. 1994;3:83-94. Godde R, Akkad DA,Aming L, et al. Electrophoresis of DNAin human geneticdiagnostics - state-of-the-art, alternatives and future prospects. Electrophoresis 2006;27:939-946. Kemp G. Capillary electrophoresis: a versatile family of analytical techniques. Biotechnol Appl Biochem. 1998;27(I):9-17. Maniatis T, Jeffrey A, van deSande H. Chain lengthdetermination of small double-and single-stranded DNAmolecules by polyacrylamide gel electrophoresis. Biochemistry 1975; 14:3787-3794. Nataraj AJ, Olivos-Glander I, Kusukawa N, et al, Single-strand conformation polymorphism and heteroduplex analysisfor gel-based mutation detection. Electrophoresis 1999;20:1177-1185. Olson MV.Separation of largeDNAmolecules by pulsed-field gel electrophoresis. A review of the basic phenomenology. J Chromatogr. 1989;470:377-383. Pourzand C, Cerutti P. Genotypic mutation analysisby RFLP/PCR. Mutat Res. 1993;288:113-121 . Raymond S, Weintraub L. Acrylamide gel as a supporting medium for zone electrophoresis. Science 1959;130:711. Schwartz DC, Cantor CR. Separation of yeast chromosome-sized DNAs by pulsedfield gradientgel electrophoresis. Cell 1984;37(1 ):67-75. Serwer P. Sievingof double-stranded DNAduringagarosegel electrophoresis. Electrophoresis 1989;10:327-331.
Protein Detection Methods Dunbar B. Troubleshooting and artifacts in two-dimensional polyacrylamide gel electrophoresis. In: DunbarBS, ed. Two-Dimensional Electrophoresis and Immunological Techniques. NewYork, NY: Plenum; 1987: 173-195. Fenyo D, Qin J, Chait BT. Proteinidentification using mass spectrometric information. Electrophoresis 1998;19:998-1005. Gillespie PG, Hudspeth AJ. Chemiluminescence detectionof proteins from singlecells. Proc Natl Acad Sci USA 1991 ;88:2563-2567. Hames BD, Rickwood D, eds, Gel Electrophoresis of Proteins: A Practical Approach (2nd ed). NewYork, NY: Oxford University Press; 1990. Harlow E, Lane D. Immunoblotting in Antibodies: a Laboratory Manual. Cold Spring Harbor: NewYork, NY: Cold Spring HarborLaboratory Press: 1988;471-510. Hochstrasser DF, Harrington MC, Hochstrasser AC, et al. Methods for increasing the resolution of two-dimensional proteinelectrophoresis. Anal Biochem. 1988;173:424-435. Patterson SD. Matrix-assisted laser-desorption/ionization mass spectrometric approaches for the identification of gel-separated proteins in the 5-50 pmol range. Electrophoresis 1995;16:1104-1114.
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Porstmann T, KiessigST. Enzyme immunoassay techniques, an overview. J Immunol Methods. 1992;150(1-2):5-21. Smith JA. Electroelution of proteinsfrom stainedgels, In: Ausubel FA, BrentR, Kingston RE, et al, eds. Current Protocols in MolecularBiology. NewYork, NY: John Wiley & Sons; 1993:10.15.1-10.15.5. Stults JT. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Curr Opin Struct Bioi. 1995;5:691-698.
Sample Collection and Processing Methods O'Leary TJ, Brindza L, Kant JA, et al.lmmunoglobulin and T-cell Receptor Gene Rearrangement Assays; approvedguidline. Wayne, PA: NCCLS; publication MM2-A: 1995;15(18). Rainen L, Arbique JC, Asthana, et aI. Collection, Transport, Preparation, and StorageofSpecimens for MolecularMethods; approvedguideline. Wayne, PA: Clinicaland Laboratory Standards Institute; publication MMI3-A: 2006;25(31).
Sequencing of Nucleic Acids Brown PR, Robb CS, Geldart SE. Perspectives on analyses of nucleic acid constituents: the basis of genomics. J Chromatography A 2002; 965(1-2):163-173. Dolnik V. DNAsequencing by capillaryelectrophoresis (review). J Biochem BiophysMethods 1999;41(2-3):103-119. Dovichi NJ. DNAsequencing by capillaryelectrophoresis. Electrophoresis 1997;18:2393-2399. Heller C. Principles of DNAseparation with capillaryelectrophoresis. Electrophoresis 2001 ;22(4):629-643. Maxam AM, Gilbert W. Sequencing end-labeled DNAwith base-specific chemical cleavages. MethodsEnzymol. 1980;65(1 ):499-560. Sanger F, Nicklen S, Coulson AR. DNAsequencing with chainterminating inhibitors. Proc Natl Acad Sci USA 1977;74(12):5463-5467.
Signal Detection Methods Thelwell N, Millington S, Solinas A, et al. Modeof action and application of Scorpion primersto mutation detection. NucleicAcids Res. 2000;28( 19):3752-3761 . Tyagi S, Kramer FR. Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 1996;14(3):303-308. Wittwer CT, Ririe KM, Andrew RV, et aI. The LightCycler: a microvolume multisamplefluorimeter with rapid temperature control. Biotechniques 1997;22(1):176-181.
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4
Tissue Microarrays and Biomarker Validation Martina Storz, ss and Holger Moch,
MD
CONTENTS I. Tissue Microarray Technology General Benefits Limitations Examples
II. Method
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Organization of a TMA Creation of a TMA Map Punching the Array Cutting the Array
III. Suggested Reading
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Collection and Selection of Tissue Probes........4-2
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Molecular Genetic Pathology
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TISSUE MICROARRAY TECHNOLOGY General • The technology of tissue microarrays (TMA) was developed against the background of decoding the human genome and the widespread application of high-density cDNA microarrays • TMA facilitate comprehensive molecular profiling of cancer specimens with minimal tissue requirements
• Novel web-based database structures allow to handle clinical and pathology data for each patient in a TMA, easily facilitating intra- and interinstitutional collaborations • There are systems allowing secure and reliable evaluation of TMA images over the internet
Limitations
• Multiple specimens can be simultaneously investigated with different in situ techniques under identical laboratory conditions
• Because of the small size of the individual array tissue samples (diameter usually 0.6 mm), the specimens are not totally representative of their donor tumor due to tumor heterogeneity. It is important to realize that the TMA technology has been designed to examine tumor populations, and not to survey individual tumors. The impact of tissue heterogeneity can be studied by taking multiple different punches from one tumor or by constructing replica arrays. Most, if not all associations between molecular changes and clinical end points have been verified by TMA studies
Benefits
• Tissue spots on a TMA-slide can be non-informative because of floating during the staining procedures or due to non-informative tissue (mispunching)
• TMAs are useful for rapid and high-throughput discovery and validation of biomarkers, assessing their prognostic and predictive value, and identifying novel therapy targets
• The results obtained from TMAs depend on the quality of the archival tissues. Differences in using buffered formalin or a variability in fixation can influence TMAs composed of archival tissue
• TMAs are a cost-effective tool for quality control and standardization in immunohistochemistry (IHC)
• The analysis of novel biomarkers frequently requires availability of antibodies for paraffin-embedded tissues. TMAs constructed from frozen tissue allow widespread in situ analysis of RNA and proteins. The construction of frozen TMAs is time-consuming and difficult
• A TMA is a paraffin block composed of multiple tissue specimens • TMA sections provide targets for parallel in situ detection of DNA, RNA, and protein targets allowing the rapid analysis of hundreds of molecular markers in the same set of specimens
• TMAs can be used to evaluate sensitivity and specificity of antibodies, tissue fixation methods, and antigen-retrieval methods • TMAs help to save reagents, manpower, and money • The technology is less exhausting for the original donor material because only minor tissue samples obtained from valuable materials or rare tumors are required and the basic tissue is not destroyed • Collaborative studies or setup of collaborative networks is facilitated by TMAs • Rapid translation of results from cell lines, xenographs, and animal models to human cancer is achieved • Digital images can be stored in relational databases, allowing handling the rapidly increasing amount of the generated data • Automatic image analysis will be more and applied for different TMA systems
Examples • Multitumor-TMAs are composed of samples from multiple tumor-types . These arrays are used to screen different tumor-types for molecular alterations of interest • Progression TMAs have been used to study molecular alterations in different stages of one particular tumor • Prognostic TMAs contain samples from tumors of patients for whom clinical follow-up data and clinical end points are known • TMAs are constructed also for research in other fields such as inflammatory, cardiovascular, and neurologic diseases • TMAs can be used for cell lines, xenograft tumors, or tissues from animal model systems
METHOD Collection and Selection of Tissue Probes A design of a TMA is based on the aims of the planned study. According to the aim of the study a list of cases to be included into the TMA should be prepared:
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• Collect all the hematoxylin and eosin (H&E)-stained slides and the corresponding paraffin-embedded blocks of interest • Make sure the H&E slides show the current state of the block or cut fresh H&E sections. This will ensure that
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TMAs and Biomarker Validation
Fig. I. H&E slide with differentially labeled tumor areas. the desired tissue observed on the H&E slide ends up on the TMA • Check all the collected H&E slides • Mark the areas of interest with a felt tip pen directly on the cover slip • Different areas on a tissue block may be of interest, for example, tumor tissue as well as normal tissue. Choose a color code and mark the different areas of interest with different colors. As example : tumor red, normal tissue blue, and other areas black • Mark the largest possible area that contains the area of interest. It will be easier to find the desired area to be biopsied and it enables the investigator to take more than one or two cores • The minimum size of the circled area should be at least
3 mm• To draw the areas of interest onto the H&E slides is an important part of the project. The rule is: the TMA can only be punched as precisely as it has been marked (Figure 1) • Compare the circled H&E slides with the corresponding paraffin-embedded blocks. Do not include thin tissue blocks into the TMA . These cores will be missing after cutting a few sections of the TMA block • The thickness of the designated tissue should be at least 3mm
Organization of a TMA • Arrange the corresponding block onto the H&E slide and put them on a tray by ascending numbers. This is important for an array with a large number of cases and will help later on if one has to look for a certain case • It is recommended to include about 600 cores in a TMA . Although up to 1000 cores can be arranged on a block the tension will increase and can result in cracks at the edges of the TMA block and in technical problem s when cutting sections
creates a coordinate system, in which each core gets its own x- and y localization. We recommend preparing a punch file and a picture file (Figures 2 and 3) The generation of the punch file is an important part in a TMA project. A mistake on this file will also appear on the TMA and on all immunostainings performed on the TMA slide. The picture file is a visual representation of the array. Different diagnoses or tissue types can be shown in colors. The marking spots on the left are extremely important for the correct orientation of the slide. • Decide on the number of cores per tumor block . For heterogeneous tumors 2-4 cores per donor block are recommended. The construction of several replicates is advised if many studies are planned • Insert all the block numbers into a punch file, prepare the picture file • Prepare a blank recipient block using a standard tissue cassette and paraffin with a melting point of 50-60°C. Use only recipient blocks completely filled with paraffin, discard blocks with holes or cracks
Punching the Array The following punch procedure is based on using a manual Beecher Instruments arrayer (Beecher Instruments, Sun Prairie, WI [http://www.beecherinstruments.com]) (Figure 4) • Put the recipient block into the magnetic recipient block holder • Tighten the screws in the block holder carefully • Make sure that the block holder is precisely touching the location bars • Move the needle with the micrometer screw to the edge of the recipient block and adjust the depth to which the needle goes into the recipient block. The needle should not touch the plastic cassette • Set up the location of the very first core xl' y \. Make sure that all cores will fit on the recipient block and try to maintain the same distance to all four edges of the block. Press the zero buttons on the micrometer screw; make sure that the display shows 0.000 • Punch a hole into the recipient block with the smaller needle. Put the donor block bridge over the recipient block and place the donor block on top. Punch a tissue cylinder out of the desired area using the bigger needle (Figures 5 and 6)
• Add normal tissues as control
• Remove the donor block bridge and push the tissue cylinder carefully into the hole of the recipient block
Creation of a TMA Map
• Be careful not to push the tissue cylinder all the way into the recipient block, leave about 0.5 mm exposed . Place a clean glass slide on the surface of the array and gently push the remaining 0.5 mm of the tissue cylinder into the recipient block. The goal for a good array is to have all the cores at the same level. Maintaining this procedure for every single core will lead to the best result
Individual maps can be created according to the study. Arrays should be organized indifferent quadrants; for 640 cores four quadrants are recommended. A space between quadrants helps to minimize the tension in the block and facilitates evaluation. Each quadrant has 20 rows (x-axis) and eight columns (y-axis) . The rows and columns get different numbers and that
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Spot #
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
SP number
B 1993.01979 B 1993.02020 B 1993.02803 B 1993.02929 B 1993.04960 B 1993.07547 B 1993.08372 B 1993.08905 B 1993.10878 B 1993.11206 B 1993.14664 B 1993.15096 B 1993.16595 B 1993.17139 B 1993.19866 B 1993.20272 B 1993.22750 B 1993.24118 B 1993.25328 B 1993.25661 B 1993.26148 B 1993.26680 B 1993.26828 B 1993.27254
Block 10
Tissue type
OX name
2
Kidney
Chromophob
1
1
010
1
Kidney
Other
2
1
80010
1
Kidney
Clearcell
3
1
160010
3
Kidney
4
1
240010
2
Kidney
5
1
320010
7
Kidney
Clearcell Papillary type Papillary type
6
1
400010
2
Kidney
Clearcell
7
1
480010
3
Kidney
Clearcell
8
1
560010
4
Kidney
Clearcell
1
2
0/800
2
Kidney
Clearcell
2
2
800/800
4
Kidney
Clearcell
3
2
1600/800
1
Kidney
Clearcell
4
2
2400/800
1
Kidney
Clearcell
5
2
3200/800
1
Kidney
6
2
4000/800
2
Kidney
Oncocytoma Papillary type
7
2
4800/800
2
Kidney
Clearcell
8
2
5600/800
8
Kidney
Clearcell
1
3
0/1600
6
Kidney
2
3
800/1600
2
Kidney
Chromophob Papillary type
3
3
1600/1600
1
Kidney
Clearcell
4
3
240011600
6
Kidney
Clearcell
5
3
3200/1600
2
Kidney
Clearcell
6
3
4000/1600
Kidney
Clearcell
7
3
4800/1600
Kidney
Clearcell
8
3
5600/1600
2
Localization Localization y x Coordinates
Fig. 2. Example of a punch file. In an Excel spreadsheet all information are included for punching and evaluating the stained TMA slide. The specimen number and the ID number of the donor block, tissue type and diagnosis, the x- and y localization , and the coordinates of the micrometer screw on the manual arrayer. Other parameters can be included. • For the next core, move one of the micrometer screws about 800 urn when using the 600 urn needles, this results in a 200 urn space between two cores • Continue the punch procedure by following the punch file Put the completed array in an oven at 40-50°C for 15-30 minutes . This is necessary to better adhere the tissue cylinders
136
to the paraffin of the recipient block. Place a clean glass slide on the surface of the array and slowly press down. Be aware that this is a dangerous step. If the TMA remains too long in the oven, the paraffin will melt and the array will be damaged. To alleviate and shorten the punching procedure semiautomated tissue arrayer (
[email protected]) are available as shown in Figure 7.
TMAs and Biomarker Validation
x x x x x x x x 1 2 3 4 5 6 7 8
I
Y
4-5
x x x x x x x x 10 11 12 13 14 15 16 17
x x x x x x x x 19 20 21 22 23 24 25 26
x x x x x x x x 27 28 29 30 31 32 33 34
1
Y 2 Y Y Y Y Y Y Y
3 4 5 6 7 8 9
Y
10
Y 11 Y 12 Y 13 Y Y Y Y Y Y
Y
14 15 16 17 18 19 20 Total :640 •
Clear cell
•
Normal
•
Marking spots
•
Pap. type I
D D
Pap. type II
•
Chromophobe
Oncozytoma
•
Other
Fig. 3. Example of a picturefile.
Fig. 4. Manual tissue arrayer.
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Molecular Genetic Pathology
Fig. 5. Punching procedure with donor and recipient block.
Fig. 6. Example of a TMA containing 640 cores .
Cutting the Array • General: - Cutting the array is a critical step and only a trained person should be in charge to prevent destruction of theTMA - The tape transfer system from Instrumedics (Instrumedics Inc., St. Louis, MO [http://www.instrumedics.com]) is a tool to cut the array. The procedure is as follows: • Place a tape window on the surface of the block and slowly cut the section of array and tape
138
• Take a slide covered with a special glue, remove the Mylar cover, and roll the section of array and tape onto the slide • Place the slide under a ultraviolet lamp for 30 seconds, put the slide in a solvent bath, and remove the tape • Let the slide air-dry • Benefits: - Each core stays in the intended location. Using a water bath could lead to a shift of the rows and columns of the array and this could be difficult when evaluating the stained slide
TMAs and Biomarker Valid ation
4-7
Fig. 7. Semiautomated tissue arrayer. The glue on the slide makes it possible to perform a wide range of heat pretreatment steps for IHC, fluorescent hybridization, and in situ hybridization without tissue loss This system allows cutting consecutive slides. No tissue is wasted trying to get a slide without crinkles and all cores present • Limitations: The glue on the slide can result in background staining but this does not effect the staining of the marker The glue can interfere with procedure for fluorescence in situ hybridi zation Daylight can polymerize the glue and the tape will not stick on the slide anymore. Make sure to always close the lid of the slide box
Automated IHC systems are disturbed by the glue. Section s performed with the tape transfer system should be manually processed • Example: Use the tape transfe r system to cut the TMA and perform IHC manually Without the tape transfer system, construct arrays with not more than 300 cores. Cut the array with the traditional method and the application of an automated IHC system is possible Store the slides at +4°C, regardless of whether the tape system was applied or not. Use the slides within 3 months, after that the staining intensity of several antibodies will decrease
SUGGESTED READING Berman JJ, Datta M, Kajdacsy-Balla A, et al, The tissue microarray data exchange specification: implementation by the Cooperative Prostate Cancer Tissue Resource. BMC Bioinfo rmatics 2004;5:19.
Dell'Ann a R, Demi chelis F, Barbareschi M, et al. An automated procedure to properly handle digital images in large scale tissue microarray experiments. Comput Methods Programs Biomed. 2005;79: 197- 208.
Bolli M, Schultz-Thater E, Zaj ac P, et al. NY-ESO-IILAGE- I coexpression with MAGE-A cancer/testis antige ns: a tissue microa rray study. Int J Cancer 2005;115:960-966.
Diaz LK , Gupta R, Kidwai N, et al. The use of TMA for interlaboratory validation of FISH testing for detection of HER2 gene amplification in breast cancer. J Histochem Cytochem. 2004;52:501-507.
Bubendorf L, Kolme r M, Kononen J , et al, Hormone therapy failure in human prostate cancer: analysis by compleme ntary DNA and tissue microarrays. J Natl Cancer Inst. 1999;91:1758- 1764.
Fowler JM, Ramirez N, Cohn DE, et al. Correlation of cyclooxygenase-2 (COX-2) and aromatase expression in human endometrial cancer: tissue microarray analysis. Am J Obstet Gynecol. 2005;192:1262-1 271; discussion 1271- 1273.
Bubendorf L, Kononen J , Koivisto P, et al , Survey of gene amplifications during prostate cancer progression by high-throughout fluorescence in situ hybridization on tissue microarrays. Cancer Res. 1999;59:803-806.
Garcia JF, Camacho FI, Morente M, et aI. Hodgkin and Reed-Sternberg cells harbor alterations in the major tumor suppressor pathways and cell-cycle checkpoints: analyses using tissue microarrays. Blood 2003;101:68I--Q89.
Bub endorf L, Nocito A, Moch H, et al, Tissue microarray (TMA) technology: miniaturized pathology archives for high-throughput in situ studies. J Pathol. 2001;195:72-79 (review article).
Gulmann C, Loring P, O'Grady A, et at. Miniature tissue microarrays for HercepTest standardisation and analysis. J Clin Pathol. 2004;57:1229- 1231.
Chung GG , Yoon HH , Zerkowski MP, et al, Vascular endothelial growth factor. FLT-I, and FLK-I analysis in a pancreatic cancer tissue microarray. Cancer 2006;106:1677-1 684.
Hendriks Y, Franken P, Dier ssen JW, et al, Conventional and tissue microarray immunohistochemical expression analysis of mismatch repair in hereditary colorectal tumors. Am J Pathol. 2003;162:469-477 .
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Howard BA, Zheng Z, Campa MJ, et al, Translating biomarkers into clinical practice: prognostic implications of cyclophilin A and macrophage migratory inhibitory factor identified from protein expression profiles in non-small cell lung cancer. Lung Cancer 2004;46:313-323.
Molecular Genetic Pathology
Moch H, Schraml P, Bubendorf L, et al, High-throughput tissue microarray analysis to evaluate genes uncovered by cDNA microarray screening in renal cell carcinoma. Am J Pathol. 1999;154:981-986.
Kallioniemi OP, Wagner U, Kononen J, et al, Tissue microarray technology for high-throughput molecular profiling of cancer. Hum Mol Genet. 2001; 10:657---662 (review article).
Nielsen TO, Hsu FD, O'Connell JX, et at. Tissue microarray validation of epidermal growth factor receptor and SALL2 in synovial sarcoma with comparison to tumors of similar histology. Am J Pathol . 2003; 163:1449-1456 .
Kay E, O 'Grady A, Morgan JM, et al. Use of tissue microarray for interlaboratory validation of HER2 immunocytochemical and FISH testing. J Clin Pathol. 2004;57:1140-1144.
Nocito A, Bubendorf L, Tinner EM, et al, Microarrays of bladder cancer tissue are highly representative of proliferation index and histological grade. J Pathol. 2001;194:349-357 .
Kettunen E, Nicholson AG, Nagy B, et at. L1CAM, INPIO, P-cadherin, tPA and ITGB4 over-expression in malignant pleural mesotheliomas revealed by combined use of cDNA and tissue microarray. Carcinogenesis 2005;26: 17-25.
Packeisen J, Korsching E, Herbst H, et al. Demystified tissue microarray technology. Mol Pathol. 2003;56: 198-204 (review article).
Kim R, Demichelis F, Tang J, et al. Internet-based Profiler system as integrativeframework to support translational research. BMC Bioinformatics 2005;6:304. Kocher T, Zheng M, Bolli M, et al. Prognostic relevance of MAGE-A4 tumor antigen expression in transitional cell carcinoma of the urinary bladder: a tissue microarray study.Int J Cancer 2002;100:702-705 . Kononen J , Buhendorf L, Kallioniemi A, et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 1998; 4:844-847. Koynova OK, Tsenova VS, Jankova RS, et al, Tissue microarray analysis of EGFR and HER2 oncogene copy number alterations in squamous cell carcinoma of the larynx. J Cancer Res Clin Oncol. 2005; 131:199-203 . Kuefer R, Hofer MD, Gschwend JE, et al, Tissue microarrays. Highthroughput procedures to verify potential biomarkers. Urologe A. 2004;43:659-667 (review article).
Rubin MA, Dunn R, Strawderman M, et al. Tissue microarray sampling strategy for prostate cancer biomarker analysis. Am J Surg Pathol. 2002; 26:312-319. Rubin MA, Mucci NR, Figurski J, et al. E-cadherin expression in prostate cancer : a broad survey using high-density tissue microarray technology. Hum Pathol. 2001;32:690-697 . Schraml P, Kononen J, Bubendorf L, et al, Tissue microarrays for gene amplification surveys in many different tumor types. Clin Cancer Res. 1999;5:1966-1975. Schraml P, Schwerdtfeger G, Burkhalter F, et at. Combined array comparative genomic hybridization and tissue microarray analysis suggest PAKI at Ilql3.5-qI4 as a critical oncogene target in ovarian carcinoma. Am J Pathol. 2003;163:985-992. Simon R, Sauter G. Tissue microarrays for miniaturized high-throughput molecul ar profiling of tumors. Exp Hematol. 2002;30 :1365-1372 (review article) . Simon R, Sauter G. Tissue microarray (TMA) applications: implications for molecular medicine. Expert Rev Mol Med. 2003;2003:1-12 (review article).
Linke SP, Bremer TM, Herold CD, et at. A multimarker model to predict outcome in tamoxifen-treated breast cancer patients. Clin Cancer Res. 2006;12:1175-1183 .
Simon R, Mirlacher M, Sauter G. Tissue microarrays. Methods Mol Med. 2005;114:257-268 (review article).
Liu CL, Montgomery KD, Natkunam Y, et al, TMA-Combiner, a simple software tool to permit analysis of replicate cores on tissue microarrays. Mod Pathol. 2005;18:1641-1648 (review article).
Staller P, Sulitkova J, Lisztwan J, et al. Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL. Nature 2003;425:307-311.
Liu X, Minin V, Huang Y, et aI. Statistical methods for analyzing tissue microarray data. J Biopharm Stat. 2004;14:671---685 (review article).
Struckmann K, Schraml P, Simon R, et al, Impaired expression of the cell cycle regulator BTG2 is common in clear cell renal cell carcinoma. Cancer Res. 2004;64:1632-1638 .
Lusis EA, Chicoine MR, Perry A. High throughput screening of meningioma biomarkers using a tissue microarray. J Neurooncol. 2005;73:219-223. Manley S, Mucci NR, De Marzo AM, et al, Relational database structure to manage high-density tissue microarray data and images for pathology studies focusing on clinical outcome: the prostate specialized program of research excellence model. Am J Pathol. 2001; 159:837-843 (review article).
Torhorst J, Bucher C, Kononen J, et at. Tissue microarrays for rapid linking of molecular changes to clinical endpoints. Am J Pathol. 2001; 159:2249-2256. Tzankov A, Pehrs AC, Zimpfer A, et al. Prognostic significance of CD44 expression in diffuse large B cell lymphoma of activated and germinal centre B cell-like types: a tissue microarray analysis of 90 cases. J Clin Pathol.2003 ;56:747-752 .
Mengel M, Kreipe H, von Wasielewski R. Rapid and large-scale transition of new tumor biomarkers to clinical biopsy material by innovative tissue microarray systems. Appl lmmunohistochem Mol Morphol. 2003;II :26 1- 268 (review article).
Tzankov A, Zimpfer A, Lugli A, et al. High-throughput tissue microarray analysis of G I-cyclin alterations in classical Hodgkin's lymphoma indicates overexpression of cyclin EI. J Pathol. 2003;199:201-207.
Mirlacher M, Kasper M, Storz M, et al. Influence of slide aging on results of translational research studies using immunohistochemistry. Mod Pathol. 2004;17: 1414-1420.
Varga Z, Theurillat JP, Filonenko V, et al, Preferential nuclear and cytoplasmic NY-BR-I protein expression in primary breast cancer and lymph node metastases. Clin Cancer Res. 2006;12:2745-2751 .
Moch H, Kononen T, Kallioniemi OP, et al, Tissue microarrays: what will they bring to molecular and anatomic pathology? Adv Anat Pathol. 200 I; 8:14-20 (review article).
Went P, Vasei M, Bubendorf L, et al. Frequent high-level expression of the immunotherapeutic target Ep-CAM in colon, stomach, prostate and lung cancers. Br J Cancer 2006;94:128-135.
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5 Laser Capture Microdissection Matthew Kuhar,
MD
and Liang Cheng,
MD
CONTENTS I. Overview of Laser Capture Microdissection (LCM) ......•.....•.........5-2 Principles and Procedural Overview Conceptual Importance of Procuring Pure Cell Populations
II. Laser Capture Microdissection Systems (LCM) Specimen Considerations and Handling Types of Specimens Specimen Processing Considerations Post-Processing Specimen Considerations System Components Key Characteristics of the Laser and Transfer Film Benefits and Limitations of Laser Capture Microdissection for Cell Isolation
III. LCM Laboratory Protocols Slides Preparation Formalin-Fixed, Paraffin-Embedded Slides Frozen Section Slides Laboratory LCM Procedures
5-2
Extraction of Genomic DNA From FormalinFixed, Paraffin-Embedded Tissue 5-9 Procedure and Protocol Resources 5-10
IV. Other Cellular Isolation Techniques 5-2
5-2 5-2 5-2 5-3 5-4 5-5 5-5 5-7
5-8 5-8 5-8 5-9 5-9
5-10
Non-LCM Microdis section Methods 5-10 Manual Extraction of Cells Under Direct Light Microscope Observation .5-10 Selective Ultraviolet Radiation Fractionation (SURF) 5-10 Laser Microbeam Microdissection (LMM)/Laser Cutting (LC) 5-10 Non-Microdissection Method s 5-10 Xenograft Enrichment 5-10 Cell Line Cultures 5-11 Cell Sorting 5-11
V. Methods of Analysis and Potential Applications of LCM Methods of Molecular Analysis DNA Analytic Techniques RNA Analytic Techniques Protein Analytic Techniques Potential Applications of LCM Diagnosis Progno sis Drug Discoveries Scientific Inquiry
VI. Suggested Reading
5-11 5-11 5-11 5-11 .5-12 5-12 5-12 5-13 5-13 5-13
5-13
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5-2
Molecular Genetic Pathology
OVERVIEW OF LASER CAPTURE MICRODISSECTION (LCM) • LCM is a technique developed by the National Institute of Health employing a laser to dissect individual cells or small clusters of cells selected by concurrent light microscopy • Objective: to obtain a pure sample comprised only of the specific cells of diagnostic, prognostic, or research interest
• The lysis or digestion buffer digests the cells in the film on the underside of the cap and thereby releases a pure sample suspension with macromolecules (DNA, RNA, and proteins) suitable for molecular analysis
Principles and Procedural Overview
Conceptual Importance of Procuring Pure Cell Populations
• The cells of interest are identified using light microscopy
• It was originally asserted by Virchow (1821-1902) that
(Figure 1) • A specialized centrifuge cap with an attached thin thermoplastic transfer film is placed over the area of interest with the film coming in direct contact with the tissue on an uncoverslipped, uncharged glass slide • A near infrared laser with a thin beam is passed through the transfer film and the underlying cell(s) of interest • The transfer film rapidly heats and focally melts • The melted transfer film permeates the empty spaces in the tissue immediately under the laser beam • The transfer film cools to form a new polymer with the selected cells - The heating and cooling process completes in milliseconds - The new bond between the selected cell(s) and the transfer film is stronger than the bond between the glass slide and the cell • The cell-film polymer is retracted from the surrounding tissue by lifting the cap • The transfer film can be moved to multiple additional areas to harvest many areas of interest on a single film • When all desired harvesting is complete, the cap and attached transfer film are removed from the setup and placed on a standard 0.5-mL microcentrifuge tube containing a lysis buffer or a digestion buffer
the cell, rather than the tissue, represents the most basic unit of disease • A single cell cannot be adequately analyzed without a means of reliably isolating it from adjacent cells • Accurate and sensitive detection of molecular changes in malignant or pre-malignant cells usually requires that only cells of interest are examined - Genetic material from contaminating cells can mask a finding with conflicting data • Even rare unintended cells' genetic material can become amplified during polymerase chain reaction techniques • Using non-microdissected material often underestimates the actual incidence of genetic alterations - Human tumors are heterogeneous with admixed cell populations • Normal parenchyma that the tumor is invading or in which the tumor developed • Blood vessels supplying the tumor • Inflammatory cell infiltrate that often accompanies malignancy • Stromal cells and/or desmoplastic connective tissue in response to an invasive tumor • LCM is capable of extremely selective sampling (Figure 2)
LASER CAPTURE MICRODISSECTION (LCM) SYSTEMS
Specimen Considerations and Handling Types of Specimens (Figure 3) • Paraffin-embedded specimens Formalin-fixed • Most common archival tissue in surgical pathology • Neutral buffered formalin fixation is acceptable; however, it causes extensive cross-linking of nucleic acids and protein, which makes polymerase chain reaction (PCR) amplification more difficult • Typical DNA fragments range from 100 to 1500 bp
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• RNA may be too poor a quality to be useful, though recent advances have overcome some technical difficulties • Over-fixation worsens macromolecule quality o Biopsies should fix <12 hours Large specimens should fix <48 hours - Metal salt-based fixatives should be avoided o
- Bouin's and B5 fixatives are even more damaging than formalin and should be avoided whenever possible - Alcohol fixation is the most desirable method for paraffin-embedded specimens
5-3
Laser Capture Microdissection
Individual cells of interest
1
A
B
Cap - { o - -Transfer film I
I
c O " -Isolated cells of interest
D
E
-
O.5 ml Microcentrifuge tube filled with lysis butter
F
Fig . 1. The step s of laser capture microdissection are shown in part s A-F. The cell s are identified using the light microscope to observe the uncover-slipped slide (A) . The cap and transfer film are then placed directly on the area of interest (B). Near infrared light is passed through the cap, tran sfer film , and cells of interest (C), which forms a polymer between the struck cells and the overlying transfer film. With removal of the cap (D), the cells adherent to the transfer film are successfully extracted. Finally, the cap is placed on a microcentrifuge tube with lysis buffer (E). After the lysis buffer dige sts the cells in the transfer film, a pure suspension is achieved (F).
• Frozen specimens - Specimens frozen and embedded in optimal cutting temperature compound (OCT) and then sectioned with a microtome provide the best genomic and molecular preservation - Requires foresight since a different processing procedure is used Microscopic morphology is the least desirable in frozen specimens compared with all the other processing techniques • Identifying the cells of interest is more difficult - Specimens must be stored at -80°C until moment of LCM procedure • Cytology specimens - Isolating cell s is relatively easy since the predominance of cells are already single or in small clusters comprised of identical cells - Fixed with methanol or ethanol
-
Archival specimens Provides excellent genomic and molecular preservation, particularly since the nuclei are intact (i.e., not sectioned by a microtome or cryostat)
• Archival stained and cover-slipped slides -
A wide variety of archival specimens may be procured using LCM , including immunolabeled and FISH hybridized cells
- The cover slip needs to be removed before the tissue can be used
Specimen Processing Considerations • For all specimens, freezing or fixation should occur as quickly after the cessation of perfusion as possible to avoid degradation by endogenous RNase ses, nucleases, and proteases
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A
c
Fig. 2. (A) Cutaneous malignant melanoma with a prominent central group of melanocytes, as observed under light microscope prior to laser capture microdissection. (B) The central group of melanocytes has been removed without alteration to the adjacent tissue in this image following LCM . (C) An image of the group of isolated melanocytes following extraction.
• RNase-free techniques should be employed for RNA extraction
• Staining (also see Laboratory Protocol section) - May be stained with hematoxylin and eosin (H&E)
- Disposable gloves
• H&E does reduce PCR efficiency
- RNase-eliminating cleaner on instruments, including microtome, between each sample
• H&E needs to be light and balanced
- Use RNase -free solutions, glassware, and plastics
• Using only eosin (i.e., withholding hematoxylin) improves results Papanicolaou and Wright stains yield good results
Post-Processing Specimen Considerations • Histologic sections should be 5-10 11m thick • Cut onto uncharged, uncovers lipped glass slides
144
Tissues may be unstained • Makes visual morphologic identification of the cells of interest vastly more difficult
5-5
Laser Capture Microdissection
Staining (H&E, Eosin, Wright, Pap, unstained, immuno, and so on.)
LCM
Fig. 3. A wide variety of specimens and stains are suitable for use with the LCM technique.
- May also be stained with immunohistochemical markers • This can be incredibly useful in locating the cells of interest in selected studies • Precursor lesions • Borderline neoplasms • Early, low-grade neoplasms • Neoplasms with multiple components
System Components • Light microscope (Figures 4 and 5) • Near infrared laser with laser control unit • Electronically controlled (or joystick-controlled) microscope stage with vacuum immobilization of the slide • Camera capable of relaying real-time microscopic video • Computer system or color monitor
• Mutifocal cancers • Other circumstances when morphology alone is not conclusive
Key Characteristics of the Laser and Transfer Film
• Can be useful in identifying cells during LCM since their morphologic appearance can be distorted due to the lack of a cover slip
• The transfer film is composed of ethylene vinyl acetate and is 100 11m thick
• May be used to stain for possible contaminating cells to avoid • An example is using CD34 to identify endothelial cells within a tumor • May require an altered immunohistochemical staining protocol to decrease RNA damage
- The film has a diameter of approximately 6 mm - It is mounted upon an optically clear cap for ease of withdrawing it from the specimen after polymerization (Figure 6)
- The film does not transfer heat well to adjacent tissue and the near infrared light is closely matched
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Molecular Genetic Pathology
5-6
Fig. 4. Leica laser capture microdissection system (LMD6000) (Leica Microsystems, Wetzlar, Germany).
e
J~
Fig. 5. Artcurus laser capture microdissection system (PixCell lIe) (Arcturus Bioscience Inc., Mountain View, California, USA).
to the absorption spectrum of the dye-impregnated transfer film • Therefore, almost all energy is absorbed by the transfer film with minimal heating (and resultant possible damage) of the underlying tissue • The tissue under the transfer film reaches a maximum temperature of approximately 90°C for only a few milliseconds
146
• The various biologic macromolecules are capable of enduring the process without alteration • Only the film directly struck by the laser photons melts, deforms and polymerizes with the underlying cells (Figure 7) - The laser's diameter (and therefore diameter of ethylene vinyl acetate that can be melted) can be adjusted from 7.5 to 30 11m • This can accommodate cells of various sizes or even small clusters of cells
Laser Capture Microdissection
5-7
Fig. 6. In this close-up of the Arcturus PixCell lIe, the cap can be seen resting on the glass slide (Arcturus Bioscience Inc., Mountain View, California, USA).
Fig. 7. The isolated cells are seen adherent to the transfer film in this scanning electron micrograph (Arcturus Bioscience Inc., Mountain View, California, USA).
Benefits and Limitations of LCM for Cell Isolation • Benefits - Overall, the least time-consuming method of cell isolation • Can be performed in a matter of a few minutes - Capable of isolating individual cells; excellent precision - Does not destroy adjacent tissue - Cells are attached firmly to transfer film with little risk of loss - Allows for examination of cells removed from their natural, in vivo surroundings - Less advanced lesions (e.g., dysplasia) that do not form tumors can be sampled • Limitations - Cells may be more difficult to lift off tissue if a fixed specimen is inadequately dehydrated or if a
frozen specimen is allowed to air-dry for a prolonged period - Does not work well on hard tissues such as bone and cartilage - Some cells are smaller than the minimum laser size (7.5 urn) • Therefore , if a single small cell is targeted, its adjacent cells may also be struck by the laser beam and adhere to the transfer film More expensive than manual microdissection methods Potential for contamination by non-specific weak attraction of the transfer film to cells not heated by the laser • Newest capltransfer films are available that have no direct contact with underlying tissue to minimize the possibility of contamination
(Figure 8)
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Molecular Genetic Pathology
A
Transfer film does not contact tissue
Rails suspend cap above tissue
B
-
•
-
Laser beam
•
J,
I
-
-,-,
I Transfer film deforms/sa gs to contact and bond to underlying soft tissue when melted by the laser beam
Fig. 8. Advanced cap/transfer films use built-in rails to suspend the transfer film just above the tissue (A). When the film is struck by the laser beam, the transfer film melts and deforms to contact and bond to the underlying tissue (B). This cap minimizes the possibility of contamination by non-specific binding of unintended cells to the transfer film.
LCM LABORATORY PROTOCOLS
• The following were protocols used in our laboratory
Formalin-Fixed, Paraffin-Embedded Slides Deparaffinize/Hydrate Xylene
10 minutes
Slides Preparation
Xylene
10 minutes
• Uncharged slides will yield the best results
100%ETOH
2 minutes
100%ETOH
2 minutes
• Tissue sections should be centered on slides
85% ETOH
1 minute
70% ETOH
1 minute
HzO
1 minute
• 4-5 urn sections are typically used
148
5-9
Laser Capture Microdissection
Stain Hematoxylin
30seconds
HzO rinse 30 seconds
Eosin
HzO rinse Dehydrate 70% ETOH
I minute
85% ETOH
I minute
100% ETOH
5 minutes
100% ETOH
5 minutes
Xylene
5 minutes
Frozen Section Slides Fix Cold 100% methanol or acetone at -20°C for 10 minutes Distilled water rinse twice for2 minutes
Stain 30 seconds
Hematoxylin DEPC H20 rinse Eosin
30 seconds
DEPC H20 rinse
(DEPC, diethyl pyrocarbonate, a nuclease inhibitor)
• Initiate the vacuum to immobilize the slide on the stage for cell harvesting • Align a cap on the right side of the stage on the load line • Swing the transfer arm to the cap, firmly pull arm up to load the cap on the arm • Swing the arm toward the specimen, and it will automatically lower over the selected area of the slide • Enable the laser (a small red indicator light will appear in the center of the monitor screen) • Use the laser focus wheel to focus the laser into a small, sharp, bright red dot • Afterfocusing the laser, the machine is readyto harvest cells • Choose appropriate laser size using the small arm located above the laser focus wheel • Continue using hand held control and joystick to move around the selected field, harvesting the cells of interest until enough cells have been collected for appropriate analysis • Lift the swing arm off the slide and place the cap into the cap holder located to the right of the loader • Use the cap transfer tool to pick up the cap from the cap holder, and insert the cap into a 0.5-mL tube containing 50 ul, appropriate digested solution • Be sure to label the cap and the tube
Extraction of Genomic DNA From Formalin-Fixed, Paraffin-Embedded Tissue • Secure pure cell population of interest using LCM • Put the tissue into 0.5-mL Eppendorf tube containing 50 ul. of digestion solution - Note : digestion solution
Dehydrate 70% ETOH
I minute
Tris-HCI
20mM
85% ETOH
I minute
KCI
50mM
100% ETOH
5 minutes
MgCI 2
5mM
100% ETOH
5 minutes
Proteinase K
5 mg/mL
Xylene
5 minutes
Step-by-Step Procedures Using the Arcturus LCM System • Tum on the machine using the main power switch • Ensure that the monitor is on • Center the joystick to make sure that the joystick is vertical and perpendicular to countertop surface • Place a slide on the stage • Find the area of interest, either by looking at the monitor or through the ocular lenses
• • • •
Incubate at 37°C overnight Boil the tube for 10 minutes to inactivate the proteinase K Cool down the tube with ice for 2 minutes Take 2 ul, of the solution from the tube as a genomic DNA template for PCR reactions • Estimated yields from formalin-fixed paraffin-embedded tissues DNA
5 pg percell
RNA
20-50pg percell (1-5% of which is mRNA)
Protein
10-100 pg percell
149
5-10
Molecular Genetic Pathology
Procedure and Protocol Resources
- http://dir.nichd .nih.gov/l cm/lcm .htm
• Detailed procedures and protocols for the handling and processing of specimens as well as protocols for molecular analysis can also be found at:
- http://www.arctur.com - http://www.leica-microsystems.com
OTHER CELLULAR ISOLATION TECHNIQUES
Non-LCM Microdissection Methods Manual Extraction of Cells Under Direct Light Microscope Observation • Concept is to manually extract small portions of tissue (often <1 mm in size) from paraffin-embedded tissue using modified Pasteur pipets or tungsten wire needles under direct observation - This requires fairly advanced malignancies since sizable clusters of cells need to be present to manually obtain fairly large clusters en bloc - Works better for mass lesions (e.g., carcinoma) than precursor lesions (e.g., dysplasia or carcinoma in situ) • Malignancies are increa singly detected at far smaller sizes due to improved computed axial tomagraphy technology and serologic studies including carcinoembryonic antigen , cancer antigen 125, and prostate-specific antigen • Genetic characteristics of cells in large mass lesions may be significantly different from cells in smaller lesions
• • • •
• Cannot be used to study malignancies like Hodgkin lymphoma since Reed-Sternberg cells are rare and scattered in an abundant reactive inflammatory cell infiltrate No expensive procurement equipment needed Very tedious and time consuming Susceptible to operator variability and room airflow Sample is not completely pure since admixed cells may be included
Laser Microbeam Microdissection (LMM)/ Laser Cutting (LC) • The main modem rival to LCM • Uses a highly focused UV laser beam to photoablate a thin rim of tissue around the cells of interest • After the cells of interest are cut from the adjacent cells , they are transferred into their media using either a needle tip or laser pressure catapulting - This step has increased potential for loss of the specimen and require s more technical skill than LCM • Advantage of LMMILC compared with LCM - Has the advantage of working on hard tissues, which have stronger intercellular bonds • Bone • Cartilage - Avoids the potential for non-specific cell adhesion to the transfer film that is possible with LCM - Faster than LCM for isolating relatively large areas of tissue • Disadvantages of LMMILC compared with LCM - Less adept at isolating single cells - Destroys adjacent tissue - More technically challenging - Typically requires more time • Some instruments are capable of performing both LCM and LMMILC techniques (Figure 9)
Non-Microdissection Methods
Selective Ultraviolet Radiation Fractionation (SURF)
Xenograft Enrichment
• Concept is to use ultraviolet (UV) radiation to destroy all the unwanted tissue in the paraffin-embedded block
• Serial passage of tissues through immunodeficient rodents to obtain human tumor cell populations whose non-malignant cells are rodent in origin
• Achieved by protecting the areas of interest with ink dots • Cells that are not protected from the radiation then have damaged DNA, which is unsuitable for PCR • The technique is able to isolate cells of interest ; however, the remaining tissue is completely destroyed - Cells of interest cannot be readily compared with surrounding stromal cells - No tissue archiving possible
150
- Essentially limitless numbers of cells can be obtained - Self-propagating - Extensive expertise is required - An animal facility is required - The cell population require s up to 6 months to establish - The tumor cells are susceptible to additional genetic changes over time
5-11
Laser Capture Microdissection
Fig. 9. The Arcturus Veritas Series (Arcturus Bioscience Inc., Mountain View, California, USA) is capable of performing both LCM and LMMILC technique. lesions or lesions whose diagnosis would have prognostic impact on the patient
• A subset of cells with evolutionary advantage (i.e., more aggressive) may propagate - Rodent cells may still contaminate the human tumor cells
- The in vivo microenvironment is significantly different from the artificial in vitro microenvironment
Cell Line Cultures
• Different exogenou s factors are available for the cell to utilize
• Allowing human cells to mitotically divide in vitro in culture media
• Different interaction s may cause various up and down regulation of genes with resultant change s in protein expression profiles
- Many cells can be obtained - Takes time to establi sh cell lines - A fair degree of experti se is required - The tumor cells are susceptible to additional genetic change s over time - Most cell lines are derived from late stage malignancies • Genetic alterations in these cells may be significantly different from earlier, more diagnostically difficult
Cell Sorting • Using cell density or ability to bind specific labeled antibodie s to sort the cells by their characteristics • Require cells to be in solution - Works relatively well for hematologic disorder s, but not solid tumors
METHODS OF ANALYSIS AND POTENTIAL APPLICATIONS OF LCM Methods of Molecular Analysis • A wide variety of techniques are available for molecular analysis - DNA, RNA, and prote ins are the macromolecules of analytic interest
• • • •
Direct DNA sequencing Single strand conformation polymorphi sm Restriction fragment length polymorphi sm Comparative genomic hybridization
• Southern blot
• Each can be altered in different disease states
• Others
• PCR can be employed to amplify the desired DNA and RNA
RNA Analytic Techniques
DNA Analytic Techniques
• Complementary DNA (cDNA) array and expression profiling
• Microsatellite instability
• • • • •
• DNA methylation
• Others
• DNA array and dideoxy fingerprinting • Loss of heterozygosity • X-chromo some inactivation
cDNA sequencing Northern blot Real-time PCR Genetic cloning MicroRNA fingerprinting
151
5-12
Molecular Genetic Pathology
Cell selection by light microscopy
~
Cap/transfer film placement on tissue
~
Laseractivation
~
Cell extraction by cap/transfer film removal
I ~
/
\
~
Sample preparation
I
t EJ
\
~
/
I Proteins
I
I Molecular analysis I
Fig. 10. Steps of LCM and molecular analysis of macromolecular isolates .
Protein Analytic Techniques • Immunoprecipitation for protein purification • Antibody screening and drug discovery • • • • •
Western blotting Peptide sequencing Mass spectrometry UV spectrometry Chromatography
• Others
152
Potential Applications of LCM Diagnosis • Discriminating between precursor lesions and early malignancy - Malignancy has typically been defined based upon an array of cytologic and architectural abnormalities observed under a light microscope, yet before these morphologic manifestations, there are cellular and genetic alterations, which may prove to be a better definition of malignancy
Laser Capture Microdissection
5-13
• Carcinogenesis is a multi-step process , often with a sequential order of genomic alterations appreciable in precursor lesions • Diagnosing lesions with challenging morphologic criteria - Examples include histologically deceptive and benignappearing variants of carcinoma in various anatomic sites such as bladder, skin, and soft tissues - Some tumors are currently classified by size criteria, such as papillary adenoma of the kidneys , which is distinguished from papillary renal carcinoma purely based on an arbitrary size cutoff • Conceptually, if cancer is a clonal proliferation derived from a single cell, carcinoma could exist at a very small, even single cell, level • Genetic profiling may be more accurate in tumor classifications - Many hematologic malignancies are very difficult to classify based upon light micro scopy, and molecular techniques can prove essential • Also, using LCM can avoid the difficulties in establishing cell line cultures that can frequently occur using standard methods for karyotyping • Ascertaining tumor origin of unknown primary tumor site - Fairly frequently, the difficulty arises of a metastasis becoming clinically apparent before its primary tumor • Determining the primary tumor can be quite difficult • Comparing the unknown's genetic profile to a library of known tumor characteristics may prove diagnostic
Prognosis • Predicting how aggressively a malignancy is likely to behave - For some malignancies, aggressive behavior is known to be correlated with specific translocations or ploidy • Examples include t(9;22) BCR-ABL in some hematopoietic malignancies and ZAP70 in chronic lymphocytic leukemia/small lymphocytic lymphoma Morphologic grading of tumor differentiation is quite subjective, and therefore not consistent • The degree of macromolecular derangement may prove to be a more accurate and more objective measure
• Assessing future malignancy risk - Patient's risk for developing additional malignancies may be gleaned from the initial presenting malignancy • Examples include multiple endocrine neoplasm (MEN) syndromes, some of which are associated with specific gene mutations - Familial risk for developing a malignancy may also be better assessed by genetic change s • Examples include BRCA I and BRCA2 in breast, ovarian, and prostate carcinoma • Predicting the utility of targeted therapy - Currently testing for specific protein (over-)expression may aid in therapy • An example include s HER2/neu over-expression in breast carcinoma • Determining the nature of multiple tumors - In some instances, multiple morphologically similar tumors cannot be definitively classified using light microscopy as representing multiple independent primary tumors or intraglandular metastasis
DrugDiscoveries • As a larger molecular database is collected, we may be able to find targets for additional drug mechanisms of action - HER2/neu, EGFR, and c-Kit are just a few early examples of molecular characteristics that allow tumors to receive tailored treatment modalities
Scientific Inquiry • Identifying the genetic difference between normal, premalignant, and malignant tissues by evaluating various macromolecular traits increases our overall knowledge of disease and will contribute to future diagnostic, prognostic, and treatment modalities - The National Cancer Institute is currently working on the Cancer Genome Anatomy Project, which is attempting to achieve this goal by creating a cDNA library based on cell protein expression profiles • http://www.ncbi.nlm.nih.gov/projects/CGAP/
SUGGESTED READING Bonner RF, Emmett-Buck M, Cole K, et al, Laser capture microdissection: molecular analysis of tissue. Science 1997;278:1481-1483. Cheng L, Gu J, Eble IN, et al. Molecular genetic evidence for different clonal origin of components of human renal angiomyolipomas. Am J Surg Pathol.2001 ;25:1231-1236. Cheng L, Gu J, Ulbright TM, et al. Precise microdissection of human bladder carcinomas reveals divergent tumor subclones in the same tumor.
Cancer 2002;94:104-110.
Cheng L, Jones TD, McCarthy RP, et aI. Molecular geneti c evidence for a common clonal origin of urinary bladder small cell carcinoma and coexisting urothelial carcinoma. Am J Pathol.
2005;166:1533-1539. Cheng L, MacLennan GT, Zhang S, et al. Laser capture microdissection analy sis reveals frequent allelic losses in papillary urothelial neoplasm of low malignant potential of the urinary bladder. Cancer
2004;10I:183-188.
153
Molecular Genetic Pathology
5-14
Cheng L, Song SY, Pretlow TG , et al. Evidence of independent origin of multiple tumors from prostate cancer patients. J Natl Cancer Inst.
1997;90:233- 237. Craven RA, Banks RE . Laser capture microdissection and proteomics: possibilities and limitation. Proteomics 200I; I: 1200-1204. Craven RA, Banks RE . Use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis. Methods
Enzymol. 2002;356:33-49 . Craven RA, Totty N, Harnden P, et al. Laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis: evaluation of tissue preparation and sample limitations. Am J Pathol. 2002;160:815-822. Curran S, McKay JA , Mcleod HL , et al. Laser capture microscopy. J Clin
Pathol: Mol Pathol. 2000;53:64-68. Emmert-Buck MR, Bonner RF, Smith PO, et al. Laser capture microdissection. Science 1996;274:998-1001. Espina V, Wulfkuhle JD, Calvert VS, VanMeter A, et al. Laser caption microdissection. Nat Protoc. 2006;I :586-603. Fend F, Emmert-Buck MR, Chuaqui R, et al. Immuno-LCM : laser capture microdissection of immunostained frozen sections for mRNA analysis. Am J Pathol. 1999;154:61-66.
154
Fend F, Raffeld M. Laser capture microdissection in pathology. J Clin
Pathol. 2000;53:666-672. Fink L, Bohle RM. Laser microdissection and RNA analysis. Methods Mol
Bioi. 2004;293:167-1 86. Luo L, Salunga RC, Guo H, et al. Gene expression profiles of lasercaptured adja cent neuronal subtypes. Nat Med. 1999;5: I 17-1 22. Maitra A, Gazdar A. Tissue microdissection and processing. Cancer Treat
Res. 2001;106:63-8 4. Maitra A, Wistuba II, Virmani AK, et al. Enrichment of epithelial cells for molecular studies. Nat Med. 1999;5:459-4 63. Schutze K, Lahr G. Identification of expressed genes by laser-mediated manipulation of single cells. Nat Biotechnol. 1998;16:737-742. Simone NL, Bonner RF, Gillespie JW, et al. Laser capture microdissection: opening the microscopic frontier to molecular analysis. Trends Genet.
1998;14:272-276. Wittliff JL, Erlander MG. Laser capture microdissection and its applications in genomics and proteomics. Methods Enzymol.
2002;356:12-25.
6
Clinical Flow Cytometry Magdalena Czader,
MD, PhD
CONTENTS I. Definition and Applications of Flow Cytometry II. Technical Aspects of Flow Cytometry Principle and Instrumentation Sample Processing Selection of Antibody PaneL
6-2 6-2 6-2 6-4 6-4
III. Analysis and Interpretation of Flow Cytometric Data
6-6
IV. Basic Cell Populations Identified by Flow Cytometry
6-7
Granulocytic Lineage Monocytic Lineage Erythroid Lineage Megakaryocytic Lineage Lymphoid Lineage B Cell Lineage T Cell Lineage Natural Killer (NK) Cells
6-7 6-7 6-7 6-8 6-9 6-9 6-9 6-10
V. Flow Cytometric Analysis of Myeloid Disorders Acute Myeloid Leukemias AML with Recurrent Cytogenetic Abnormalities AML Not Other wise Categorized Chronic Myeloproliferative Disorder s and Myelod ysplastic Syndrome s
6-10 6-11 6-11 6-12 6-13
Myelod ysplastic Syndrome s Chronic Myeloproliferative Disorders
6-14 6-16
VI. Flow Cytometric Analysis of lymphoid Neoplasms (Acute lymphoblastic leukemia [All] and Mature lymphoid Neoplasms) Precursor B ALLlLymphobl astic Lymphom a (Pre-B ALL) Pre-B ALL with Rearrangements of IIq23 (MLL Gene) Pre-B ALL with BCR/ABL Translocation Pre-B ALL with TELlAMLI Translocation Precursor T ALLlLymphoblastic Lymphom a (Pre-T ALL) Mature Lymphoid Neoplasms Mature B Cell Neoplasms Mature T- and NK-Cell Lymphom as
VII. Other Clinical Applications of Flow Cytometry Primary and Secondary Immunodeficiencie s Paroxysmal Nocturnal Hemoglobinuria (PNH) Stem Cell Transplantation Novel Applications of Flow Cytometry
VIII. Suggested Reading
6-17 6-17 6-17 6-17 6-17 6-18 6-19 6-20 6-23
6-26 6-26 6-26 6-27 6-27
6-28
155
Molecular Genetic Pathology
6-2
DEFINITION AND APPLICATIONS OF FLOW CYTOMETRY • Flow cytometry is the technique that measures the physical and antigenic properties of particles • Any particle that can be suspended in a fluid, i.e., cells , chromosomes, and individual molecules, can be detected and characterized by flow cytometry • The most significant discovery that led to the advancement of flow cytometry and its subsequent widespread application to clinical practice was the development of monoclonal antibodies, for which Georges J.E Kohler and Cesar Milstein received a Nobel Prize in 1984 • Currently, immunophenotyping using fluorochrome conjugated monoclonal antibodies is the most common clinical application of flow cytometry • In contrast to other applications of monoclonal antibodies, such as immunohistochemistry or Western blotting, flow cytometry examines antigens in their native (non-fixed) state • Multi-color immunophenotyping is the current standard in clinical flow cytometry. Most commonly 4-6 antibodies are analyzed at the same time. However, technical advances allow for the simultaneous detection of up to 17 antigens on an individual cell • The most common applications of clinical flow cytometry include:
- Diagnosis and sub-classification of malignant hematologic disorders including leukemias and lymphomas - Detection of minimal residual disease in acute leukemia - Enumeration of T cell subsets for follow-up of HIVpositive patients - Determination of immunophenotypiclfunctional abnormalities in congenital immunodeficiencies - Enumeration of hematopoietic stem cells for bone marrow transplantation - Diagnosis of platelet disorders -
Detection of fetal hemoglobin in feto-maternal hemorrhage
• The technique of flow cytometry can also be applied to: - Cell sorting - Detection of chromosomal abnormalities based on in situ hybridization or polymerase chain reaction (PCR) - Functional assays : • Proliferation • Apoptosis • Calcium efflux • Phosphorylation (cell signaling)
TECHNICAL ASPECTS OF FLOW CYTOMETRY Principle and Instrumentation • Flow cytometry measures light scattering and fluorescence of individual particles as they are illuminated by a light (laser) source - In flow cytometer, individual particles are suspended in a fluid and pass one by one in front of a light source (Figure 1) - As particles are illuminated, they scatter light and emit fluorescent signals • Light scattering • Forward scatter signal (FSC) is roughly proportional to size of a particle • Side scatter signal (SSC) reflects the internal complexity of a cell (cytoplasmic granules, vacuoles, and organelles) . Cell size and refractive index may contribute to SSC characteristics • Fluorescence • The principle of fluorescence is illustrated in the simplified Jablonski diagram (Figure 2): as an electron in its ground state absorbs light, it is raised to the excited state. The excess energy is emitted as non-radiative transition in a process of
156
internal conversion and vibrational relaxation. Upon subsequent return to the ground state, the fluorescence is emitted. Since some energy is lost during non-radiative transitions, the energy content of emitted fluorescence is lower than the energy absorbed resulting in the emission at a longer wavelength than the absorption (Stokes shift) • Each fluorochrome is characterized by a distinct spectral pattern of absorption and emission (fluorescence) . The fluorochromes must be specifically selected to absorb a certain wavelength of light emitted by the laser available in the instrument (some flow cytometers are equipped with more than one laser). Currently, a wide variety of monoclonal antibodies conjugated to various fluorochromes are available, which allows simultaneous detection of multiple antibodies bound to a single cell. However, great care should be taken to select fluorochromes with minimal overlap in the emission spectra to optimally resolve individual antibodies • Flow cytometer consists of fluidics, a light source (laser), a detection system, and a computer (Figure 1)
Clinical Flow Cytometry
6-3
Sheath fluid
Fluorescence detectors
Fse detector
- - - - - - - - - -0 . . . . . .
Laser beam
-- ----- -- -
~
,,
1_ _...... _ _ 1" --_ _ 1
, ' ,
'
" "
, '
,,0
~
~
sse (RALS) detector
Fig. 1. Diagramof flow cytometer. Cell suspension is injectedinto sheath fluid under pressure,whichpositionsthe cells in a single file in the center of the stream for interrogation by the laser. FSC and SSC signals and fluorescent signals of specific wavelength are recorded by separate detectors.
Vibrational relaxation
Excited - - - + - - - - -.l.",..<:--i - - - - state
Abso rption
Fluorescence
Electronic Electron ground ----'- - - - - - - - - ' - - - state
Fig.2. Jablonski diagram. As a resultof energy absorption, electrons are raisedto the excited state. The energyis emitted in a process of internal conversion or vibrational relaxation, and subsequently as fluorescence when electrons return to their groundstate.
157
6-4
Molecular Genetic Pathology
• Steps in the flow cytometric analysis of the sample include :
Table 1. Lineage-Associated Markers Commonly Analyzed in Clinical Flow Cytometry
- Aspiration of the stained cell suspension into a stream of sheath fluid Alignment of a single cell file centrally in the sheath fluid through the hydrodynamic focusing Illumination of cells passing individually in front of the laser source Registration of light scatter and fluorescence signals from individual cells by dedicated photodetectors (separate detectors for light scatter and each fluorochrome; partitioning into different wavelengths is achieved by a series of dichroic mirrors) As the sample is run, the data is digitized and simultaneously displayed and stored for subsequent analysis
Sample Processing • Any specimen in a form of single cell suspension is suitable for flow cytometric analysis • Most common clinical samples analyzed by flow cytometry include: - Bone marrow and peripheral blood (collected with an anticoagulant i.e., sodium heparin, ethylenediamine tetra acetic acid) - Solid tissues (lymph nodes and extranodal sites suspected to harbor hematologic malignancy should be submitted in culture media to maintain viability, or on saline moistened gauze and subsequently, mechanically dissociated usually by mincing with a scalpel to yield a single cell suspension) - Body cavity fluids • Quality of the sample is critical for accurate analysis: - Prolonged transport or transport in inappropriate conditions may render a sample unsuitable for analysis - Peripheral blood and bone marrow specimens should be processed within 24-48 hours from the time of collection and, if transported, should be kept at room temperature - Certain samples, such as body cavity fluids or specimens with a high proliferation rate may require even shorter time intervals between collection and processing • Steps in sample processing: - Hypotonic lysis for specimens with an admixture of red blood cells - Determination of the cellularity and viability of all submitted samples • Cell count can be obtained using flow cytometry with standardized beads or using automated cell counters • Flow cytometry of a sample stained with DNAdyes (e.g., propidium iodide, 7-AAD) or a manual method utilizing trypan blue exclusion can be used to test viability
158
Immature CD34 COl 17
Granulocytic! monocytic
Erythroid CD71 CD235a
TdT
CD33 CDB COl 5 CDl6 CD14
Megakaryocytes
B cell
T cell
CD41 CD42b CD61
CD19 CD20 CD22 x-Iight chain A.-light chain
CD2 CD3 CD4 CD5 CD7 CD8
- Preparation of a cytospin for the morphologic inspection of the cell suspension - Staining with a cocktail of fluorochrome-conjugated monoclonal antibodies (both surface, i.e., membrane bound, and intracellular antigens can be analyzed)
Selection of Antibody Panel • Comprehensive antibody panels with multiple markers for myeloid and lymphoid lineage are recommended by the US-Canadian Consensus Project in LeukemialLymphoma Immunophenotyping • Antibody panels are designed to identify multiple cell sub-populations expected to be present in the sample . Both terminally differentiated cell populations and successive developmental stages should be covered • Numerous hematopoietic cell antigens and the corresponding antibodies have been cataloged by Workshops on Human Leukocyte Differentiation Antigens (HLDA) held regularly since 1982 • These workshops provide a forum for reporting new antigens/antibodies and defining a cluster of antibodies, which recognize the same antigen (cluster of differentiation [CD], Tables 1 and 2). Consecutive numbers are assigned to each new reported antigen . The recent, VIII HLDA workshop, currently known as HCDM for Human Cell Differentiation Molecules, brought the number of characterized antigens to 339 • The selection of an antibody panel is based on the properties of the antibodies and fluorochromes - The selection of an antibody clone is often critical because antibodies can recognize different epitopes on the same antigen with different distributions in hematopoietic cells (e.g., antibodies against CD34 antigen recognize
6-5
Clinical Flow Cytometry
Table 2. Hematolymphoid Antigens Commonly Used in Clinical Flow Cytometry Cluster of differentiation
Cellular expression
CDIa
PrecursorT cells, Langerhanscells
CO2
Precursor and mature T cells, NK cells, thymic B-cells
cm
Precursor and mature T cells
CD4
Precursor T cells, helper T cells, monocytes
CDS
Precursor and mature T cells, subset of B cells (Bla cells)
CD7
Precursor and mature T cells, NK cells
CD8
PrecursorT-cells, suppressor/cytotoxic T-cells, subset of NK cells
CD10
Precursor B cells, germinal center B-cells, granulocytes
CDIlb
Granulocytic and monocytic lineage, NK-cells, subset of T- and 8 cells
CDB
Granulocytic and monocytic lineage
CDI4
Mature monocytes, neutrophils(dim expression)
CDIS
Granulocytic and monocytic lineage
CDI6
Granulocytic and monocytic lineage, NK cells
CDI9
Precursor and mature 8 cells
CD20
Precursor and mature 8 cells
C022
Precursor and mature B cells
CD31
Megakaryocytes, platelets, leukocytes
cm3 cm4 cm6 cm8
Granulocytic and monocytic lineage
CD41
Megakaryocytes, platelets
CD42b
Megakaryocytes, platelets Hematopoietic cells
CD45
Hematopoietic stem cells Megakaryocytes, platelets, erythroid precursors, monocytes Hematopoietic cells including activated lymphocytes and plasma cells
CD56
NK-cells, subset of T cells
CD61
Megakaryocytes, platelets
CD62P
Megakaryocytes, platelets
CD63
Megakaryocytes, platelets
CDM
Granulocytic and monocytic lineage
CD71
High density on erythroid precursors, low densityon other proliferating cells
CD79a
Precursor and mature 8 cells
CDII7
Hematopoietic stem cells and mast cells
three different epitopes of this molecule). Additionally, clones may differ in binding capacity - The choice of the fluorochrome should be related to the density of a given antigen . For example, when only a few molecules of the antigen are expected , one should select the antibody coupled with a strong fluorochrome to enhance the detection
• Selected markers most commonly analyzed by flow cytometry in hematopathology are presented in Tables 1 and 2 • Dependent on the laboratory, a comprehensive antibody panel can be analyzed upfront. Alternatively, a limited screening panel is utilized initially with the subsequent addition of selected markers
159
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Molecular Genetic Pathology
ANALYSIS AND INTERPRETATION OF FLOW CYTOMETRIC DATA • The evaluation of flow cytometric data is based upon analysis of the pattern s of antigen expression presented graphically in the form of scattergrams and histograms
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• The detailed knowledge of immunophenotypic characteristics of normal hematopoietic differentiation , as well as normal variations (e.g., age-related) is critical for optimal interpretation of flow cytometric data • Both qualitative (positive/negative; homogeneous vs heterogeneous expression) and semi-quantitative information on antigen expression (low/moderate/high intensity) should be recorded, as patterns of antigen expression, rather than percentage of positive cells, are diagnostically significant
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In the gating process, a population of interest is selected (outlined with a cursor or computer mouse) for further analysis (i.e., display of antigen expression for the selected-"gated" population) Gating can also be applied at the time of data acquisition (so called live-gating ) to selectively collect high number of cells from a specific subpopulation, for example , CD 19 positive cells to facilitate the detection of a small number of monoclonal B cells - For diagnostic purposes the data is most commonly collected ungated, i.e., all events detected by a flow cytometer are recorded, to comprehensively analyze the entire sample and retain internal positive and negative controls. A separate portion of the sample can be live-gated to better visualize specific cell population s (e.g., lymphocytes and plasma cells) • Steps in the analysis of flow cytometri c data: - Inspection of dot plots presenting cell size (FSC), internal complexity (SSC), and the expression of panhematopoietic antigen CD45 • Specific cell populat ions can be identified based on their size and cytoplasmic comple xity (granules/vacuoles) (Figure 3A) • The identificat ion is confirmed and further resolved on the display of CD45 antigen and SSC (Figure 3B). This scattergram provides information on the relative proportion of specific cell populations in the flow cytometric sample and is of particular value when analyzing bone marrow/peripheral blood specimens • CD45 is a surface protein expressed at different levels on all hematopoietic cells
160
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• The concept of gating : Cells with similar physical properties (size, internal complexity, and the presence/absence of a specific antigen) form clusters on the displays of flow cytometric data - Gate is a borderline that identifies these clusters of cells
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Fig. 3. Main hematopoietic populations of normal bone marrow. (A) Scattergram of FSC (cell size) vs SSC (internal comple xity) reflects the hetero geneit y of bone marrow. Lymphocytes as smallest with negligible amount of cytoplasm are located closest to the origins of the axes (shown in aqua). Monoc yte s are slightly larger with occa sional granules and vacuoles (green). Granulocytic series shows prominent granularity (navy). (B) Differential densitie s of panhematopoieti c marker CD45 on marrow leukocytes. Lymphocyte s (aqua) and monocytes (green) show highest density of CD45 antigen. Intermediate expre ssion of CD45 is seen in granulocytic population (navy) and blasts (black). Late erythroid precursors (red) are negative for CD45 outigen.
Clinical Flow Cytometry
6-7
• Lymphocytes show the highest density of CD45 expression with approximately 10% of the cell membrane occupied by this antigen
• Residual normal cells present in a sample can be used as an internal negative and positive control and to gauge the intensity of staining
• Granulocytic series including myeloid blasts, B cell precursors, and proerythroblasts show intermediate CD45 density
• The intensity of staining is dependent on technical variables including antibody clone and type of fluorochrome. Thus, levels defining bright, moderate, and dim expression should be established by individual laboratories taking into account specific antibodies and fluoro chromes used, and previous experience
• Late erythroid precursors along with megakaryocytes are negative for the CD45 antigen - Focused analysis of the patterns of antigen expression including both qualitative data (antigen present/absent) and fluore scence intensity (on the logarithmic scale) as a relative measure of the antigen density on the cell surface
• The autofluorescence and non-specific background staining due to Fe receptors should be taken into account when evaluating antigen expression
BASIC CELL POPULATIONS IDENTIFIED BY FLOW CYTOMETRY • Genetically controlled differentiation program and bone marrow environment govern the expression of surface and cytoplasmic molecules that define hematopoietic cell populations
• Further maturation to myelocytes results in loss of CD1l7 and down-regulation of CD 13 antigen . The decrease in the density of CD33 is also seen at this stage. CD15 and CD II b are positive
• Specific morphologic stages of development are accompanied by distinct changes in immunophenotypes. However, even though approximate morphologic-immunophenotypic correlates exist, transitions between immunophenotypes of various developmental phases are best described as a continuum
• Finally, as myeloid cells near the band stage CD 16 and CD I0 are acquired and the density of CD 13 increases. Further decrease of CD33 intensity is also noted
• All hematopoietic progeny are derived from pluripotent stem cells
Monocytic Lineage
- These cells are morphologically unrecognizable and are defined by their functional and antigenic characteristics - They usually express a combination of CD34, CD 117 (c-kit) , CD38 , and HLA-DR • As hematopoietic cells mature they lose stem cell markers and acquire lineage -specific antigens • Neoplastic hematopoietic cells to a certain extent mimic normal maturation stages; however, they frequently display aberrant antigenic patterns
Granulocytic Lineage • The differentiation of granulocytic lineage, as defined by the expression of specific antigens, correspond closely to the morphologic maturation stages as depicted in Figure 4 • Myeloblast is the first morphologically recognizable cell committed to the myeloid lineage and typically expresses immature cell markers CD34, CD38 , HLA-DR, and stem cell factor receptor CD 117, and pan-myeloid markers, CD 13 and CD33 • As the myeloblast matures to a promyelocyte, it loses CD34 and HLA-DR and gradually acquires the CD15 antigen
• The segmented neutrophil is characterized by highdensity CDI3, CDllb, and CDI6, and dim CD33
• The immunophenotype of the earliest stage of monocytic development, a monoblast, overlaps with that of myeloblast and includes the expression of CD34, HLA-DR, CD 117, and pan-myeloid markers CD33 and CD 13 (Figure 4) • Further monocytic maturation is marked with the increase in density of CD33 and CD 13, appearance of CD64, CDl1b, and subsequently CD15 (promonocyte) • Subsequent acquisition of CD14 and further increase in density of CD45 defines the transition point to a mature monocyte • The expression of CD 163 and CD68 antigens is strongest on tissue macrophages
Erythroid Lineage • Erythroid precursors are characterized by a gradual decrease in the density of CD45 antigen to the undetectable level in reticulocytes. Thus , late erythroid precursors are one of the few cells in the bone marrow that express a negligible number of CD45 molecules • The earliest marker of erythroid differentiation is the transferrin receptor, CD71 (Figure 5). This marker increases in density starting from the proerythroblast stage and is rapidly downregulated in reticulocytes. Mature erythrocytes are negative for the CD71 antigen
161
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Molecular Genetic Pathology
Promonocyte
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Fig. 4. The maturation of myeloid series is a genetically driven developmental program characterized by the continuum of phenotypic and functional changes. Discrete morphologic stages correspond to specific immunophenotypes.
Proerythroblast CD117 CD45 dim CD71 bright
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Erythrocyte CD235a
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Fig. 5. The development of erythroid series is defined by stepwise loss of CD45 along with acquisition of erythroid markers.
• The decrease in CD45 intensity seen in basophilic erythroblasts is accompanied by the emergence of CD235a (glycophorin A). The latter marker persists through erythroid maturation and is also present in erythrocytes
CD41 CD61
Megakaryocyte CD41 CD61 CD42 CD63 CD62P
Platelets CD41 CD61 CD42 CD63 CD62P
Fig. 6. The sequence of immunophenotypic changes of megakaryocytic lineage is characterized by early appearance of CD41 and CD61, i.e., gpIIbllIIa complex.
on a small subset of CD34 and CD 117 positive cells believed to represent early megakaryoblasts
Megakaryocytic Lineage
• CD31 and CD36, although not entirely specific for megakaryocytic lineage, are also present on megakaryoblasts
• The identification of megakaryocyte population by flow cytometry is not done routinely due to the rarity of the population
• As megakaryoblasts mature to megakaryocytes and platelets, additional antigens appear including CD42b, CD62P, and CD63
• CD41 and CD61 (gpIIbllIIa complex) appear as the first markers of megakaryocytic differentiation and are present
• CD41 and CD61 persist through the megakaryocyte differentiation (Figure 6)
162
6-9
Clinical Flow Cytometry
Lymphoid organs
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Fig. 7. The early stages of B cell maturation are completed in the bone marrow. Subsequent maturation of B cell lineage occurs in lymph nodes and extranodallymphoid tissues , and results in the production of plasma cells and memory B cells.
• The precise sequence of expression of megakaryocyteassociated antigens is not yet well studied
Lymphoid Lineage • The Band T lymphocytes are derived from lymphoid progenitors expressing CD34, terminal deoxynucleotidyl transferase (TdT), and HLA-DR • The number of CD45 molecules steadily increase with B cell maturation and reach characteristic high-density expression at the level of mature B cells . Early B cell precursors show low-density CD45 • The lymphoid differentiation represents a continuum of changes in the expression of surface and cytoplasmic antigens
B Cell Lineage • The schema of B cell differentiation is presented in Figure 7 • The earliest B cell markers include cytoplasmic CD22, CD 19, and cytoplasmic CD79a • As B cell precursors proceed in their maturation, they acquire the CDIO antigen, which is initially expressed at high levels • Subsequent appearance of the CD20 antigen is accompanied by the decrease in CD 10 intensity and its subsequent loss
• The u-heavy chain, a portion of immunoglobulin (lg) molecule, is initially expressed in the cytoplasm and eventually transported to the cell surface where it forms a B cell receptor (BCR) • The mature naive B cell population expresses heterogeneous (polyclonal) surface light chains, and in this respect, differs from neoplastic B cells, which are restricted to only a single 1(- or A-light chain • The mature B cells circulate to the secondary lymphoid organs including lymph nodes, spleen, and lymphoid tissues of extranodal sites, where they settle in the follicles and marginal zones • Further differentiation of mature naive B cells occurs upon antigen exposure and includes passage through germinal centers marked by the signature co-expression of CD I0 and Bcl-6 antigens • Plasma cells, the terminal stage of B cell differentiation, lose CD20 and surface Ig chains, and can be identified by the high-density expression of CD38 and CD 138 (syndecan-l)
T Cell Lineage • Early stages of T cell development take place in the bone marrow (Figure 8) • The first committed T cell precursor (prothymocyte) expresses immature markers (CD34, TdT, and HLA-DR)
163
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Molecular Genetic Pathology
Bone marrow
Thymus
I
Prothymocyte C034 TdT HLA-OR C02 CD? cytC03
Immature thymocyte TdT C02 CD? cytC03 C025 TCR-r
Common thymocyte C02 CD? Pre-TCR Including C03 dim C0 5 C0 1a C0 4 and C08
Mature thymocyte/naIve Tcell C02 CD? TCR C03 C05 C04orC08
Activated Tcell C0 2 CD? TCR C0 3 C05 C04 or C08 C0 25 HLA-OR
Fig. 8. The early T cell precursors are generated in the bone marrow and migrate to thymu s to complete their maturation.
and T-cell-associated antigens including CD2, CD7, and cytoplasmic CD3 • Prothymocytes migrate to the thymus to complete T cell development • Successive steps of T-cell receptor (TCR) gene rearrangement with the production of TCR -complex, expression of CD Ia, CD5, and co-expression of CD4 and CD8 antigens, define immature and common thymocyte stages • As the double-positive (CD4+, CD8+) common thymocyte matures, the density of CD3 antigen increases and CD4 or CD8 are lost, giving rise to mature helper (CD4+) and suppressor (CD8+) T cells
Natural Killer (NK) Cells • NK cells are positive for CD2, CD7, CD56, and CD 16 • Different densities of surface antigens allow for the separation of two functionally distin ct NK subsets:
- Cytotoxic NK cells show expre ssion of CD56dim, CD16bright, KIRbright, and CD94INKG2Adim(90% of NK population) - Immunoregulatory NK cells show expression of CD56bright, CD16 diml-, CDI17, KIRdim, and CD94INKG2Abright (10 % of overall NK population; however, represent the majority of the NK cells in lymph nodes) • Published data suggests that the development of NK cells starts in the bone marrow from CD34+ progenitor cell • It has been suggested that the later stages of development of immunoregulatory NK subset occur in the lymph nodes from CD34dim progenitor cells • The site and exact sequence of the development of cytotoxic NK cells are unknown
FLOW CYTOMETRIC ANALYSIS OF MYELOID DISORDERS • In myelo id malignancies, flow cytometry can be used for the initial diagnosis, follow-up, and prognostication (specific immunophenotypes are associated with prognostically significant cytogenetic abnormalities) • The majority of myeloid malignancies are regarded as disorders of stem cells • In acute myeloid leukemias (AML) , the maturation arrest leads to the accumulation of a homogeneous population of cells demonstrating an immature myeloid immunophenotype, thus the blast region , best demonstrated on a
164
CD45/SSC scattergram, is densely populated in most cases (Figure 9A, compare with Figure 9B) • In myelodysplastic syndrome (MDS) and chronic myeloproliferative disorders, a certain degree of maturation is preserved, thus both evaluations of immature and maturing cells is considered mandatory for the diagnosis
• In this chapter, the immunophenotypic features of AML and chronic myeloid disorders are briefly discussed in the context of the World Health Organization (WHO) classification
Clinical Flow Cytometry
6-11
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• The WHO classification introduced new categories of AML defined by recurrent cytogenetic abnormalities. These leukemias often show specific immunophenotypes, thus they will be presented separately in the following outline
Acute Myeloid Leukemias AML with Recurrent Cytogenetic Abnormalities AML with t(8;21)(q22;q22);(AMLl/ETO) • Majority of cases show an immature myeloid immunophenotype with a high density of CD34 and coexpression of low-den sity CDI9 (Figure 10) • In addition, numerous myeloid antigen s, including CD33 , CD 13, and myeloperoxidase are expressed • Frequently, there is asynchronous co-expression of CD34 and CDl5 • The presence of TdT is common • The co-expre ssion of CD56 has been reported to be associated with worse progno sis
AML with inv(16)(p13q22) or t( 16; 16)(p13;q22)/(CBF-~/MYH1l)
• The co-expression of CD2 , antigen normally seen on T- and NK cells, is common (Figure 11)
Acute Promyelocytic Leukemia (APL) (AML with t( 15;17)(q22;q12)) • In contra st to most less differentiated myeloid leukemias, APL presents with high SSC reflecting the granular cytoplasm of leukemic cells (Figure 12) • A constellation of immunophenotypic features used to diagnose APL include lack of CD34 and HLA-DR antigen s • Expre ssion of homogeneou s bright CD33 along with myeloperoxidase and variable expre ssion of CD 13 and CD 15 are present • CD2 and higher incidence of CD34 expression have been reported in APL with microgranular morphology
AML with 11q23 (MLL Gene) Abnormalities • Constitute a heterogeneous group most commonl y presenting with monocytic differentiation
• Immature cells show expression of CD34, COlI?, and TdT
• The immunophenotypic feature s are not specific and can be seen in any acute myelomonocytic or monocytic leukemias
• Sub-population of maturing cells expre ss monocytic (C0l4, CDllb, CD4 dim ) and granulocytic (CDI5) markers
• Most commonly, these leukem ias are negative for CD34 and positive for CD33 , CD13, CDI4, CD4 dim , COllb, and CDM
165
6-12
Molecular Genetic Pathology
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AML not Otherwise Categorized
• Myeloperoxidase is negative or only expressed in a minority of cells
AML Minimally Differentiated and AML Without Maturation
AML with Maturation
• Blasts show low-density CD45 antigen expression and display low SSC reflecting their relatively agranular cytoplasm (Figure 9A)
• In addition to primitive hematopoietic and early myeloid antigens, more mature myeloid markers such as CDl5 and myeloperoxidase are often expressed
• The majority of even least differentiated AMLs express myeloid markers such as CD 13, CD33, and/or CD 117
• Occasionally, there is an asynchronous co-expression of antigens, which in normal hematopoietic cells are not expressed simultaneously (e.g., exclusive early or late myeloid markers such as CD34 and CDI5)
• Primitive hematopoietic antigen s, CD34 and HLA-DR, are often seen
166
Clinical Flow Cytometry
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• Co-expression of markers associated with other lineages, for example, lymphoid, may be seen on the myeloid blasts. The most common example is the CD? antigen
Acute Leukemias with Mono cytic Differentiation (Acute Myelomonocytic Leukemia and Acute Monoblastic/Monocytic Leukemia) • The SSC and CD45 expression in acute leukemias with monocytic differentiation is variable and dependent on the relative proportion of primitive myeloid blasts and the degree of different iation of neoplastic monocytes. Patterns with a distinct and separate population of blasts and monocytes or a large merging cluster of cells, starting in the blast region and extending upwards to monocyte region, can be seen (Figure 13) • In acute myelomonocytic leukemia a population of primitive myeloid blasts is often distinct • The expression of myeloid markers and antigens associated with monocytic lineage such as CD 14, CD4, CDIlb, and CD64 is commonly seen • Despite the CD 14 antigen being present on all mature monocytes, it can be negative in monocytic leukemias. Frequently, a heterogeneous pattern of CDl4 expression is seen reflecting a maturation spectrum of neoplastic monocytes • Immature monocytic markers such as CD64 are more consistently expressed
Acute Erythroid Leukemias • Acute erythroid leukemias are categorized into two subtypes: pure erythroid leukemia and erythroid/myeloid leukemia (erythroleukemia)
• In the erythroleukemia, both primitive myeloid blasts and erythroid precursors are present • Erythroid markers CD?I, glycophorin A, and hemoglobin can be present • When glycophorin A and hemoglobin are absent, the diagnosis is based on the absence of myeloid markers, the presence of bright CD?I, and scatter characteristics of leukemic cells
Acute Megakaryobla stic Leukemia • Usually shows low SSC and dim to absent CD45 • Early megakaryocytic markers, CD4I and CD6l, are frequently expressed (Figure 14) • Occasionally, the late megakaryocytic marker, CD42b, is present • There is variable expression of stem cell markers, CD34 and HLA-DR, on the population of leukemic megakaryoblasts
Chronic Myeloproliferative Disorders and MDS • The abnormalities detected by flow cytometry in chronic myeloid disorders reflect abnormal morphologic features and abnormal maturation • Both qualitative (presence or loss of a particular antigen) and quantitative changes (differences in the number of antigen molecules) are used for diagnostic purpo ses • A detailed review is beyond the scope of this text; however, a few examples are presented next to illustrate the contribution of flow cytometry to diagnosing these diseases
167
6-14
Molecular Genetic Pathology
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Fig. 12. Acute promyelocytic leukemia. (A) Typical SSC pattern of APL corresponds to prominent granularity of leukemic cells (in red). Residual lymphocytes are presented in aqua. (B) Leukemic promyelocytes are positive for CD33 (bright as compared with low-density CD33 of residual normal neutrophils, in navy) and negative for HLA-DR. (C) CD34 is negative and myeloperoxidase is expressed by the majority of cells. (D) Low-density CDIS antigen can be present.
Myelodysplastic Syndromes • MDS is characterized by ineffective hematopoiesis with abnormalities in maturation , and often, decreased survival of hematopoietic progeny • The diagnosis of MDS is based on the morphologic , immunophenotypic, and genetic features as well as clinical manifestations
168
• Flow cytometry contributes to the diagnos is of MDS through the immunophenotypic identification of aberrant maturation • The following features make the flow cytometric analysis an important component of MDS diagnosis : Aberrant immunophenotypes demonstrated by flow cytometry are seen in up to 98% of MDS cases and
6-15
Clinical Flow Cytometry
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can be routinely analyzed in granulocytic, monocytic, and erythroid lineages
flow cytometry was predictive of future cytogenetic abnormalities, and thus, diagnostic of myelodysplasia
It has been previously reported that even in cases with minimal or no morphologic features indicative of MDS,
- Recent studies underscored the prognostic significance of specific immunophenotypic abnormalities for the
169
6-16
Molecular Genetic Pathology
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increased expression of CD34 , CD 13, and CD II c was reported on myeloid blasts
natural course of the disease or the outcome after bone marrow transplantation • The categories of abnormalities detected by flow cytometry in MDS include: - Aberrant SSC reflecting, morphologically identified dysplasia: hypogranulated neutrophils can be in up to 70% visualized by their abnormally low of cases (Figure 15A) - Changes in the relative proportion of cells at specific stages of myeloid maturation: the high-grade MDS usually demonstrates increased number of immature cells. A significant left shift in granulocytic maturation and an increase in blast percentage can be demonstrated by flow cytometry (Figure ISA)
sse
- The disruption of normal maturation patterns as reflected by the asynchronous expression of myeloid markers : • Appearance of late myeloid markers inappropriately early in the differentiation (i.e., CDI5 on myeloblasts) • Persistent expression of immature markers in late granulocytic stages (e.g., retention of CD34 and HLA-DR on mature granulocytes) • Uncoupling of the normal sequence of CD 13 and CD 16 expression in granulocytic differentiation - Abnormal density of normally occurring antigens: among others, the decreased density of CD45 and
170
-
Aberrant expression of lineage-associated markers: The inappropriate expression of lineage-associated markers such as CD7 and CD56 on myeloid blasts is common (Figure 15B)
Chronic Myeloproliferative Disorders • The utility of flow cytometry in chronic myeloproliferative disorders is less well established
Chronic Myelogenous Leukemia • Application of flow cytometry as a diagnostic tool in chronic myelogenous leukemia is limited to the accelerated phase or blast cri sis, in which the lineage of an expanding blast population needs to be determined • In the chronic phase, presence of the Philadelphia chromosome (as seen on conventional karyotyping or molecular analysi s) remain s the defining feature of this disorder
Philadelphia Chromosome Negative Chronic Myeloproliferative Disorders • In general, flow cytometric abnormalities are seen in the majority of cases with cytogenetic abnormalities • However, no consistent set of abnormalities to routinely subclassify neoplastic myeloproliferative states was described
Clinical Flow Cytometry
6-17
FLOW CYTOMETRIC ANALYSIS OF LYMPHOID NEOPLASMS (ACUTE LYMPHOBLASTIC LEUKEMIA [ALL] AND MATURE LYMPHOID NEOPLASMS)
• The diagnosis of lymphoid malignancies relies on the presence of lineage-associated markers corresponding to specific stages of lymphoid development • No single marker can be used for a definite diagnosis; thus the presence of several B- or T-cell-associated antigens is used for lineage assignment • The sentinel feature of mature B- and T cells is the presence of surface receptor complexes • As the immune system has to respond to a wide array of antigens, in healthy individuals, B- and T-cells express a great diversity of surface receptor complexes (Ig and TCRs). This diversity defines reactive polyclonal lymphoid populations • On the contrary, the neoplastic lymphoid cells are characterized by monoclonal B- and T-cell receptors. In the majority of cases, the presence of clonality is a definite confirmation of the malignant nature of lymphoid proliferation • Lymphoid precursors often show an absence of surface receptor complexes. Thus, in precursor-derived neoplasms, the homogeneous expression of specific markers on lymphoblast population, rather than the presence of clonal surface receptors, is considered diagnostic of malignancy. The following paragraph presents the key immunophenotypic features of lymphoblastic and mature lymphoid malignancies
Precursor B ALL/Lymphoblastic Lymphoma (Pre-BALL) • Pre-B ALL shows expression of markers seen in normal B cell differentiation such as CD 19, CD22 (cytoplasmic or membranous), CD79a, HLA-DR, CD34, and TdT
(Figure 16) • High-density CDI0 antigen is frequently seen
(Figure 16B) • Surface Ig light chains are not present; however, cytoplasmic u-chain or IgM may be detected • Immunophenotypes of leukemic lymphoblasts resemble stages of normal B cell differentiation. However, even though maturation sequence is roughly reproduced, the majority of cases show aberrant expression of selected markers. In addition, the categorization according to maturation stage is of limited practical value as clinical behavior is influenced mostly by clinical features and genetic abnormalities • Frequently, specific immunophenotypes correlate with cytogenetic and clinical features . However, in routine practice, the confirmation of cytogenetic abnormality with either conventional karyotyping
and/or molecular techniques is necessary. Selected examples of immunophenotypic-genotypic associations are presented below
Pre-B ALL with Rearrangements of llq23 (MLL Gene) • This rearrangement commonly occurs in infant ALL, while the frequency of MLL involvement in older children and adults is much lower «5 %) • The most frequent fusion partner for MLL gene in ALL is AF4 gene on chromosome 4q21 (t[4;11]) • Rarely other genes are involved in pre-B ALL including ENL (19p13.3) and AF9 (9q21-22) • CDI9, CD34, and TdT are positive . The more mature B cell marker, CD20, is negative • Contrary to the majority of ALL cases, blasts in this leukemia are negative for the CD 10 antigen, which indicates the early stage of B cell maturation • Myeloid markers, CD 15 and CD65 are frequently positive
Pre-B ALL with BCRlABL Translocation • Philadelphia chromosome (t(9;22); BCR/ABL) is a hallmark of chronic myelogenous leukemia. However, a BCR/ABL translocation with a breakpoint in the minor breakpoint region (m-BCR) occurs in both pediatric and adult pre-B ALL • Cases of ALL with BCR/ABL translocation carry a particularly dismal prognosis, thus their prompt identification is essential for effective treatment decision making • Most BCR/ABL cases have a classic intermediate (common) ALL immunophenotype with the expression of CDl9 and TdT • The homogeneous CD10 and CD34, and dimlheterogeneous CD38 expression are seen • The expression of myeloid markers, especially CDI3, is common • The expression pattern of CD34 and CD38 allow differentiation between BCR/ABL positive and negative ALL in multivariate analysis
Pre-B ALL with TEUAMLI Translocation • TEUAMLl translocation occurs in 25% of childhood pre-B ALL cases and is associated with a favorable prognosis • As routine karyotyping can detect t(12;21) in only a minority of cases, other means of screening for the
171
6-18
Molecular Genetic Pathology
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presence of TEUAMLl fusion are necessary. The identification of this subset of pre-B ALL can be accomplished using flow cytometry • The immunophenotype is that of a precursor B cell with expression ofCD19, CD34, CDlO, and TdT. CD20 antigen is negative • The CD45 is more commonly positive and the aberrant co-expression of CD 13 is frequent • The most specific immunophenotypic features predictive of TEUAMLl fusion are: negative or partially positive CD9 and negative CD20
172
Precursor T ALL/Lymphoblastic Lymphoma (Pre-T ALL) • Both pre-TALL and lymphoblastic lymphoma are derived from immature cells committed to T cell lineage • Similar to pre-B ALL, dependent on the primary site of involvement, i.e., bone marrow or lymph node, the designation of leukemia or lymphoma is used • The most specific marker of T cell differentiation is the CD3 antigen. Similar to normal T cells, in pre-TALL this antigen is initially seen in the cytoplasm before the
6-19
Clinical Flow Cytometry
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transfer to the cell surface as a portion of the TCR complex • Other T cell antigen s include CD2, CD7 , CDS, CDla, CD4, and CD8 • Pre-TALL mimics the normal maturation of T cells; however, aberrant antigen densities and/or antigen loss are frequent (Figure 17) • CD34 and CD 10 may also be present ; however, HLA-DR is typically absent
• In pre-TALL, the correlation of the immunophenotype with specific genetic lesions is not clear and the stage of differentiation of leukemic T cells is not utilized for progno stication
Mature Lymphoid Neoplasms • The flow cytometri c diagnosi s of lymphomas is based on the presence of clonal lymphoid population bearing numerous lymphoid markers
173
Molecular Genetic Pathology
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Fig. 18. Surface Ig light chain expression in reactive and malignant B cells. (A) Reactive B cells show heterogeneous (polyclonal) expression of x and A. (B) Mature B cell neoplasms are monoclonal with the entire lymphoma population expressing only one type of Ig light chain . • Gating based on SSC/FSC characteristics is most commonly used • On FSC/SSC dot plots, neoplastic lymphoid cells are seen in the area of small or large lymphocytes. In large cell lymphomas or hairy cell leukemia, the neoplastic population may overlap with the monocyte region • The display of SSC vs CD45 is not typically used for gating of lymphomas as most mature lymphoid malignancies display high-density CD45 antigen. Only rare cases of lymphoma show slightly dimmer CD45 , or even more infrequently, are negative for the CD45 antigen as in lymphoma with plasmablastic differentiation • The designation "clonal" implicates that the entire lymphoma population is derived from a single lymphoid cell that underwent malignant transformation. Thus, all neoplastic cells should demonstrate similar genetic and immunophenotypic features . The clonality is best represented as an expression of uniform (monoclonal) surface light chain or TCR. This stands in stark contrast to the highly variable, polyclonal immunophenotype of normal lymphocytes, which reflects a random receptor gene rearrangement and a subsequent response to a variety of antigenic stimuli
Mature B Cell Neoplasms • Normal precursor B cells randomly rearrange Ig heavyand light chains. As a result, mature B cells show a polyclonal pattern of Ig heavy and light chains (Figure 18A). In contrast, a monoclonal surface light chain expression (exclusively x or A) is seen in the majority of B cell lymphomas (Figure 18B)
174
• The light chain monoclonality along with the expression of pan-B cell markers is diagnostic of B-cell lymphoma. The sub-classification is based on the presence and specific density of selected lymphoid markers • Rarely, mature lymphoid neoplasms lose their surface Igs, a feature not commonly seen in normal mature B cells • On the contrary, neoplastic plasma cells typically lack surface Igs and show only cytoplasmic expression of K or A
Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLUSLL) • CLL and SLL are derived from re-circulating CD5+, IgM+, IgD±, B cells normally present in the peripheral blood (Figure 19) • The WHO classification considers CLL and SLL as one entity with different presentations. The diagnosis is based on the predominant site of involvement (bone marrow/peripheral blood vs lymphoid organs) • CLLlSLL is positive for pan-B cell antigens including CDI9 and C020 (dim expression, Figure 19B and C) • In addition, the expression of CD5, C023, and weak surface monoclonal (x or A) light chain is seen • The absence of FMC? and cyclin DI, and presence of CD23 are features distinguishing CLLlSLL from mantle cell lymphoma (MCL) • Currently, two groups of CLLlSLL are recognized: - One, corresponding to the pre-germinal center phenotype (naive, showing no mutations in the variable region ofIg heavy chain [VH ] gene) - The second type is derived from memory B cells (post-germinal center, mutated VH gene)
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- The sub-classification roughly corresponds to the expression of ZAP-70 (Figure 19D) and CD38 molecules, which can be quantified by flow cytome try
Mantl e Cell Lymph oma (MCL) • MCL dem onstrates expression of pan-B cell markers (CD I9, CD20) and clo nal surface light chains • There is co-expre ssion of CDS antige n; however CD23 is negative
• In contrast to CLLlSLL, the high-density CD20 and light chains are seen, and there is co-express ion of FMC7 , the antigen that is invariabl y negative in typical cases of CLLlSLL • The defining feature of MCL is the t(l I ;14), in which the eye/in D J gene is translocated into the proximity of the Ig heavy chain gene prom oter, resulting in the constitutive expression of this protein. The presence of cycl in D 1 can be demonstrated by flow cytometry
175
6-22
Molecular Genetic Pathology
Follicular Lymphoma (FL)
Plasma Cell Neoplasms
• The immunophenotype reflects the follicle center cell origin ofFL
• Plasma cell neoplasm s are characterized by a monoclonal proliferation of terminally differentiated B cells, i.e., plasma cells
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• Pan-B cell markers (CDI9, CD20) are present along with the co-expression of CD I and clonal surface Ig • The co-expression of CD 10, similar in density to that seen in reactive follicular hyperplasia, and relative lowdensity CD 19 are characteristic features of FL • In contrast to reactive germinal centers, neoplastic follicular cells express BCL-2, which results in decreased sensitivity to apoptosis and allows for the accumulation of neoplastic lymphocytes. The expression of BCL-2 by FL cells is due to the t(l4;18)(q32 ;q21), which places the BCL-2 gene under a promoter of the Ig heavy chain gene
Marginal Zone Lymphomas • Three subtypes of marginal zone lymphomas are recognized: nodal, extranodal (mucosa-associated lymphoid tissue lymphoma), and splenic . They all share similar immunophenotypes • Marginal zone lymphomas express the B cell markers (CDI9, CD20, and CD22) and clonal surface light chains • CD5 and CD 10 are absent • No specific surface markers, routinely analyzed by flow cytometry, allow for sub-classification of this lymphoma based on immunophenotype; therefore the diagnosis is based on the absence of immunophenotypic features specific for other lymphoma subtypes
Lymphoplasmacytic LymphomalWaldenstrom Macroglobulinemia • Lymphoplasmacytic lymphoma is a B-cell Iymphoproliferative disorder composed of a heterogeneous proliferation of small B cells, Iymphoplasmacytoid lymphocytes, and plasma cells • The defining feature is the demonstration of monoclonal IgM protein in the serum • B-cell associated antigens, including CD 19, CD20, and CD22, are consistently expressed • In the majority of the case s, the B cell immunophenotype is non-de script • Frequency of co-expression of other antigens seen in B-celllymphomas such as CD5, CD23 , and FMC7 is variable . CD lOis most frequently negative
• These disorders can present as a localized or disseminated process most commonly involving bone marrow, bone, and more infrequently, extramedullary sites and peripheral blood • Neoplastic plasma cells demonstrate a highly heterogeneous immunophenotype, different from that of their normal counterpart • Due to overlap with various hematopoietic populations on SSCIFSC and SSC/CD45 , the identification of plasma cells is best accomplished through their expression of CD138 and unique bright CD38 (Figure 20A) • The majority of neoplastic plasma cells, unlike their normal counterpart, show a decreased density of CD45 antigen (negative to weakly positive, Figure 20B) . Only about 20% of plasma cell myelomas demon strate homogeneous bright to heterogeneous expression of CD45, similar in pattern to that of normal plasma cells • Neoplastic plasma cells are most often negative for the pan-B cell markers, CD 19 and CD20. The expression of CD20 is retained in approximately 20% of cases. CDl9 is seen in <5% of neoplastic plasma cell proliferations • Neoplastic plasma cells present with monoclonal cytoplasmic and occasionally surface Igs • The CD56 antigen is seen in 70% of myeloma cases and its absence has been associated with adverse clinical outcome • The presence of myeloid markers including early antigens , such as CD 117, is frequently reported
Diffuse Large BCL (DLBCL) • The defining morphologic feature of DLBCL is a large cell size that can be appreciated on the displays of FSC vs SSC • As in other B-celllymphomas, pan-B cell antigens are expressed including CDI9, CD20, and CD22 • DLBCL can originate from different stages in B cell development; hence, the co-expression of other markers is heterogeneous. The CD5, CD 10, BCL-6, CD30 , and CD 138 can all be present
• The surface IgM expression can be demonstrated in all cases
Burkitt Lymphoma (BL)
• The majority of cases show dim positivity for CD25 antigen
• This lymphoma is composed of medium-sized, highly proliferating lymphoid cells with basophilic vacuolated cytoplasm. The WHO classification distinguishes three variants of this lymphoma: endemic (occurring predominantly in Africa), sporadic, and immunodeficiency associated. All variants show similar immunophenotypic features
• In a proportion of cases a second subset of monoclonal B cells is identified corresponding to Iymphoplasmacytoid lymphocytes (intermediate FSC and SSC, positive for CDI9, CD20, FMC7 , and bright CD38 , and negative for CD138) • In addition, as reported previously, a minute monoclonal plasma cell component can be identified in the majority of cases
176
• The immunophenotype of BL reflects germinal center origin :
6-23
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- CDI9, CD20, CDlO, and BCL-6 antigens are positive - BCL-2 is negative • As in the majority of mature B cell neoplasms, there is surface expression of monoclonal Ig light chains • Even though the immunophenotype of BL alone is not specific enough for the definitive sub-classification of this lymphoma , the confirmation of high-proliferative activity by the expression of CD71 can be used to support the diagnosis . This feature is linked to the constitutive expression of MYC gene (cell cycle gate-keeping gene) due to its translocation under the promoter of Ig heavy or light chain genes (t(8;14), t(2;8), t(8;22» . The above translocations are pathognomonic of BL
Mature T- and NK Cell Lymphomas • Lymphomas derived from mature T- and NK cells are much less common than the previously discussed mature B cell neoplasms and show greater geographic and ethnic variability • The immunophenotypic features of T- and NK cell malignancies are overlapping and frequently less specific than those seen in B-celllymphomas • Expansion of the specific T cell subset (e.g., CD4 or CD8) with loss or altered intensity of T-cell-associated markers (most commonly CD7, CD3, and CD5) is seen in the majority of T cell lymphomas (TCL) • The aberrant immunophenotype is a reliable diagnostic feature only when the neoplastic population is significant, as small numbers of T cells with unusual antigen makeup can also be seen in inflammatory conditions,
autoimmune disorders, or viral infections . Thus, the aberrant immunophenotype alone cannot be considered pathognomonic of a T cell malignancy • Considering these factors, an integration of morphologic, immunophenotypic, cytogenetic, molecular, and clinical information, as stressed by the WHO classification, is of particular importance in diagnosing T- and NK cell malignancies • Until recently, the demonstration of clonality in T cell proliferations was limited to molecular methods detecting TCR gene rearrangements (PCR or Southern blot analysis) . The development of multiple V~-family antibodies directed against the variable region of the TCR ~-chain allows for the determination of T cell clonality by flow cytometry. The determination of clonality is based on the preferential usage of a single V~-family and has close to 90% sensitivity and specificity in diagnosing T-celllymphoproliferative disorders. The results of this assay must be correlated with additional immunophenotypic and clinical data, as rare cases with V~-family expansion have been reported in patients with no diagnosis of malignant lymphoma
T-Cell Prolymphocytic Leukemia • This is a mature TCL derived from helper T cells • The majority of cases retain the mature T cell immunophenotype with expression of CD4 antigen • Rare cases (-10%) can be double-positive or doublenegative for CD4 and CD8 • CD25 can be co-expressed with the density similar to that of activated mature T cells
177
6-24
T-Cell Large Granular Lymphocytic Leukemia (T-LGL) • T-LGL is an indolent lymphoproliferative disorder derived from cytotoxic T cells • The demonstration of a monoclonal aberrant T cell population in bone marrow and peripheral blood in a patient with cytopenias is a hallmark of the disease • Considering the variable morphology of T-LGL, flow cytometric immunophenotyping plays a key role in identifying the aberrant cytotoxic immunophenotype of this disease • The immunophenotype is similar to CD8+ T cells. Varying degrees of loss or decreased density of CD?, CD2, and CD3 were reported • CD5? is positive in the majority of cases . A small number of cases express CD56 and/or CDI6 • NK receptors for class I major histocompatibility molecules (both of killer cell Ig-like receptor type, CD158 antigens; and C-type lectin type, CD94 , and NKG2 molecules) showed aberrant expression in the majority of reported cases supporting the diagnosis ofT-LGL
Aggressive NK Cell Leukemia • This is a rare neoplasm of NK cells typically presenting with systemic involvement and an aggressive clinical course
Molecular Genetic Pathology
• Rare cases showing positivity for TdT and CD34 were reported • The presence of T cell, B cell, and myelomonocytic lineage-associated markers should be excluded
Adult T Cell Leukemia/Lymphoma • Adult T cell leukemia/lymphoma is a mature T cell neoplasm caused by human lymphotropic virus type I(HTLV-l) • Even though classically it has been considered to be a neoplasm of helper T cells, recent studies demonstrated its immunophenotypic and functional similarity to regulatory T cells • In addition to pan-T cell markers , CD3, CD5, and CD2, the expression of CD4, bright CD25, and regulatory T cell marker FoxP3 can be demonstrated by flow cytometry • CD? antigen and cytotoxiclNK cell markers are absent
Hepatosplenic TCL • Hepatosplenic TCL is a disseminated TCL originating T cells from
yo
• The neoplastic cells are positive for surface CD3 and associated TCR. Only rare cases of a~ TCR type were also reported
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• Typically CD5, CD4, and CD8 antigens are negative
• As in normal NK cells , surface CD3 and CD5 are absent • The CD2, CD56 , and CD 16 are present in the majority of cases . CD5? can be absent
• CD56 and CDl6 are expressed in some cases . CD5? is negative
• A varying degree of CD? loss is seen
Angioimmunoblastic T-Cell Lymphoma (AILT)
Extranodal NK/T-Cell Lymphoma, Nasal Type • Nasal type NK/T-celllymphomas are EBV positive extranodal proliferations derived from NK or cytotoxic T cells. Both upper respiratory tract and other extranodal sites can be involved • The immunophenotypic features include positivity for CD56, CD2 , frequently CD?, and the cytopla smic e-chain of CD3 antigen
• AILT is most commonly diagnosed by the combination of morphologic, immunophenotypic, and clinical features. The hypothetical origin of this lymphoma is a CD4+ T cell from the germinal center • The pathologic diagnosis can be challenging due to morphologic heterogeneity and immunophenotypically mixed T cell proliferation with a significant admixture of reactive component
• CD8 and CD4, as well as other T-cell-associated markers, are negative
• Even though the number of malignant T cells may be low, flow cytometry demonstrates the immunophenotypically aberrant T cell population in >90% of cases
• Rare cases demonstrate cytotoxic T cell immunophenotype
• The immunophenotype is that of CD4+ mature T cells with a varying loss of the CD3 and CD? antigens
Blastic NK Cell Lymphoma (Agranular CD4+, CD56+ Hematodermic Neoplasm)
• CD8 and CD56 antigens are absent • Characteristic co-expression of CD lOis seen in the majority of cases
• Initially, this neoplasm was thought to have originated from NK cells, but most current evidence suggests its origin from the plasmacytoid dendritic cells
Mycosis Fungoides (MF) and Sezary Syndrome (55)
• The immunophenotype reflects the cell of origin : CD56, CD4, HLA-DR, and COl 23 antigens are positive. The latter, along with recently reported expression of blood dendritic cell antigens 2 and 4, is highly specific for dendritic cells and their precursors
178
• MF is the most common cutaneous lymphoma. SS presents as a disseminated disease with widespread skin involvement, lymphadenopathy, and circulating lymphoma cells • Flow cytometry is most commonly utilized to demonstrate circulating MF/SS cells with a specific
6-25
Clinical Flow Cytometry
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T cell immunophenotype that includes the expression of pan-T cell markers CD3, CD5, and CD2 along with CD4 antigen • An important feature is the absence of the CD? antigen. Other T-cell-associated antigens such as CD2, CD3, and CD5 can also be negative (Figure 21)
Enteropathy- Type TeL • Enteropathy-type TCL is derived from intraepithe1ial T cells and involves predominantly small bowel • Flow cytometric immunophenotype was reported in rare cases and demonstrated neopl astic lymphoid cells positive for CD3, CD2 , CD7 , CDllc, and CDI03 with
179
6-26
Molecular Genetic Pathology
variable loss of pan- T cell antigens, most commonly CD5 • Reported cases were positive for CDS or double-negative for CDS and CD4
Peripheral T-Cell Lymphoma, Unspecified (PTCL) • This is morphologically heterogeneous group of lymphomas with mature T cell phenotype • The majority of the cases are derived from CD4+ T cells and retain this immunophenotype. Approximately 10% of cases show CDS expression. Double-negative cases have also been reported • Variable loss of pan-T cell antigens is seen. Most frequently CD7 (in 50% of cases) and surface CD3 are lost
Anaplastic Large Cell Lymphoma (ALCL) • ALCL is composed of large pleomorphic cells, which as other lymphomas with this morphology, often fall in the gate overlapping with large lymphoid cells and/or monocytes (Figure 22)
• The presence of CD30 antigen and, in up to 70% of cases, ALK-l protein is the defining immunophenotypic features of this lymphoma and can be demonstrated by flow cytometry. The overexpression of ALK-l is most often due to t(2;5)(p23 ;35), between ALK gene and nucleophosmin gene. Alternative fusion partners for the ALK translocation have also been identified • Various combinations of the CD30 antigen and T cell markers are seen including CD2, CD4, CD3, CD7, CD5, and CDS • CD7 antigen is lost most frequently followed by CD5 , CD3, and CD2 • CD25 antigen is positive in up to 90% of cases • In ALK positive cases, ALK protein can be demonstrated by flow cytometry • Interestingly, the expression of myeloid markers (CD 13, CD33, and CDI5) has also been reported, which especially in the rare cases with leukemic involvement, can bring a myeloid neoplasm into the differential diagnosis
OTHER CLINICAL APPLICATIONS OF FLOW CYTOMETRY
Primary and Secondary Immunodeficiencies • Flow cytometry is commonly used to diagnose and immunophenotype primary and secondary immunodeficiencies. The detailed discussion of this topic is beyond the scope of this text. The following examples are provided to illustrate the most common applications - Immunophenotyping: the loss of specific antigens, such as ~2 integrins, is easily demonstrated by flow cytometry and used to diagnose leukocyte adhesion deficiencies - Functional defects: an absence or low levels of NADPH oxidase, an enzyme involved in oxidative burst, occurs in chronic granulomatous disease . The level of enzymatic activity can be assayed using flow cytometry and correlate with specific genetic lesions (Figure 23) • In the presence of adequate levels of NADPH oxidase the non-fluorescent compound, dihydrorhodamine 123, converts to fluorescent rhodamine. The phorbol 12-myristate-13-acetatestimulated granulocytes from healthy volunteers serve as a positive control (Figure 23B) • The X-linked recessive form of the disease most frequently results in the complete absence of the enzymatic activity (Figure 23C), whereas the autosomal recessive-type such as defect of p47phox enzyme subunit present with markedly decreased level of fluorescence (Figure 23D)
180
- Enumeration of CD4+ helper T cells in human immunodeficiency virus infection is performed using flow cytometry and serves as an indicator of disease progression and response to treatment. The absolute number of helper T cells in peripheral blood correlates to the stage of the disease and patient prognosis. The enumeration of T cells and their subsets is easily accomplished by flow cytometry using a simple combination of antibodies against CD3, CD4, and CDS antigens. The absolute numbers are derived either from a routine white blood cell count of the concurrent peripheral blood specimen (dual platform) or from calibrating beads run simultaneously with the patient sample (single-platform method)
Paroxysmal Nocturnal Hemoglobinuria (PNH) • PNH is caused by an absence or decreased numbers of membranous glycosylphosphatidylinositol (GPI) anchor, which results in a loss of GPI-linked proteins • Diagnosis of PNH by flow cytometry relies on the demonstration of decreased expression of two GPIanchored proteins on two cell populations. Antibodies against CD55 and CD59 antigens are most commonly used - The analysis of CD59 expression on red blood cells provides the best discrimination between type I, II, and III cells (Figure 24) - Granulocytes or monocytes are frequently analyzed in addition to red blood cells
6-27
Clinical Flow Cytometry
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Fig. 23. Chronic granulomatous disease. (A) Unstimulated neutrophils from a healthy volunteer show baseline level of dihydrorhodamine 123 fluore scence. (B) Normal granulocytes with adequate activity of NADPH oxidase show a distinct shift in fluorescence intensity upon phorbol 12-myristate-13-acetate stimulation. (C) X-linked recess ive chronic granulomatous disease shows complete loss of enzyme activity (no fluorescence shift) . (D) Low-level enzyme activity in a patient with p47-phox deficiency corresponds to decreased fluore scence intensity as compared with normal control.
Stem Cell Transplantation • Flow cytometry is utilized for enumeration of CD34+ stem cells and cell sorting - CD34 population can be enriched using flow cytometric sorting. Using this approach, heterogeneous populations can be physically separated into cell subsets with different physical or immunophenotypic properties - High-speed flow cytometric sorting is achieved through charging of droplets containing individual cells with a specific polarity. As the charged droplet
passes through the electrostatic field, it is isolated from the remainder of the sample and collected into a separate container
Novel Applications of Flow Cytometry • Flow cytometry-based molecular testing : detection of PCR target amplicons using liquid bead array systems • Tissue typing • Assaying response to medications, for example, monitoring platelet activation after anti-platelet therapy
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B
A
10°
10'
10°
10 3
102
10 1
10 2
10 3
CD59-PE
CD59-PE
c
10°
102
10'
10 3
CD59-PE
Fig. 24. The diagnosis of PNH is based on the absence of GPI-anchored molecules . Type I (normal), II (partial deficiency) , and III (completed loss) cells are best recognized among red blood cells using antibody against CD59 antigen. (A) Red blood cells from healthy volunteer, used as a positive control, show high intensity CD59 (correspond to type I cells) . (B) Type I and II cells (small peak with decreased expression) in a patient with PNH. (C) Granulocytes with a complete loss of CD59 (Type III cells) in a patient with PNH supported by red blood cell transfusion.
SUGGESTED READING Adriaansen H, Boekhorst PAW, Hagemeijer AM, et al. Acute myeloid leukemia M4 with bone marrow eosinophilia (M4Eo) and inv(l6)(pI3q22) exhibits a specific immunophenotype with CD2 expression. Blood 1993;81:3043- 3051.
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Andrieu V, Radford-Weiss I, Troussard X, et aI. Molecular detection of t(8 ;21)/ AMLl-ETO in AML M 11M2: correlation with cytogenetics, morphology and immunophenotype. Br J Haematol . 1996;92:855-865 .
Clinical Flow Cytometry
Beck RC, Stahl S, O'Keefe CL, et al, Detection of mature T-cell leukemias by flow cytometry using anti T-cell receptor V beta antibodies . Am J Clin Pathol. 2003;120:785-794. Borowitz MJ, Rubnitz J, Nash M, et al, Surface antigen phenotype can predict TEL-AMLI rearrangement in childhood B-precursor ALL: a Pediatric Oncology Group study. Leukemia 1998:12:1764-1770. Chen I-M, Chakerian A, Combs D, et al. Post-PCR multiplex fluorescent ligation detection assay and flow cytometry for rapid detection of genespecific translocations in leukemia. Am J Clin Pathol. 2004;122:783-793. Chen W, Kesler MV, Karandikar NJ, et al. Flow cytometric features of angioimmunoblastic T-cell lymphoma. CytometryPartB (Clin Cyto) 2006;70B:142-148. Ciudad J , Orfao A, Vidriales B, et al, Immunophenotypic analysis of CD 19+ precursors in normal human adult bone marrow: implications for minimal residual disease detection. Haematologica. 1998;83:1069-1075. Domingo-Claros A, Larriba I, Rozmann M, et al. Acute erythroid neoplastic proliferations. A biological study based on 62 patients. Haematologica. 2002;87:148-153. Edwards BS, Oprea T, Prossnitz ER, et al. Flow cytometry for highthroughput . high-content screening . Curr Opin Chem Bioi. 2004;8:392-398. Gorczyca W. Differential diagnosis ofT-celllymphoproliferative disorders by flow cytometry multicolor immunophenotyping. Correlation with Morphology. Methods Cell Bioi. 2004;75:595-621. Harbott J, Mancini M, Verellen-Dumoulin C, et al, Hematological malignancies with a deletion of Ilq23: cytogenetic and clinical aspects.
Leukemia 1998;12:823-827. Helleberg C, Knudsen H, Hansen PB, et al. CD34+ megakaryoblastic leukaemic cells are CD38-. but CD61+ and glycophorin A+: improved criteria for diagnosis of AML-M7? Leukemia 1997; II :830-834. Human cell differentiation molecules--collaborative research on cellular markers. [http://www.hlda8.org) . Jaffe ES, Harris NL, Stein H, et al, eds. In: World Health Organization Classification of Tumours: Pathology and Genetics: Tumours of Haematopoietic and LymphoidTissue, Lyon: IARC Press; 2001. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495-497. Kohno T, Yamada Y, Akamatsu N, et al. Possible origin of adult T-cell leukemia/lymphoma cells from human T Iymphotropic virus type-Iinfected regulatory T cells. Cancer Sci. 2005;96:527-533. Krasinskas AM, Wasik MA, Kamoun M, et al. The usefulness of CD64, other monocyte-associated antigens , and CD45 gating in the subclassification of acute myeloid leukemias with monocytic differentiation. Am J Clin Pathol. 1998;110:797-805. Kussick SJ, Wood BL. Four-color flow cytometry identifies virtually all cytogenetically abnormal bone marrow samples in the workup of nonCML myeloproliferative disorders . Am J Clin Pathol. 2003; 120:854-865.
Li S, Eshleman JR, Borowitz MJ. Lack of surface immunoglobulin light chain expression by flow cytometric immunophenotyping can help diagnose peripheral B-cell lymphoma . Am J Clin Pathol. 2002;118:229-234. Lo Coco F, Avvisati G, Diverio D, et al, Rearrangements of the RARalpha gene in acute promyelocytic leukaemia: correlations with
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morphology and immunophenotype. Br J Haematol. 1991;78 :494-499. Loken MR, Shah YO, Dattilio KL, et al. Flow cytometric analysis of human bone marrow: I. normal erythroid development. Blood 1987;69:255-263. Macedo A, Orfao A, Ciudad J, et al, Phenotypic analysis of CD34 subpopulations in normal human bone marrow and its application for the detection of minimal residual disease . Leukemia 1995;9:1896-190I. Matreo G, Castellanos M, Rasillo A, et al. Genetic abnormalities and patterns of antigenic expression in multiple myeloma. Clin CancerRes. 2005;11:3661-3667. Ogata K, Nakamura K, Yokose N, et al. Clinical significance of phenotypic features of blasts in patients with myelodysplastic syndrome.
Blood 2002;100:3887-3896. Perfetto SP, Chattopadhyay PK, Roederer M. Seventeen-colour flow cytometry : unraveling the immune system. Nat Rev Immunol. 2004;4:648-655. Petrella T, Bagot M, WilIemze R, et al, Blastic NK-ceillymphomas (Agranular CD4+CD56+hematodermic neoplasms). Am J Clin Pathol. 2005;123:662-675. San Miguel JF, Vidriales MB, Ocio E, et al. Immunophenotypic analysis ofWaldenstrom's macroglobulinemia. Semin Oncol. 2003;2:187-195. Stelzer GT, Shults KE, Loken MR. CD45 gating for routine flow cytometric analysis of human bone marrow specimens . Ann NY Aca Sci. 1993;677:265-281. Stetler-Stevenson M, Arthur DC, Jabbour N, et al, Diagnostic utility of flow cytometric immunophenotyping in myelodysplastic syndrome . Blood 200 1;98:979-987. Stewart CC, Behm FG, Carey JL, et al. U.S.-Canadian consensus recommendations on immunophenotypic analysis of hematologic neoplasia by flow cytometry : selection of antibody combinations .
Cytometry 1997;30:231-235. Tabemero MD, Bortoluci AM, Alaejos I, et al, Adult precursor B-ALL with BCRlABL gene rearrangements displays a unique immunophenotype based on the pattern of CD 10, CD34, CD 13 and CD38 expression . Leukemia 2001 ;15:406-414. Terstappen LWMM, Huang S, Picker LJ. Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow. Blood 1992;79:666-677. Terstappen LWMM, Safford M, Loken MR. Flow cytometric analysis of human bone marrow Ill. Neutrophil maturation . Leukemia 1990;4:657-663. van Lochem EG, van der Velden VHJ, Wind HK, et al. Immunophenotypic differentiation patterns of normal hematopoiesis in human bone marrow. Reference patterns for age-related changes and disease-induced shifts. Cytometry 2004;60B:1-13 . Wells DA, Benesch M, Loken MR, et al. Myeloid and monocytic dyspoiesis as determined by flow cytometric scoring in myelodysplastic syndrome correlates with the IPSS and with outcome after hematopoietic stem cell transplantation . Blood 2003;102:394-403. Wu JM, Borowitz MJ, Weir EG. The usefulness of CD71 expression by flow cytometry for differentiating indolent from aggressive CD 10+ B-cell lymphomas . Am J Clin Pathol. 2006; 126:1-8 .
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Conceptual Evolution in Cancer Biology Shaobo Zhang, MD, Darrell D. Davidson, MD, PhD, and Liang Cheng, MD
CONTENTS I. Stem Cells Overview Cancer Model s Old Cancer Model New Cancer Model Cancer Stem Cell (CSC) Definition and Properties of CSCs CSC Pathways CSC Markers CSC Functional Profiling Clonal Proliferation of CSCs Clinical Implications of CSC s Embryonic Stem Cell (ESC) Fetal Stem Cells (FSC) Adult Stem Cell (Somatic Stem Cell)
II. Epigenetics Overview Gene Silencing Other Epigen etic Processes
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Polymerase Chain Reaction-Based Method s Non-PCR-Based Method s
IV. Microsatellite Instability (MSI)
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Overview Definition and Mechan isms of MSI.. DNA MMR Enzymes and Regulation Factors Implicati ons of MSI Bethesda Panel for MSI MSI in Hereditary Non-Polyposis Colorectal Cancer (HNPCC) Methods for MSI Analysi s
7-10 7-1O 7-10 7-10 7-10 7-II 7-11
V. Chromosomal Instability (ClN)
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Overview Mechanisms of CIN Clinical Implication s of CIN CIN Syndrome s Method s for CIN Analysis
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VI. Gene Imprinting •..•......•......•..........•..•7-13
III. DNA Methylation Overview DNA Meth ylation Enzymes DNA Meth ylation and Geneti c Regulation DNA Methyl ation and Cancer Methylation and Potent ial Clinical Interventions Method s for DNA Methyl ation Analy sis
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Overview Regulation of Gene Impri nting H19 Gene Imprinting Imprintin g and Disease Imprinting Syndrom es Imprinting and Human Cancer
VII. MicroRNA (miRNA) 7-8 7-8
Overview Clinical implications Method s for miRNA analysis
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VIII. RNA Interference Overview Biologic and Clinical Implications of siRNA
IX. Telomere Overview Telomere Structure and Maintenance Ce ll Senesce nce and Telomere Shorte ning
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Telomere Shortening and Cance r Telomerase Summary Telomerase and Cance r Diagnostic and Therapeu tic Implications of Telomerase , Anti-Telomerase Therapy Methods for Telomere Analysis
X. Suggested Reading
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Conceptual Evolution in Cancer Biology
STEM CELLS Overview • Stem cells are the found ation cells for every organ, tissue, and cell in the body • Stem cells have unique properties: - They can renew themselves through division to maintain a population of cells with the same properties as the original cells - Stem cells propagate by asymmetric differentiation allowing a stem cell to generate one stem cell and one differentiated progenitor cell (Table 1, Figure 1) - Potency refers to the ability of stem cells to differentiate into different cell types - Unipotent stem cells produce only one cell type but still possess the property of self-renewal - Multipotent stem cells can differentiate into a limited number of mature cell types - Pluripotent stem cells can differenti ate into a variety of cell types, includin g cell types from each of the three embryonic layers - Totipotent stem cells can differentiate into any cell type in the organism. Embryo s at early stages consist of totipotent stem cells
Table 1. Characteristic of Stem Cells and Progenitor Cells Stem cell
Progenitor cell
Self renewal
Unlimited
Can be limited
Plasticity
Pluripotent
Unipotent or multipotent
Progenitor cell
• Stem cells are the source of cell renewal in individual organs • Stem cells are divided into different groups according to their origin • Stem cells can be used to gener ate healthy and functional specialized cells, which can then replace diseased or dysfunctional cells • Cancer stem cells (CSC) are the origin of cancer
Cancer Models Old Cancer Model • All tumor cell s can form new tumors and are therefore equally tumorigenic • Unregulated growth is due to serial acquisition of genetic events leading to the expression of genes that promote cell proliferation with concomitant silencing of growth inhibitory genes and blunting of cell death genes • Cancer is a proliferative disease • Any cell type could be targeted in carcinogenesis
Fig. I. Stem cells may renew themselves through division to maintain a population of cell s with the same properties as the original cells . They also may propagate with asymmetric differentiation, depending on environmental factors and on whether the organism needs to expand or maintain a constant stem cell population. A cycle with asymmetri c differentiati on yields one stem cell and one differentiated progenitor cell. The progenitor cells can further differentiate into various committed cell types.
Cancer Stem Cells (CSC)
New Cancer Model
Definition and Properties of CSCs
• Only a minorit y of tumor cells can form new tumors • Unregulated cell growth in tumor s results from disruption in the regulatory mechani sm of stem cell self-renewal • Cancer is a regulatory disorder in stem cells and not a simple augmentation of proliferation signals
• CSCs are the cellular source of cancer and have stem cell propertie s (self-renewal and asymmetric differentiati on)
• Only stem cells or progenitor cells are carcinogenic targets possessing the self-renewal pathway
• CSCs maintain themselves through self-renewal • CSCs propagate by asymmetric differentiation • A normal stem cell may become a CSC by genetic mutation giving it unambiguous transformed genomic characteristics
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Table 2. Selected ._-_
_-_
__ .. __
..
__ .. __
......• Cancer stem cells
Tumor
Breast stem cell
esc Markers Tissue-specific marker
CDM +CD24-/lo w,
CD29 hiCD24+ ESA+, ABCG2
-......
Differentiated somat ic cells
.....•
Differentia ted cancer cells
Fig. 2. CSCs are the cellular source of cancer and have the characteristics of stem cells. A CSC may result from transformation of eithera normalstemcell or progenitor cell through genomic mutation. CSCs proliferate and expand the population of malignant cells related to the original malignant clone. Dashedlines indicated the possible pathways of carcinogenesis. • CSCs possess the character of both stem cells and cancer - CSCscan self-renew and differentiate into the heterogeneous clonesthat makeup a malignant neoplasm - CSCs can be maintained in culture in an undifferentiated state - CSCs proliferate and expand the population of malignant cells related to the original malignant clone - CSCs initiate tumor growth after xeno-transplantation in mice • Cancer cells derived from a single CSC have the clonal characteristics of that stem cell - Isolated CSC differentiates into new cancers that are phenotypically indistinguishable from the original tumor - Although most CSCs originate from transformed normal stem cells, some CSC may arise from mutation of committed progenitor cells - Derivation of CSC fromcommitted progenitor cellscould result fromreactivation of self-renewal mechanisms CSC transformation is the result of genetic and epigenetic changes in oncogenes and tumor-suppressor genes (Figure 2)
esc Pathways • Normal or somatic stem cells may be transformed into CSCs through dysregulation of self-renewal and differentiation pathways, usually resulting in wanton proliferation and autonomous differentiation - Bmi-I is a transcriptional repressor activated in lymphoma, in neural stem cells, and in CSC of pediatric brain tumors
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Neuroectodermal stem cell
Nestin, Sox2
Brainstem cell
CDl33
Prostate CSC
CD133, CD34, CD24+CD49f-, SCA-l
Hematopoietic stem cell
CD34+CD38-, CD133, ABCm
Leukemia stem cell
CDM+,Oct4
Testicular germinal stem cell
Oct4, STELLAR, NANOG, GDF3 SSEA
- Notch pathway regulates proliferation in hematopoietic, neural, and mammary stem cells. Components of the Notch pathway may act as oncogenes in mammary and hematopoietic neoplasia. Notch is a transmembrane hetero-oligomer with a large extracellular portion and a small intracellular region. Notch ligandbinding upregulates genes related to cell proliferation Sonic hedgehog homolog is the ligand of the hedgehog signaling pathway, which regulates vertebrate organogenesis and embryonic morphogenesis - Wnt signalingpathway is a complex network of proteins involved in embryogenesis, cancer, and normal adult physiologic processes. The name Wnt was a combination of Wg and lnt , two genes associated with fruitfly development and murine carcinogenesis • Both genetic and epigenetic changes transform normal stem cells to CSC
esc Markers • CSC retains the marker of the stem cell it derives from • CSC markers are not related to carcinogenesis per se • CSCs are moreaccurately defined by functional dysregulation thanby specific phenotypic markers (Table2) - Hoechst 33342 dye efflux defines a small and homogeneous population of cells consistent with stem cells
esc Functional Profiling • CSCs produce all the malignantcells in a primary tumor • CSCs compose the small reservoir of drug-resistant cells that are responsiblefor relapse after chemotherapy • CSCs can give rise to distant metastasis • CSCs may acquire features associated with tumor progression - CSCs may become genetically unstable
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- The second daughter celI retains the ability to selfrenew by asymmetrical differentiation • Mutations accumulated in the self-renewing population allow progression toward increasingly malignant behavior • Progenitor celIs sometimes regain self renewal ability due to new mutations in self-renewal and differentiation pathways (Figure 2) Self-renewal
•
Clinical Implications of CSCs • The growth of tumors depends upon the presence of neoplastic stem celIs • CSC-targeting therapy aims to deplete the CSC pool to cure the cancer - Surface molecule -targeting therapy uses a cytotoxic drug conjugated to antibodies against CSC surface molecules - Another type of therapy targets specific oncoproteins expressed by CSCs, such as HER21neu, ras, kit, bcr/abl, and PMURARa. - Other therapies would target of CSC pathway downstream signal molecules , such as Bmi-l, Notch , Wnt, and Sonic Hedgehog
Fig. 3. Niche is a specialized microenvironment that provides CSCs with the support needed for self-renewal. Niche is necessary for the CSC population to expand and form a cancer. Under the specialized microenvironment CSCs process selfrenewal and differentiation. Signals from the stem celI niche may determine whether stem celIs differentiate asymmetrically or expand symmetricalIy.
• ChalIenges for CSC targeted therapy - Need to selectively eradicate CSCs without harming normal stem cells - Need to develop functional assays for CSCs to monitor the effectiveness of targeted treatment - Need more knowledge of mechanisms by which CSCs evade or become resistant to targeted therapy
Embryonic Stem Cell (ESC) - CSCs resist several chemotherapeutic agents through efflux of the drugs by drug transporter proteins - CSCs empower distant metastasis - CSCs are relatively radio-resistant - CSCs express angiogenesis factors, such as vascular endothelial growth factor and CXCR4 (Chemokine [c-x-c motif] receptor 4) • Niche is a specialized microenvironment that provides CSC with the support needed for self-renewal. Niche is necessary for CSC to form a cancer (Figure 3)
Clonal Proliferation of CSCs • CSCs represent 2-3% of the overalI tumor celI populations • CSCs can differentiate into heterogeneous cancer celI lineages • Asymmetric differentiation is a characteristic of CSC replication, through which a CSC divides into a new CSC and a progenitor celI - The progenitor celI loses self-renewal capacity but gains the ability to proliferate and further differentiate into the various celI types of the malignancy
• ESCs are the stem cells derived from the inner cell mass of a blastocyst, 4-5 days after fertilization • ESCs are pluripotent and could potentially differentiate into celI types from alI three primary germ layers, ectoderm, endoderm, and mesoderm - ESCs are potential sources for tissue replacement after injury or disease because of pluripotency - ESCs remain undifferientated through the actions of transcription factors such as Nanog, Oct4, and Sox2 • ESC regenerative therapies have been difficult to develop because of risk for neoplastic transformation in celIs with highly activated proliferation and differentiation pathways • ESCs are similar to adult stem celIs with added properties of pluripotency and high regenerative capacity
Fetal Stem Cells (FSC) • FSC populations are prominent in the fetus but dwindle with the development of the baby • FSCs are easily isolated and induced to differentiate into mature cell types
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- FSCs have the ability to self-renew - FSCs readily differentiate in vitro and retain these cell lineage properties when transplanted in vivo - FSCs express the adhesion molecule, AA4, in three major cell types • FSCs can be isolated from different origins From fetal blood
• Adult stem cells from different organs possess different tissue-specific markers • Adult stem cells are instrumental for organogenesis, tissue homeostasis, and carcinogenesis • Adult stem cells share the common properties of all stem cells - Self-renewal allows adult stem cells to experience numerous cycles of asymmetric cell division, reproducing themselves and propagating progenitor cells with differentiation plasticity
- From hematopoietic organs in early pregnancy - From a variety of somatic organs - From amniotic fluid and placenta throughout gestation • FSCs proliferate more rapidly than adult stem cells • FSCs may thus represent an intermediate cell type in the current debate focusing on dichotomized adult vs ESCs, and may prove advantageous as a source for regenerative and replacement therapies • FSCs havesimilarproperties to adultstemcells, withhigher proliferative rates and greater plasticity (see Stem Cell Overview)
Adult Stem Cell (Somatic Stem Cell) • Adult stem cells are a tiny population of reserve cells in organs with regenerative capacity, which divide to replenish dying cells and to replace damaged tissues • Adult stem cells self-renew indefinitely and generate all the functional cell types of the organ from which they originate
Adult stem cells have multi-differentiation potential (multi-potency) through which they may differentiate into progeny of several distinct cell types - Adult stem cells have the property of multi-drug resistance. They are capable of actively pumping a diversity of organic molecules out of the cell - Tissue-specific adult stem cells can generate a spectrum of cell types of other tissues. This is the basis of metaplasia and transdifferentiation, in which the metaplastic cells cannot revert to the original cell type • Comparison between normal stem cells and CSCs - Proposed markers of normal stem cells are also expressed in CSCs - CSC proliferation is due to dysregulation of pathways involved in normal stem cell self-renewal
EPIGENETICS
Overview • Epigenetics refers to stable changes in gene expression that are not due to mutation or DNA rearrangement • Specific epigenetic processes of interest include gene silencing, paramutation, bookmarking, imprinting, X chromosome inactivation, position effect, reprogramming, transvection, maternal effect, and histone modification
Gene Silencing • Gene silencing describes the phenomenon of "switching off" of a gene by any mechanism other than genetic mutation (Figure 4) - Transcription level gene silencing • DNA methylation impedes transcription by adding a methyl group to the number 5 carbon of cytosine in promoter regions (see DNA methylation) • Histone modifications include : • Acetylation, substitution of an acetyl group for an active hydrogen atom of a hydroxyl group. As a consequence, the condensed chromatin is transformed into a transiently relaxed structure,
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which allows genes to be transcribed. Deacetylation leads to gene silencing • Methylation is the transfer of methyl groups to histones. Histones which are methylated on certain residues can act epigenetically to repress or activate gene expression • Phosphorylation of histone proteins is crucial for chromosome condensation and cell cycle progression, and enables the transcription of genes that are activated as a consequence of a variety of cell-signaling events • These modifications serve as signals for whether a region of the chromosome to be packed into silent heterochromatin or to remain active as euchromatin - Post-transcription level gene silencing involves RNA interference (RNAi) machinery to stop messenger RNA (mRNA) translation and to induce mRNA degradation (see MicroRNA section) - Gene silencing protects the genome from transposons and viruses. It thus represents an intracellular system protecting from infectious DNA particles
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Conceptual Evolution in Cancer Biology
--. r>"=1......~......~........f--. .--tlt--...- t1- - - • Inactive gene
• Maternal effect is the phenomenon in which the genotype of the mother is expressed unaltered in the phenotype of the offspring. This generally occurs when maternal mRNA or mitochondrial DNA influences early stages of embryonic development • Pathologic epigenetic changes are non-sequence-based alterations, which interrupt gene function and cause disease. Examples include: - Hypermethylation of promoter regions for tumorsuppressor genes
Fig. 4. Epigenetic effects are stable changes in gene expression not due to genomic mutation. One important epigenetic process is gene silencing, the phenomenon of gene "switching off" by any mechanism other than genetic mutation. DNA methylation is the most frequent mechanism for gene silencing. This process impedes transcription by adding a methyl group (purple dots) to cytosine (larger blue dots) in the promoter region of a gene to eliminate its expression.
Other Epigenetic Processes • Paramutation is an interaction between two allele s of a single locus, resulting in a heritable change of one of the allele s. Thi s phenomenon, which violates Mendel' s law of independent transmi ssion of traits , allows for varying penetrance or continuous variation of a monogenetic trait • Bookmarking is an epigenetic mechanism for transmitting cellular memory of gene expression pattern to daughter cells. This biologic phenomenon causes daughter cells to maintain the phenotype of a cell lineage • Imprinting causes a subset of all the genes to be expressed according to their parental origin • X-chromosome inactivation is a DNA methylation process causing one of the two X-chromo somes in a female mammal to be randomly inactivated. This compensates for the gene dose doubling of X-chromosome genes in females • Position effect is the expression effect of gene location in a chromosome. Expression is often changed by translocation • Reprogramming refers to demethylation and reestablishment of DNA methylation during mammalian fetal development • Transvection is an epigenetic interaction between an allele on one chromosome and the corresponding allele on the homologous chromosome. This interaction may lead to either gene activation or repression
- Hypomethylation of promoter switches on oncogenes - Histone modification causing heterochromatin in regions of tumor-suppressor gene loci - Loss of gene imprinting is associated with many pediatric tumors. Loss of IGF2 imprinting accounts for half of all Wilms' tumors • Epigenetic carcinogens are not mutagenic but result in increased incidence of tumors . Many of these have previously been classified as chemical carcinogen promoters . Examples include diethylstilbestrol , arsenite, hexachlorobenzene, and nickel compounds • Teratogens influence fetal development by epigenetic mechanisms - Reversibility of epigenetic changes expo ses pathologic epigenetic change s to possible cure by targeted therapy • Examples of "epigenetic therapy" include : - DNA methyltran sferase inhibitors • Nucleoside analogs with a modified cytosine ring that is resistant to DNA methylation • Non-nucleoside analogs that inhibit DNA methylation by binding to the catalytic region of the methyltransferase enzyme - Histone deacetylase inhibitors • Short-chain fatty acids that inhibit cell growth and induce apoptosis by inhibiting histone deacetylase • Hydroxamic acids (R-CONHOH) are potent inhibitors of histone deacetylase. They induce cell differentiation and inhibit proliferation • Cyclic tetrapeptides are irreversible histone deacetylase inhibitors • Benzamides are anti-tumor agents that bind to the catalytic region of histone deacetylase - Epigenetic therapies may be applicable whenever aberrant heterochromatic regions in tumor chromosomes involve tumor-suppressor genes or other critical regulatory genes
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Molecular Genetic Pathology
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DNA METHYLATION Overview • DNA methylation is an epigenetic chemical modification of DNA which impedes transcription of a gene. Methyl groups are added to the number 5 carbons of cytosine pyrimidines in a CpG island (Figure 5) • CpG islands are DNA regions with frequent cytosineguanosine dinucleotide pairs in the 5/-3/ direction. CpG islands mark the start of about 50% of human exons • Only cytosines in CpG dinucleotides are methylated - Only I % of DNA bases are subject to DNA methylation
• However, an overall methyl deficit is observed in tumor cells consistent with global hypomethylation of tumor cell DNA and increased expression of many nonsuppressor genes - This occurs despite high levels of DNA methyltransferase expression - Hypomethylation leads to increased gene transcription, frequently causing increased expression of oncogenes - Hypomethylation induces chromosomal instability (CIN)
In adult somatic tissues, DNA methylation typically occurs only in CpG dinucleotide pairs
• Hypermethylation and hypomethylation are frequently found in promoter regions of genes in cancer cell
Non-CpG methylation is prevalent in ESCs
• Changes of global level and regional methylation patterns are among the most frequent and earliest events to occur in cancer
When a CpG site is methylated, it is methylated on both strands In many disease processes, including familial cancers, gene repression due to promoter hypermethylation is a heritable trait
DNA Methylation Enzymes • In humans , the process of DNA methylation may be carried out by one of three isoenzymes: - DNA methyltransferase 1 (DNMTl) - DNA methyltransferase 3a (DNMT3a) - DNA methyltransferase 3b (DNMT3b)
DNA Methylation and Genetic Regulation • DNA methylation is an important contributor to gene silencing - Methylation of DNA may itself physically impede the binding of transcriptional proteins to the gene, blocking initiation of transcription The methylated DNA binds proteins known as methyl-CpG-binding domain proteins (MBDI), which recruit additional proteins to the promoter and silence transcription DNA methylation also affects histone modification and chromosome structure, which can alter gene expression
DNA Methylation and Cancer • Many tumor-suppressor genes in cancer cells are silenced by hypermethylation of CpG islands • The high incidence of C-to-T transitions found in the p53 tumor-suppressor gene is attributed to the spontaneous deamination of 5-methylcytosine residues (Figure 6) • Observations linking DNA methylation to cancer also suggest a model in which there is a high rate of mutation
192
at CpG dinucleotides due in part to methyltransferasefacilitated deamination
• Both mutational and epigenetic changes alter DNA methylation and have a direct impact on neoplastic transformation
Methylation and Potential Clinical Interventions • One of the most characteristic features of cancer is the inactivation of tumor-suppressor genes by hypermethylation of the CpG islands located in their promoter regions • Compounds with DNA-demethylating capacity have potential for treatment of cancer • Discovery of DNA-demethylating capacity was a decisive event in the clinical trials that has merited the approval of 5-azacytidine by the US Food and Drug Administration for the treatment of myelodysplastic syndrome
Methods for DNA Methylation Analysis Polymerase Chain Reaction-Based Methods • Methylation-specific polymerase chain reaction (PCR) is a frequently used method for studying DNA methylation (Figure 7) - Genomic DNA is modified using sodium bisulfite to deaminate non-methylated cytosine to uracil prior to PeR - The methylated cytosine in genomic DNA cannot be converted - Methylation-specific primers are designed according to non-convertible sequences - Only methylated sequences can be amplified by methylation-specific primers • PCR amplifies bisulfite-modified genomic DNA using strand-specific primers and the PCR product is sequenced to determine the uracil content that represents nonmythylated cytosine
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Conceptual Evolution in Cancer Biology
Thymine
5-Methylcytosine 5' - CpG -
3'
Fig. 6. Spontaneou s deamination of 5-methylcytosine is responsible for the high incidence of C-to-T transitions found in the p53 tumor-suppressor gene in malignancy. When an amino group is removed from 5-methylcytosine, the base changes from cytosine to thymine. In DNA, this reaction cannot be corrected by the DNA repair mechanisms.
3' - G pC - 5'
Fig. 5. DNA methylation is a chemical modification of cytosine pyrimidines in a CpG island in which a methyl group is added to the number 5 carbon of the base. Methylated DNA generally is "turned off" and does not undergo transcription.
A
Genomic DNA isolation
,...
+ + +
Bisulfite treatment
PCR amplification with either U or M primers
+ +
-
Bisulfite treatment
NNNNNNNG UGUGGN UGNGN UG (Converted genomic DNA)
4
Methylated primers(M) design CH3C H3
I I
CH3
I
CH3
I
NNNNNNNGCGCGGNCGNGN CG (Methylated genomic DNA)
Gel electrop horesis
Normal U M
NNNNNNNGCGCGGNCGNGN CG (Non-methylated geno mic DNA)
!
I I
Primer design
Non-methylated primers(U) design
!
Bisulfite treatment
NNNNNNNGCGCGGN CGNGN CG (Non-convertible genomic DNA)
Tumor U M
-
B
Cytosine
Uracil
Fig. 7. Methylation- specific PCR is a comm only used method for DNA methylation analysis. Genomi c DNA is extracted from tissue. Chemi cal conversion of any unme thylated cytosine to uracil (U) by hydroquinone and sodium bisulfite, through which all non-methylated cytosine bases can be identified (panel B). Methylation specific primers are designed according to the uracilcontaining sequences after bisulfite conversion. Methylcytosine resists deamination by sodium bisulfite, so any methylcytosine bases are unchanged in the reaction mixture . All the non-methylated cytosine would be converted into Uracil. Therefore, specific primers for methyl ation of non-methylated sequences are designed (right panel A). Specific primers for non-methyl ated sequence cannot amplify methylated (non-convertible) DNA; and the primer for methylated sequence cannot amplify converted (nonmethylated) sequences. The gel electroph oresis shows the different methylation states of tissue. In the example, normal tissue exhibits a non-meth ylated (U) PCR product whereas the tumor tissue shows a methylated (M) PCR product (panel A).
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• Bisulfite PCR followed by restriction analysis - The amplified bisulfite-modified region contains sites that are cleaved by restriction enzymes specific for uracil-guanosine-containing sequences • Real-time PCR allows quantitative analysis of DNA methylation using methylation-specific primers • Methylation-sensitive single-strand conformation analysis - Methylated and unmethylated sequences form different secondary structures and have different electrophoretic mobility
Non-peR-Based Methods • Single nucleotide primer extension uses internal primers to hybridize bisulfite-modified DNA amplicon in the
presence of a special DNA polymerase. The internal primer is extended only if the appropriate deoxynucleotide triphosphate has been added • DNA melting analysis based on melting properties of DNA in solution. Bisulfite modification changes the DNA sequence and lowers the DNA melting temperatures when a significant number of methylcytosines have been converted to uracil • Enzymatic regional methylation assay is a quantitative method for determining the methylation density of DNA region • Methylation-specific oligonucleotide array uses bisulfite-modified DNA hybridized to a methylationspecific microarray to analyze multiple methylation sites
MICROSATELLITE INSTABILITY (MSI) Overview
Implications of MSI
• MicrosateIIites are short repeated nucleotide sequences virtually unique for each individual
• The type of microsateIIite change can be detected only if many cells are affected
• The repeating sequences are usually only 4 or 5 nucleotides in length
• Detectable MSI is thus an indicator of clonal expansion typical of neoplasia
• The length of microsateIIites is also individual-specific and constant within an individual's normal somatic cells
Bethesda Panel for MSI
• DNA repair defects cause the number of repeats to vary in abnormal or transformed cells
• A 1997 National Cancer Institute consensus workshop recommended a 5 microsatellite marker panel for the detection of MSI
Definition and Mechanisms of MSI
• The panel recommended loci include (BAT)-25, (BAT)-26, D2Sl23, D5S346, and Dl7S250
• MSI is the condition of having longer or shorter microsateIIite regions than normal cells of the individual - Alteration in the length of microsatellites due to deletion or insertion of single nucleotides or repeating units is found in tumor DNA when compared with normallgermline DNA from the same locus The DNA instability is due to failure of the mismatch repair (MMR) system to correct errors in the transcription of microsatellite short sequence repeats, mistakes normally fixed by proofreading enzymes Defective MMR genes produce enzymes that cannot correct nucleotide mismatches due to insertion, deletion, or misincorporation of bases during DNA replication
DNA MMR Enzymes and Regulation Factors • Inactivation of the MMR genes, including hMLH1, hMSH2, hMSH6, hPMS, and hPMS2, results in MSI • Epigenetic inactivation of hMLH1 by promoter methylation is also one of the major causes of MSI • Tumors with MSI respond differently to treatments than those without MSI
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• The tumors were divided into three subgroups according to the MSI status - MSI-high (MSI-H): samples with instability in two or more of five markers. The majority of MSI-H tumors are near-diploid and have few karyotypic abnormalities - MSI-low (MSI-L): cancers show instability in only one of the five microsateIIite markers - MSI-stable (MSI-S): cancers show no MSI in any of the five markers. Tumors with abnormal cytogenetic analysis are frequently MSI-S • Interpretation of the Bethesda panel - Many colorectal cancers show MSI-H • MSI-H is associated with HNPCC • MSI-H is also found in 15-20% of sporadic colorectal cancers • The presence of MSI-H is associated with a more favorable prognosis - MSI-L cancers mayor may not represent a biologically distinct category • MSI-L cancers have a worse prognosis in some, but not all studies - Cancers with MSI have a significantly better prognosis than those with intact MMR (MSI-S)
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Conceptual Evolution in Cancer Biology
MSI in Hereditary Non-Polyposis Colorectal Cancer (HNPCC) (See Chapter 18 for Detailed Description)
N
T1
T2
• HNPCC is a hereditary cancer syndrome associated with increased risk for cancer, predominantly colorectal cancers • Defects in the DNA MMR system result in accelerated accumulation of mutations affecting critical genes that lead to malignant progression • The syndrome is caused by germline mutation in hMLHI and hMSH2 in 90% of cases - Mutation in one DNA MMR gene allele is inherited in the germline - MSI follows only if there is inactivation of the other MMR allele - MSI also occurs in the absence of germline MMR mutation as the result of epigenetic inactivation of MMR genes by promoter methylation • Tumors in MMR mutation carriers typically exhibit MSI. These are called MSI-H tumors if MSI is detected in multiple microsatellite markers • 85-90% of HNPCC-associated colorectal cancers are characterized by MSI • MSI status is useful for prognosis and therapeutic decision-making - Many studies have demonstrated improved prognosis for MSI-H cancer relative to the MSI-stable cancer - MSI-positive colorectal cancers are less responsive to fluorouracil (5FU)-based adjuvant chemotherapy
Methods for MSI Analysis • PCR-gel electrophoresis uses isotope-labeled nucleotides and primers designed to amplify microsatellite markers from genomic DNA of normal and tumor cells (Figure 8)
Fig. 8. MSI is the condition of having longer or shorter microsatellite regions than the normal cells of the individual. Alteration in the length of microsatellites due to deletion or insertion of repeating units is found in tumor DNA when compared with normallgermline DNA from the same locus. The figure illustrates a typical MSI pattern. N, normal control; T I, T2 are different tumors from same patient. The figure shows a lengthened upper allele of Tl and a shortened upper allele of T2. MSI does happen in cases with non-informative normal control, since double bands in tumor samples indicate microsatellite alteration of an allele .
- False-negative results are caused mainly by contamination from non-cancer cells. For a reliable MSI analysis at least 70% of the cells examined should be tumor cells • Fluorescence-based methods use labeled primers for coamplification of multiple markers subsequently separated by capillary electrophoresis • Denaturing high-performance liquid chromatography uses high-performance liquid chromatography instead of gel electrophoresis to analyze PCR amplified microsatellite DNA from tumor and normal cells • Real-time PCR followed by melting point analysis reveals alterations in the length of repetitive sequences within 2 hours
CHROMOSOMAL INSTABILITY (CIN)
Overview • CIN is a state of continuous new chromosome structure formation at a rate higher than in normal cells • CIN is not synonymous with aneuploidy • Neither cytogenetic complexity nor cytogenetic heterogeneity per se is conclusive evidence for CIN • CIN describes mutation at the gross chromosomal level, including structural and numerical instability
Mechanisms of CIN • CIN involves the number and structure of chromosomes, including chromosomal losses, gains, and rearrangements
• Structural rearrangement is caused by DNA damage and recombination - DNA double-strand breaks can result in a number of different structural rearrangements, including translocations, inversions, ring chromosome formation, insertions, and deletions - Downregulation or inactivation of cell cycle checkpoint systems, such as p53 and p21, allow a cell with damaged DNA to escape from apoptosis - Since telomere caps normally prevent crossing over of chromosome ends with chromosome mid-portions , telomere dysfunction results in segmental gains or losses of chromosomes by telomeric fusion between chromosomes
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• Break-fusion-break cycle structural abnormalities - Dicentric chromosomes have two centromeres, so they are pulled apart by the spindle during mitosis, producing two chromosome fragments with uncapped ends - The two broken chromosome ends often fuse into novel dicentrics and rings, which break again at the next cell division - Concurrent breaks in two different chromosomes may give rise to either translocations or dicentrics - Centromere malfunctions lead to numerical instability (aneuploidy) with gains or losses of entire chromosomes (Figure 9) • Asymmetrical segregation of chromosomes at the metaphase-anaphase transition - Abnormal number, structure , or function of the centrosome, consisting of the spindle apparatus and centrioles, leads to asymmetrical segregation of the chromosomes - An abnormal number of centrosomes or mitotic spindles causes uneven distribution of the chromosomes during mitosis - Failure of the centromere to bind the mitotic spindle leads to permanent loss of the chromosome in the next cell division • Inactivation of genes that control the timing of mitotic chromosome segregation results in some chromosomes being "left behind" • Failure of genome surveillance machinery, such as BRCAl-associated genome surveillance complex, allows cells with gain or loss of large genetic segments to escape apoptosis
Clinical Implications of CIN • Tumors with CIN tend to be aneuploid • Tumors with CIN are mostly microsatellite stable • Most malignant tumors show structural and numerical chromosome abnormalities • CIN and cancer - Almost all cancer cells have gains or losses of chromosomes with frequent rearrangements. However, scientists have argued for nearly a century about whether this abnormality is the cause of cancer or merely collateral damage - In most cancers tumor cells share similar cytogenetic abnormalities, indicating a stepwise accumulation of chromosomal changes has occurred during tumor growth - CIN appears to be both an epiphenomenon and a cause of cancer - CIN produces other traits favoring genomic variation, and inviting rapid selection for deathless phenotype
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Fig. 9. CIN is a state of continuous chromosome addition , deletion, or rearrangement at a rate higher than normal cells. CIN is mutation at the gross chromosomal level, including both structural and numerical instability. The figure shows a typical picture of multiple gains and losses by chromosomal painting. Chromosomes with different color bands or arms have had insertions or translocations from other chromosomes. Color coding allows the cytogenetecist to quickly identify pairs of chromosomes. If paired chromosomes have different lengths, there has been a deletion from the shorter chromosome resulting from chromosome instability.
CIN Syndromes • CIN syndromes are a group of inherited conditions associated with CIN and breakage. Each is associated with a tendency to develop certain types of malignancy - Ataxia-telangiectasia is a primary immunodeficiency disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, progressive cerebellar dysfunction, and recurrent pulmonary infections . About 20% develop cancer, usually acute lymphocytic leukemia or lymphoma Bloom syndrome is a rare inherited disorder characterized by CIN because of a mutation in the BLM gene which codes for a DNA heIicase protein essential for maintaining genomic stability during DNA unwinding for replication. Affected individuals have a 150--300 times increased risk of malignancy, usually acute leukemia, lymphoma, or gastrointestinal cancer - Nijmegen breakage syndrome is a recessive syndrome characteri zed by CIN due to mutations in the complex that manages double strand DNA breaks. It is characteri zed by microcephaly, short stature, immunodeficiency, radi ation sensitivity, and a strong predisposition to lymphoid malignancy
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Conceptual Evolution in Cancer Biology
- Fanconi anemia is an autosomal recessive condition associated with bone marrow failure and high sensitivity to DNA cross-linking chemicals . About 10% of patients develop leukemia, 6% myelodysplastic syndrome , and 10% solid malignancies of liver, esophagus, and vulva - Xeroderma pigmentosum is a defect in the nucleotide excision repair gene that renders these individuals exquisitely sensitive to ultraviolet radiation. They have a lOoo-fold increase in non-melanoma skin cancer. After coauthoring the original article describing this syndrome in 1874, Kaposi went on to name the condition in 1882 for the dry, pigmented skin changes usually seen from infancy in persons with this genetic syndrome
Methods for CINAnalysis • Metaphase karyotype analysis. Dividing cells with condensed chromosomes are swollen in hypotonic solution and gently burst open to deposit the chromosomes together on a slide • Banding karyograrn analysis. The chromosomes may be studied in greater detail by enzymatic digestion and special stains to reveal condensed and loose bands of the chromosomes • Fluorescence in situ hybridization (FISH) chromosome painting. A cocktail of fluorescent-labeled probes incubated with the chromosomes allows even more specific identification of specific chromosome segments, even if misplaced on the wrong chromosome • Comparative genomic hybridization. Equal amounts of normal and tumor DNA tagged with different fluorescent dye-binding molecules are allowed to hybridize. Special computer software detects zones of mismatch in the chromosomes after painting (Figure 10)
Fig . 10. The figure is a typical comparative genomic hybridization of tumor DNA . Chromosome spreads from a normal individual are painted with one color fluorescent probe (usually red). Tumor DNA painted with a different color probe mixture (usually green) is layered over the normal chromosomes and allowed to hybridize. DNA with neither red nor green probe painting binds a blue fluorescent dye. Loss of tumor DNA yields more red color, whereas amplification of tumor DNA results in more green color. When red and green are about equal, the net color appears yellow. Computer software is used to quantitatively analyze the gains and losses for each chromosome region. This technique is complementary to chromosome painting or banding, since it fails to show inversions, reciprocal translocations, or changes without gain or loss of DNA.
GENE IMPRINTING
Overview • Gene imprinting is an epigenetic phenomenon that results in a functional difference between homologous mammalian chromosomal regions based on their parental origin • Establishment of imprinting at a locus requires that the two alleles be differentially marked in oogenesis and spermatogenesis • The imprinting process is controlled by other genes , named imprinting centers, typically located on the same chromosome near the imprinted genes • Parental allele-specific expression involves a small subset «100 genes) of all the genes (about 30,000 genes) in the genome expressed according to their parent of origin
• The pattern of parental allele-specific expression is stably transmitted during cell division . Some imprinted genes are expressed from a maternally inherited chromosome and silenced on the paternal chromosome; while other imprinted genes show the opposite expression pattern • Hypermethylation on one of the two parental alleles is the major mechanism for most imprinting. Differential methylation of CpG island promoters causes one parentderived allele to have higher expression than the other, based on arrival through ovum or sperm • Selective gene silencing by hypermethylation of CpG island promoters or by RNAi directed toward one parental allele now explains some patterns of inheritance which were formerly difficult to classify
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Gametes
I
Erase of old imprint
Male
New imprint
I Gametes
Male
A· B
Fig. II . Gene imprinting is an epigenetic phenomenon that causes a functional difference between homologous mammalian chromosomal regions based on their parental origin. Maternal and paternal genomes are differentiall y marked and must be properly reprogrammed every time they pass through the germline to restore full maternal or paternal marking in the gametes . During gametogenesis the primordial germ cell s must have their original biparental DNA methylation patterns erased and re-established based on the sex of the transmitting parent. This process is referred to as reprogramming.
• Currently <100 imprinted genes (about 75) have been identified, most of which are protein coding genes
imprinting and gamete-specific differential gene silencing
• Reprogramming refers to erasure and re-establishment of DNA methylation during gamete development. Reprogramming occurs in the parent germ cell when egg or sperm is maturing (Figure 11)
• DNA rich in methylated CpG islands is associated with hypoacetylated histone cores and increased histone HI • DNA containing unmethylated CpG islands is associated with hyperacetylated histone cores and less histone HI
- During gametogenesis the primordial germ cells must have their original biparental DNA methylation patterns erased and re-establ ished based on the sex of the transmitting parent - The maternal and paternal genomes are differentially marked and must be properly reprogrammed every time they pass through the germline - Reprogramming is achieved through re-establishment of DNA methylation in regulatory sequences for imprinted genes - Reprogramming results in gene silencing based on the sex of the parent - Phosphorylation or other chemical modification of histone proteins also contributes to gene
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• Histone core acetylation modulates the expression of numerous genes
Regulation of Gene Imprinting • The imprinting control region is associated with regulation of imprinted genes • The imprinted region is differentially methylated on one allele - Binding of non-mythelated imprinting control region by zinc finger proteins forms a chromatin barrier
Conceptual Evolution in Cancer Biology
H19 Gene Imprinting
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• Functional grouping of genes modulated by H19 RNA indicates that cellular migration, angiogenesis, and metastasis are favored by H19 imprinting
• Prader- Willi (PWS) and Angelman syndrome (AS) - PWS and AS are two clinically distinct genetic diseases associated with genomic imprinting on chromosome 15qII-q 13 In 70% of the cases of the PWS, there is a genetic deletion of proximal chromosome 15qll-q13 • The deletion is inherited from patient's father • The genetic information in 15q II-q 13 derives only from mother In 70% of the patients with AS, there is also a genetic deletion of proximal chromosome 15qll-q 13 • The deletion is inherited from patient's mother • The patients with AS have genetic information in 15q11-q13 only derived from their father Both paternal and maternal deletions alter SNRPN (small nuclear ribonucleoprotein polypeptide N) promoter methylation and prevent expression of its paternal allele
• Parentally methylated regions in the germline are present upstream of the H 19 promoter in normal cells
Imprinting and Human Cancer
• Mutation in the methylation region for maternal H19 gene locus causes silencing of H 19 gene
• Numerous tumors are associated with the preferential loss of a particular parental chromosome, indicating the involvement of imprinted genes
• H 19 is a non-coding gene with no protein production • The characteristics of H19 gene : - It demonstrates maternal monoallelelic expression in fetal tissue
- H19 is expressed in 33 types of cancer - It is the first designated oncofetal RNA - It functions as a riboregulator causing both posttranscriptional-increased oncogene expression and decreased tumor-suppressor mRNA effective as a template
- H 19 enables tumor cell survival under stress conditions by promoting angiogenesis and cancer progression
• H19 is mapped to Ilpl5.5
Imprinting and Disease • Disturbance in the epigenetic process is an important reason for imprinting disease even when the patient has an intact DNA gene related to the imprinting disease • The imprinting diseases may also be caused by gene mutations or microdeletions in some cases • Alterations of imprinting patterns are found in a number of cancers
Imprinting Syndromes • Beckwith-Wiedemann syndrome (BWS) - BWS maps to Ilpl5
BWS is characterized by general overgrowth with symptoms including hemihypertrophy, macroglossia, and visceromegaly 10-20% of BWS individuals are predisposed to embryonal tumors , the most frequent of which are Wilms' tumor and adrenocortical carcinoma The most common molecular event occurring in BWS patients that do not have cytogenetic abnormalities is the biallelic expression of 1GF2 due to loss of imprinting Loss of imprinting at the 1GF2 locus may be accompanied by the methylation and/or silencing of the active maternal allele of H19
- Neuroblastoma is associated with imprinting disorders of maternal chromosome Ip36 and paternal chromosome 2 - Acute myeloblastic leukemia is associated with an imprinting disorder of paternal chromosome 7 - Wilms' tumor is associated with maternal chromosome Ilp15.5 • 70% of Wilms' tumors have biallelic 1GF2 expression • Loss of imprinting at the 1Gn gene in Wilms' tumor could result from loss of H 19 expression - Rhabdomyosarcoma is associated with imprinting alterations of maternal chromosome Ilp15 .5 - Sporadic osteosarcoma is associated with imprinting alteration of maternal chromosome 13 - Imprinted genes can be involved in carcinogenesis in several ways • Loss of heterozygosity at an imprinted region may result in deletion of the only functional copy of a tumor-suppressor gene • Loss of imprinting of a gene may lead to inappropriate expression of an imprinted gene that promotes cell growth • Inactivation of an imprinting center results in the aberrant expression of multiple imprinted oncogenes or tumor-suppressor genes present in an imprinted chromosomal region • Since imprinted genes are functionally haploid, they are susceptible to tumorogenic factors causing inactivation of the one allele that would eliminate tumor-suppressor gene expression
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MicroRNA (miRNA) Overview • What is microRNA (miRNA) ? - miRNAs are single-stranded, non-coding RNAs, which are 22 nucleotides in length - miRNAs regulate the expression of target genes by interfering with complimentary sites in the 3' UTR of target mRNA - miRNAs are encoded by DNA and transcribed from DNA but not translated into protein • miRNA transcription and processing - miRNA is first transcribed as a long primary transcript (pri-MIR) - Pri-MIR is subsequently processed into a 60-120 nucleotide precursor with a hairpin (stem loop) structure (pre-MIR) - Pri-MIR is processed in the cell nucleus - This processing is performed by the multi-subunit microprocessor complex, consisting of a nuclease (Drosha) and a double-stranded RNA (dsRNA)binding protein (Pasha) - Human pre-MIR molecules contain similar structural regions called "basal segments," "lower stem," "upper stem," and "terminal loop" - Most pre-MIR molecules have an imperfect dsRNA structure topped by a terminal loop - Drosha and Dicer components of the microprocessor complex are members of ribonuclease III enzyme family that recognize structural regions of pre-MIR molecules - Processed pre -MIRs are exported to the cytoplasm where they are further processed into mature miRNA - miRNA functions as a gene regulator - miRNA controls proliferation, differentiation, development, apoptosis, and stress response - miRNA is complementary to a part of one or more mRNAs (Figure 12) • Animal miRNAs usually anneal to a site in the 3' UTR • Plant miRNAs usually anneal to coding regions of mRNAs • Each miRNA may interact with multiple genes • Each mRNA is complementary to multiple miRNA s • miRNA bound mRNA either remains untranslated or is degraded by RNAi effecter complex (RNAinduced silencing complex) • Post-transcriptional gene repression is the basic function of miRNA. The annealing of the miRNA to the mRNA
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inhibits protein translation, and sometimes facilitates cleavage of the mRNA (Figure 12) - miRNAs may also increase methylation of genomic sites corresponding to targeted mRNAs - miRNA function requires several protein s collectively termed the miRNP - miRNA activity may be blocked by a locked nucleic acid oligo, morpholino oligo, or a 2'-O-methyl RNA oligo • RNA genes - RNA genes encode RNA that is not translated into a protein - The human nuclear genome contains about 3000 unique RNA genes (<10% of total gene number) - The human mitochondrial genome contains 24 RNA genes: two code for 23S and 16S rRNA subunits of mitochondrial ribosomes, the rest code for tRNAs
Clinical Implications • miRNA-related diseases - miRNA regulates insulin secretion from ~ cells, and may be involved in some cases of diabetes mellitus -
miRNA encoded by viruses suggests a potential role for miRNA in the viral infection cycle
• miRNA and cancer - miRNA tumorigenesis cluster located on 13q31 is amplified in several lymphomas and other cancers - miRNAs may cause post-transcriptional downregulation of tumor-suppressor gene expression and/or upregulation of oncogene expression - Mice engineered to produce excess lymphoma cell miRNA developed the disease within 50 days and died 2 weeks later - Two types of miRNA inhibit the E2Fl protein, a critical regulator of cell proliferation - Expression patterns of 217 miRNA genes reveal gene activity fingerprints that can distinguish developmental lineage and differentiation state of cancers • Potential miRNA-based therapies - miRNA directed at oncogenes and tumor-suppresser gene silencers may be therapeutic against cancer. For example , miRNA let-7 represses Ras oncogene and significantly inhibits the growth of lung cancer, and miRNA miR-15a and miR-16-1 repress bcl-2 oncogene and induce apoptosis in leukemic cell lines - Antagomirs (modified antisense miRNA) , neutralizing miRNAs, are present in all tissues except brain . Antagomirs block and degradate target miRNA, allowing silenced gene reactivation
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Conceptual Evolution in Cancer Biology
DNA
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Fig. 12. Post-transcriptional gene repression is the basic function of miRNA. miRNA is transcribed from RNA genes and processed to miRNA. The miRNA molecules interact with the 3' UTR of specific mRNA to suppress translation or to induce mRNA degradation. siRNA is dsRNA resembling viral RNA with similar effects toward post-transcriptional gene repression .
Methods for miRNA Analysis • Computation-driven analysis identifies candidate miRNA sequences based on known structural features. The candidate sequences must be validated by experiment • miRNA target analysis identifies "seed" nucleotide sequences that are complementary to known
mRNA 3' UTR sequences. Binding studies and functional analysis are essential to confirm true mRNA targets • De novo identification of miRNA candidates involves sequencing of size-fractioned cDNA libraries to identify 22-nucleotide RNA molecules with hairpin sequences
RNA INTERFERENCE (RNAi)
Overview
- Silencing mRNAs that are overproduced or translationally aborted
• RNAi is a mechanism conserved in related eukaryotic organisms that preserves genomic integrity, regulates gene expression, and guards against exogenous virus infection
- Guarding the genome from mutation induced by relocation of mobile genetic elements (transposons)
• RNAi is triggered by dsRNA and causes sequencespecific mRNA degradation
- RNAi machinery is involved in miRNA processing and the resulting translational repression
• The mediator of RNAi is small interfering RNA (siRNA), produced from long dsRNA by enzyme cleavage - siRNA is a dsRNA with 2-nucleotide 3' overhangs on either end - Each strand has a 5' phosphate group and a 3' hydroxyl (-oH) group • Biologic function of siRNA - Antiviral defense
• siRNA induced post-transcriptional gene silencing - siRNA interferes with the expression of a particular gene in many eukaryotes sharing a common homologous sequence RNAi is mediated by the same cellular machinery that processes miRNA - siRNA molecules are involved in large-scale gene regulation in the cell
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An siRNA
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siRNA-med iated target recogn ition
mRNA m7G (A)n
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Fig. 13. siRNAs can be either endogenous or exogenous. siRNA is a 19 nucleotide dsRNA with 2-nucleotide 3' overhangs on either end (upper panel). Each strand has a 5' phosphate group (-P) and a 3' hydroxyl group (-OH). The dsRNA chains are processed into siRNA molecules that form siRNA-protein complexes . The siRNA-protein complex can interact with mRNA to cause mRNA degradation. M7G indicates the cap site of the mRNA and (A)n refers the poly A tail.
- Before RNAi was well characterized, the phenomenon was known by other names, including posttranscriptional gene silencing, transgene silencing, and quelling (Figure 13) - siRNA is bound in the RNA-induced silencing complex and uses one of the strands as a guide to target complementary mRNA • The differences between miRNA and siRNA (Table 3)
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Biologic and Clinical Implications of siRNA • The application of siRNA - siRNA's dramatic and selective reduction of an individual protein expression makes it a valuable research tool, both in cell culture and in vivo - Post-transcriptional silencing triggers assembly of a nuclease complex that targets homologous mRNAs for degradation
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Conceptual Evolution in Cancer Biology
Table 3. Differences Between miRNA and siRNA miRNA
siRNA
Gene coding
Coded by RNA genes
Not coded by RNA genes
Structure
Single stranded
Double stranded
Processing
Post-transcriptional processing
Processing of long bimolecular RNA duplexes
Main function
Regulate gene expression
Post-transcriptional repression
Sequence conservation
miRNA sequences are conserved
siRNA sequences are not really conserved
- siRNA gene silencing may be amplified by priming the synthesis of additional dsRNA via RNA-directed RNA polymerase - siRNA can trigger transcriptional alterations at the genomic level by inducing methylation at sites of sequence homology • Role of miRNA in medicine - siRNA has been used in clinical trials for treating macular degeneration and respiratory syncytial virus - RNAi can effect the complete reversal of virusinduced liver failure in mouse models
- miRNA may inhibit viral gene expression in cancer cells - miRNA may stabilize neurodegenerative disea ses, with particular attention to the polyglutamine disease s such as Huntington's disease • Potential risk of therapeuti c siRNA in vivo - Introduction of too much siRNA can result in nonspecific activation of innate immune responses - Off-target effects may result in essential genes coincidentally similar to the targeted gene also being repressed
TELOMERE
Overview • What is a telomere? - The telomere is a ribonucleoprotein complex composed of at least seven proteins and an RNA primer sequence bound to repetitive sequence DNA at the ends of the p and q arms (Figure 14) - Telomeres are repeating simple sequences that belong to the minisatellite family (-TTAGGG-) - The repeat block extend s 10-12 kbp in length • The biologic function of telomeres - Confer stability and protect chromosome ends • The basic function of telomeres is to seal the end of chromosome arms to protect them from exonuclease degradat ion • The telomere acts to protect the ends of chromosomes from fraying or ligation to unprotected DNA • Telomeres function as a disposable buffer to protect chromosomes from gene loss during division • Telomere s also associate with other telomere s at the nuclear envelope during interphase to maintain an ordered structure of the chromatin - Count the number of cell divisions • Telomeres judge the number of cell divisions that have occurred
• Telomere determines the cellular life-span and dictate s when replicative senescence will occur - Provide a mechani sm for complete replication of DNA at the ends of chromo somes • Discontinuous replication of the lagging strand involves Okazaki fragments and loss of bases at the 3' end (Figure 16) • Telomerase adds hexamer repeat s to 3' ends, allowing DNA polymerase to complete synthesis of the laggin g strand without losing bases from coding genes
Telomere Structure and Maintenance • Telomeres are composed of both repeated DNA and specific DNA-binding proteins • Telomere s contain double- stranded DNA in a closed loop with the 3' overhang annealing to a complementary segment of the second strand • The telomere loop (T-loop) is formed by this annealing of the DNA end back on itself - T-loop formation is catalyzed by a specific enzyme - The loop is stabilized by a protein complex - The overhang is buried inside the loop; the ends will not be recognized as a break in the double strand
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Cell Senescence and Telomere Shortening
Centromere
Fig. 14. The telomeres are located on both ends of the chromosome arm s and function as a disposable buffer. Telomeres protect the chromo some from gene loss by ring chromosome formation or gain by non-reciprocal translocation during cell division.
- When the T-loop structure is disturbed, the growth of the cell is arrested and cell cycle checkpoint protein s p53 and pRb are activated • If the cell cannot pass these checkpoints the growth of the cell is arrested permanently and cell senescence occurs • If the cell cannot pass the p53 checkpoint, apoptosis occurs • If the check point s are bypassed the cell grows continuously and indefinitely resulting in genomic instability and risk of malignancy • The displacement loop (D-Ioop) is formed by the 3' Grich strand extension overhang invading the duplex telomeric repeat s (Figure 15) - The D-Ioop is about 200 bp in length • Catalytic component:
• The number of cell cycles a cell may complete is limited by telomere length • Telomere s shorten as cells repeatedly divide without primers of the lagging strand (Figure 16) • When eventually the shortening telomere reache s a senescence breakpoint, irreversible growth arrest, or apopto sis will occur • Shortened telomeres also increa se the risk of chromosomal fusions • Cell senescence arrests the growth of aged cells and prevents further mutation accumulation, thus reducing the risk of malignant transformation • Shorter telomeres are associated with shorter lives. Among people older than 60, those with shorter telomeres were three times more likely to die from heart disease and eight times more likely to die from infectious disease - Telomere shortening places a limit on replication capacity of cells - Telomere shortening can initiate CIN, which is a major driving force in malignant transformation - Long telomeres are a significant barrier to cancer formation - Telomere shortening associated with aging explain s increa sing cancer risk with age • Telomere extension - Telomere extension is catalyzed by an enzyme, telomerase - Telomerase extend s telomere s with its own octamer primer to add repeat -GGTTAG- sequences (Figure 17) - Telomerase is only active in germ cells of most multicellular eukaryotes to restore youthful telomere length in gametes - Telomerase -independent pathway (ALT) • ALT is active in 15% of the telomerase negative neoplasias • The ALT pathway is preferentially active in mesenchymally derived cells, compared with those of epithelial origin • Some ALT-positive cells are associated with malfunction of promyelocytic leukemia nuclear body, a large deoxyribonucleoprotein complex essential for chrom atin remodeling in resting cells • ALT invol ves homologous recombination between telomeres. Sequences are copied from one telomere to another by complementary annealing as a means of priming new telomeric DNA
- Human telomere reverse transcriptase (hTERT) synthesizes DNA from an RNA template in the hTERT telomerase complex - It is not expres sed in most somatic cells - Reverse transcription by hTERT synthesizes telomeric sequences lost during cell division
• Shortened telomeres are found in many cancers, including pancreatic, bone, prostate, bladder, lung, kidney, and head and neck cancers
- hTERT activity is a critical factor in stabilizing telomeres through addition of TTAGGG repeats
• Telomere shortening is associated with genetic instability and increased cancer risk
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Telomere Shortening and Cancer
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Conceptual Evolution in Cancer Biology
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Fig. 15. The structures of D-loop and T-loop are essential components of the telomere. The T-loop is formed at the 3' end of one DNA strand folding back to form a large loop. The D-Ioop is formed by the 3' end binding to the 5' end sequences of the telomere, displacing the normal complementary chain . Loop formation is catalyzed by specific enzymes and is stabilized by the protein complex. The loop structure protects the telomere.
Telomere shortening is prevalent in premalignant lesions from a variety of human epithelial tissues • Cancers with short telomeres often have high telomerase activity • Cancers with short telomeres are likely to respond quickly to anti-telomerase therapy
Telomerase Summary • Telomeraseis a reverse transcriptase (hTERn carrying its own RNA template, repetitively coding the elongatingtelomere • Telomerase is only transiently expressed during S phase in differentiated normal cells Fig. 16. Telomeres shorten during cell division. DNA polymerase requires an RNA primer to initiate synthesis in the 5'-3' direction. DNA polymerase can synthesize the leading strand to the end of the chromosome. However, for the lagging strand, DNA synthesis produces a series of fragments, each requiring an RNA primer. When DNA ligase joins the fragments, it must have an adjacent 3' DNA end to complete the conversion of the RNA primer. Since the last RNA primer has no joining DNA, a portion of the lagging strand of telomere is lost, resulting in telomere shortening.
• Telomerase adds specific DNA sequence repeats (TTAGOG) to the 3' end of DNA strands in the telomere regions • Inhibition of telomerase reduces cell proliferation and accelerates loss of the telomere 3' overhang • Cancer cells with shortened telomeres overcome the checkpoints by expression of telomerase to re-establish their telomeres • Telomerase provide s cancer cells with at least two critical functions :
- It suppresses CIN - It grants unlimited cell replication
- The mean telomere length in cancer cells is about 5 kb - Telomerae lengths between 7 and 9 kb are considered long - Telomere lengths between 3 and 5 kb are considered short • Telomere shortening is also observed in many pre-malignant lesions Marked telomere shortening is observed in 93% of prostatic intraepithelial neoplasia cases, a precursor to prostate cancer
• Telomerase is the key to cellular evasion of senescence and apoptosis in many cancer cell lines - Normally only germline and stem cells have highly active telomerase - Telomerase restores the ability of senescent cells to divide • A variety of premature aging syndromes are associated with short telomeres • Regulation of telomerase - The telomerase gene is located on the distal arm of chromosome 5p (5p1533) - Telomerase is highly active in early embryogenesis
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Molecular Genetic Pathology
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- It is downregulated during cell differentiation and has undetectable expression in differentiated somatic cells - Amplifications of 5p15 is detected in some cancers, suggesting that increased copy number may be one mechani sm for increasing telomerase expression in human tumors - The telomerase gene promoter contain s a number of regulatory sites including two c-myc binding sites, which may contribute to upregulated expression of telomerase. The oncogene c-myc is expressed in a great number of cancers - It has been suggested that the telomerase gene promoter might contain a p53-binding site that negatively regulates telomerase expression . Since the majority of human cancers are deficient in p53 protein , this might also contribute to telomerase overexpression
Telomerase and Cancer • Telomerase is expressed in many cancers but not in normal differentiated cells • Many cancer cells are considered "immortal" because telomerase activity allows them to evade senescence • Activated telomerase precludes death by chromosome instability or senescence-activated induction of apoptosis pathways • Telomerase also promotes survival by continued activation of proliferation pathways
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• Telomerase activation is observed in 90% of all human tumors , suggesting that the cellular imperishability conferred by this enzyme plays a key role in cancer development • Telomerase overexpression accounts for indefinite replication capacity in many malignant tumors • Some cancer cells have alternative lengthening of telomere s (ALT), a non-synthetic telomere lengthening pathway involving the transfer of telomere tandem repeats between sister chromatids
Diagnostic and Therapeutic Implications of Telomerase • Telomerase might playa role for screening and diagnosis of cancer - The telomerase activity is detectable in stage 0-1 breast cancer Telomerase is expressed in most urinary bladder cancers (90%), prostate cancers (80%), and kidney cancers (69%) Application of a FISH telomere assay improved the sensitivity and specificity for detecting malignant cells in cytology specimens in some studies Telomerase test s have shown promising results for improving the sensitivity and specificity of malignant cell screening in urine, pleural and peritoneal effusions, and bronchoalveolar lavage fluid
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Conceptual Evolution in Cancer Biology
anti-neoplastic drugs to induce apoptosis in certain cancer cells • Telomerase inhibition might be useful in the treatmentof any cancer with telomerase expression Telomerase inhibitors suppress telomerase activity and reduce the proliferation rate of lung, breast, liver, and prostate cancer cells Induction of differentiation, inhibition of reverse transcriptase, telomerase promoter downregulation, telomerase primer anti-sense inhibition, and blockage of telomereltelomerase interactions are different approaches for telomerase targeting treatment siRNA anti-telomerase treatmentdecreases telomerase activity and inhibits cancer cell growth in vitro
Methods for Telomere Analysis Fig. 18. FISH with telomere probes shows that each chromosome has fourtelomeres, one on the tip of eacharm. The fluorescence intensity maybe used to assaythe telomere length.
Telomerase also appears to be important as a prognostic indicator. In breast cancer there is a direct correlation between telomerase activity and higher stage, size of tumor, and nodal status Telomerase expression is low in normal peripheral blood lymphocytes and benign lymph nodes. But telomerase expression increases in malignant lymphocytes, which could be helpful for making a distinction between benign and malignant lymph tissue
Anti-Telomerase Therapy • Telomerase-inhibiting compounds would inhibit malignant cell growth, and permit other
• In terminal restriction fragment Southern blot, radiolabeled (AATCCC)n anti-sense oligonucleotide probe against the TTAGGG- telomere motif is allowed to hybridize Hinf/Rsa I digested genomic DNAon a nylon membrane. The hybridized membrane is exposed to autoradiography film and the intensity is converted into relative telomere length • FISH labeled telomeres in cytogenetic metaphase spreads. Fluorescence-conjugated telomere probes bind to the chromosome telomeres. The fluorescence intensity is converted to the relative telomere length based on the ratio with control (Figure 18) • The real-time PCR assay determines the telomereto-single copy gene (TIS) ratio, which is proportional to the average telomere length in a cell • Telomere length is generally measured relative to a control measurement of the average chromosome number, either with a normal cell population from the host or with a single copy gene in the test cell population
SUGGESTED READING Anderson S, Bankier AT, Barrell BG. Sequence and organization of the human mitochondrial genome. Nature 1981;290:457-4 65. Artandi SE, Chang S, Lee SL, et al. Telomere dysfunctionpromotes nonreciprocal translocation and epithelial cancer in mice. Nature 2000;406:641-644. Barlow DP. Methylation and imprinting: from host defense to gene regulation? Science 1993;260:309-310. Bartel DP. MicroRNAs: genetics, biogenetics, mechanism, and function. Cell 2004;116:281-297 .
Bartolomei MS, Zemel S, Tilghman SM. Parental imprinting of the mouse HI9 gene. Nature 1991;351:153-155 . Bird AP, WoltTe AP. Methylation-induced repression-belts, braces, and chromatin. Cell 1999;99:451-454.
Boland CR, Thibodeau SN, Hamilton SR, et al, A National Cancer InstituteWorkshop on Microsatellite Instability for cancer detectionand familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 1998;58:5248-5257.
Brenner S, Jacob F, Meselson M. An unstable intermediatecarrying informationfrom genes to ribosomes for protein synthesis. Nature 1961 ;190:576-581. Cheng L, Sung MT,Cossu-Rocca P,et al, OCT4: Biological functions and clinical applicationsas a marker of germcellneoplasia. J Pathol. 2007;211 :1-9. Clark MF, Fuller M. Stem cells and cancer: two faces of Eve. Cell 2006;124:1111-1115. Egger GL, Aparicio A, Jones PA. Epigenetics in human disease and propects for epigenetic therapy. Nature 2004;429:457-463 .
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Eng ler AJ, Sen SW, Sweeney HL, et al. Matrix elasticity directs stem cell lineage specification. Cell 2006; 126:677-689. Even-Ram M, Artym V, Yamada KM . Matrix control of stem cell fate. Cell 2006 ;126:645-647. Fire A, Xu S, Montgomery MK , et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditi s elegans. Nature 1998;39 1:806--8 11. Friedberg EC. DNA damage and repair. Nature 2003;421:436-440. Hamilton A, Baulcombe D. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 2006 ;286:950-952. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; I00:57-70. Hannon G. RNA interference. Nature 2002 ;418:244-25 1. Herman J , Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med. 2003;349:2042- 2054. Ionov Y, Peinado MA, Malkbosyan S, et al. Ubiquitous somatic mutations in simple repeated sequences reveals a new mechanism for colonic carcinogenesis. Nature 1993;363:558-561. Joeng KS, Song EJ, Lee KJ. Long lifespan in worms with long telomeric DNA. Nature 2004;36:607-{j11.
Molecular Genetic Pathology
Krivtsov AV, Twomey D, Feng Z. Transformation from commi tted progenitor to leukaemia stem cell initiated by MLL-AF9. Natu re 2006;442:818-822. Lapidot T, Sirard C, Vorm oor J . A cell initiating human acute myeloid leukaemia after transplantation into scm mice. Nature 1994;367:645-648. Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancer. Nature 1998;396:634-639. McKay R. Stem cells-hype and hope. Nature 2000;406 :361-364. Nowak MA, Komarova NL, Sengupta A, et al. The role of chromosome instability in tumor initiation. Proc Nail Acad Sci USA 2002;99:16226--1 6231. Polyak K, Hahn W. Roots and stem: stem cells in cancer. Nature Med. 2006; 12:296--300. Redon R, Ishikawa S, Fitch KR, et al, Global variation in copy number in the humane genome. Nature 2006 ;444 :444-454. Reya T, Morrison SJ, Clarke MF, et al, Stem cells, cancer and cancer stem cells. Nature 200 1;4 14:105- 111.
Rcdriguez-Bfgas MA, Boland CR, Hanmilton SR, et al, National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome. J Natl Cancer Inst. 1997;89:1758-1 762. Rugg-Gunn P, Ferguson-Smith AC, Pedersen R. Epigenetic status of human embryonic stem cells. Nat Genet. 2005 ;37:385-387.
Jones PA, Gonzalgo ML. Altered DNA methylation and genome instability: a new pathway to cancer? Proc Natl Acad Sci USA 1997;94:2 103- 2105.
Tan BT, Park CY, Ailles LE , et al, The cancer stem cell hypothesis: a work in progress. Lab Invest. 2006;86: 1203- 1207.
Jordan CT, Guzman ML , Noble M. Cancer stem cells. N Engl J Med . 2006;355:1253- 126 1.
Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of proxima l colo n. Scien ce 1993;260:8 16--8 19.
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8 Clinical Genomics in Oncology Hugo M. Horlings,
MO and
Marc van de Vijver,
MO, PhD
CONTENTS I. I ntroduction Clinical Genomics Human Genome Project Methodologies and Applications of Genomics Limitations
II. DNA Microarray General Micro Array Experiment Isolation and Amplific ation of RNA Different Labeling Methods of Amplified mRNA Quantifying the RNA Expression
III. Gene Expression General Dynamic and Systematic Gene Expression Profiling
IV. Data Analysis of Gene Expression Data Normalization of Gene Expression Data Statistical Analysis Unsupervised Classification Supervised Classification (Knowledge Driven)
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Classifying Tumors Based on "Functional" Gene Expression Signatures Predictive Genomic and Clinico-Genomic Decision Tree Models
V. Application in Diagnostics of Clinical Oncology General Clinical Genomics in Breast Cancer Clinical Genomics in Hematologic Malignancies Clinical Genomics in Prostate Cancer Clinical Genomics in Gastrointestinal Tumors Clinical Genomic s in Carcinoma of Unknown Primary (CUP)
VI. Interpretation of Gene Lists VII. Points of Attention in the Design of Microarray Experiments The Sample The Microarray The Statistics
VIII. Conclusions and Future Directions IX. Suggested Reading
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INTRODUCTION
Clinical Genomics
Methodologies and Applications of Genomics
• Genomics is the study of all nucleotide sequences, including structural genes, regulatory sequences, and non-coding DNA segments, in the chromosomes of an organism
• DNA microarray analysis is the most commonly used method to measure gene expression levels
• Clinical genomics can be defined as the application of large-scale, high-throughput genomics technologies in clinical settings, such as clinical trials or primary care of patients
Human Genome Project • The field of clinical genomics has grown enormously by the elucidation of the full sequence of the human genome and the characterization of all 25,000 human genes as well as the ability of large-scale surveys of gene expression, genetic polymorphisms single nucleotide polymorphisms and DNA copy numbers using microarray-based technologies
• This method has been most successfully applied to characterize human cancers in order to predict clinical outcomes and define clinically relevant subgroups of tumors
Limitations • Most analyses have used gene expression levels to define broad group differences. A consequence, there remains a considerable diversity within these groups, and thus predictions often fall short of providing accurate predictions for individual patients • An extremely important step in obtaining reliable prognostic or predictive gene expression signatures is validation in an independent, sufficiently large cohort of patients
DNA MICROARRAY
General • A microarray is any arrangement of microscopic spots attached to a solid surface such as glass, plastic, or silicon chip (Figure 1) • An array may contain thousands of spots and the corresponding probes attached to the solid support can be the complementary DNAs (cDNA), oligonucleotides of varying length, or genomic sequences • Most laboratories use fluorescent labeling with one or two dyes, Cy3 and Cy5 (excited by a green and red laser) • In dual label experiments two samples are hybridized to a microarray, one labeled with each dye. This allows the simultaneous measurement of two samples (tumor sample vs reference sample, i.e., commercially available platform from Agilent [Santa Clara, CAl [Table 1]) • In single label experiments, only one sample is hybridized to the arrays labeled with one dye . This type of hybridization has been developed by Affymetrix (Santa Clara, CA) (Table 1). For each gene, several different oligonucleotides are present on the array . The hybridization is done with RNA from the
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sample to be analyzed without the use of a reference RNA. Instead, oligonucleotides containing one mismatch in their sequence are used to correct for background hybridization.
Micro Array Experiment • Different steps of a microarray are summarized - Prepare a microarray chip by choosing probes (sequences) Isolate and amplify RNA from tumor and reference sample (Figure 2) and if a reference is used the reference is usually prepared from a mixture of cell lines, tumor samples, or normal tissues Generate a hybridization solution containing fluorescently labeled targets. Antisense cRNA is labeled in the presence of red fluorescent (Cy-5) nucleotides and can then be mixed with a greenfluorescent-labeled reference (Cy-3) (Figure 3A,B) The mixture is added to the microarray and the labeled antisense cRNA can be hybridized to the probes on the microarray
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Clinical Genomics in Oncology
Fig . 1. Printing of a glass slide with 25,000 spots (CMF, NKI, The Netherlands).
-
Detect probe hybridization: • Scan the arra y using a fluore scent scanner and store output as an imag e • Quantify each spot • Subtract back ground • In a dual label experiment the level of fluorescence is digitized and for each probe the level of gene expression , relative to the reference, is determined and tran sferred to a database (Figure 4) • Normalize data • Export a table of fluore scent intensities for each gene
- Analyze exported data - A more detailed description of each step will follow next
Isolation and Amplification of RNA • RNA is isolated totally from tumor sample (fresh frozen, cell culture, or paraffin-embedded tissue) and (if a reference is needed) reference sample (4 ug of total RNA) (Figure 2) •
1st Strand synthesis to generate first strand cDNA by rever se transcriptase with cRNA spec ific primer
• The cDNA is used as template to generate a doublestranded DNA molecule using exogenous primers
• After generation of the double-stranded DNA , it is used as template for an in vitro transcription reaction to generate many copie s of amplified antisense cRNA , which are complementary to the sequence of the original mRNA species • The amplified anti sense cRNA is ready for labeling. If a large amount of sample RNA is used, amplification is not requ ired
Different Labeling Methods of Amplified mRNA • Incorporation of modified nucleotides in cDNA or cRNA can be done using different enzymes (Figure 3A,B) • It is also pos sible to dire ctl y, chemically label DNA, RNA , or nucleotides using univer sal labeling sys tem (ULSTM, Amsterdam , The Netherlands) by forming a covalent bond on the N7 position of guanine • Labeled tumor sample (Cy5) and reference sample (Cy3) are hybridized to a microarr ay (Figure 4A)
Quantifying the RNA Expression • After hybridization of the labeled RNA, a laser scanner is used to excite the hybridi zed array at the appropriate wavelen gth. In a dual label experiment the relati ve abundance of the two transcripts is visualized in colored image by the ratio of the red to green fluore scence inten sities of each spot (Figure 4A,B)
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5000
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34--44mer oligo's
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2005
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Adapted by permission from Macmillan Publishers Ltd.: Abdullah-Sayani A, Bueno-de-Mesquita jM, van de Vijver Mj. Technology Insight: tuning into the genetic orchestra using microarrays-limitations of DNA microarrays in clinical practice. Nature Clinical Practise Oncol. 2006;3(9) :501-516.
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Table 1. Examples of Different Types of Microarrays
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Molecular Genetic Pathology
• Depending on the type of array used, the location and intensity of a color will indicate which gene is expressed • The gene expression ratios are log transformed and placed in a table in which each row corresponds to a gene and
each column corresponds to a single hybridization experiment. This is represented in a so-called "heat map" (Figure 6) • In a single label experiment (Affymetrix), a reference RNA is not required
GENE EXPRESSION General
Gene Expression Profiling
• Gene expression is a broad term used to describe the transcription of information encoded within DNA sequences (exons) into mRNA. It is assumed that for most transcripts there is a linear relationship with translation of the mRNA information into proteins that regulate cell function
• Characterization of gene expression (gene expression profiling) has been used to study infectious and immunologic disease, but the predominant focus has been on the study of cancer
Dynamic and Systematic • The gene expression pattern in any given cell is a highly dynamic and systematic process that alters with cell metabolisms, changes in environment, and in the presence of disease
• Cancer is a genetic disease where the abnormal interaction of several genes results in the development and progression ofa tumor • With gene expression profiling it has been possible to group genes to formulate "Genetic Signatures" that can potentially improve the clinical management of a cancer patient or give more insight in biologic processes of cancer
DATA ANALYSIS OF GENE EXPRESSION DATA Normalization of Gene Expression Data • Experiment normalizations are used to standardize microarray data to enable differentiation between biologic variations in gene expression levels and variations due to the measurement methods • A commonly used normalization method is the locally weighted scatter-plot smoothing algorithm
Statistical Analysis • After normalization the purpose of any data analysis of gene expression data is to group entities on the basis of similarity of features • Clustering (unsupervised and supervised) was one of the first methods to order microarray data • Other methods to analyze gene expression data include principle component analysis, self-organizing maps, and linear discriminant analysis to discover patterns of gene expression • The main methods used to identify categories of tumors based on gene expression profiles are unsupervised and
216
supervised classification (Figure SA,B); examples are given as follows
Unsupervised Classification • One of the most commonly used unsupervised classification techniques is hierarchical cluster analysis. Hierarchical cluster analysis groups genes with similar expression patterns, with the assumption that each cluster of genes is simultaneously regulated • Samples are clustered into groups based on overall similarity of their gene expression profiles • Usually the result of two-dimensional clustering is represented by the combination of a "heat map," showing expression levels of each of the genes combined with a dendrogram (Figure 6) - Example of hierarchical cluster analysis • Perou and Sorlie showed by unsupervisedclassification that similar breast tumors might now be classified into five specific subtypes (Basal, HER2 [HER2/neu or ErbB-2], Luminal A, Luminal B, and Normal-like) according to their distinct patterns of gene expression. Others have confirmed this classification
Clinical Genomics in Oncology
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Fig. 5. Unsupervised and supervised clustering. (A) Unsupervised clustering: tumor samples are clustered into groups based on overall similarity of their gene expression profiles. This approach is useful for class discovery. (B) Supervised clustering: multiple tumor samples from different known classes are used to train a model capable of classifying unknown samples. This model is then applied to a tumor set for class label assignment. (Image from C. Lai with permission.)
Supervised Classification (Knowledge Driven) • Supervised classification is the method of choice for the analysis of gene expression profiles associated with prognosis or therapy response prediction • Multiple samples from different known classes are used to train a model capable of classifying unknown samples. This model is then applied to a tumor set for class label assignment. There are several methods for supervised classification; examples are given as follows Examples of supervised classification • Van 't Veer et al. studied invasive breast carcinomas by analyzing gene expression using an oligonucleotide array containing 25,000 probes • DNA microarray analysis was performed on primary breast tumors of 78 young patients and
supervised classification was applied to identify a gene expression signature strongly predictive of a short interval to distant metastases ("poor prognosis" signature) in patients without tumour cells in local lymph nodes at diagnosis (lymph node negative) (Figure 7) • By performing supervised classification different levels of expression have been found in the 34 patients who developed distant metastases within 5 years compared with the 44 patients with no recurrence in this period • The 70-genes poor prognosis signature consists of genes regulating cell cycle, invasion, metastasis, and angiogenesis • In a subsequent validation study van de Vijver et al. investigated the prognostic power of these 70 genes in 295 patients (151 lymph node
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negative; 144 lymph node positive; age <53 years (Figure 8) • Figure 8 shows that patients with a good prognosis signature had a <15% risk of developing distant metastases over 10 years and a <10% risk of dying. Patients with a 70-gene poor prognosis signature had a 50% risk for distant metastases and a 50% mortality rate
218
Classifying Thmors Based on "Functional" Gene Expression Signatures • Classifying tumors could also be based on functional annotation of gene expression algorithms; an example is provided as follows • Chang et aI. applied this strategy to identify a gene expression signature of a wound-response and tested its role in cancer progression
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Clinical Genomics in Oncology
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Fig. 7. Expression data matrix of 70 prognostic marker genes. (A) Use of prognostic reporter genes to identify optimally two types of disease outcome from 78 sporadic breast tumors into a poor prognosis and good prognosis group. (B) Expression data matrix of 70 prognostic marker genes from tumors of 78 breast cancer patients (left panel). Each row represents a tumor and each column a gene . Genes are ordered according to their correlation coefficient with the two prognostic groups . Tumors are ordered by the correlation to the average profile of the good prognosis group (middle panel). Solid line, prognostic classifier with optimal accuracy; dashed line, with optimized sensitivity. Above the dashed line patients have a good prognosis signature, below the dashed line the prognosis signature is poor. The metastasis status for each patient is shown in the right panel: white indicates patients who developed distant metastases within 5 years after the primary diagnosis ; black indicates patients who continued to be disease-free for at least 5 years. (Adapted by permission from Macmillan Publishers Ltd: van 't Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002;415(6871) :530-536, (c)2002.
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Fig. 8. Validation study investigating the prognostic power of the 70-genes signature. Validation study investigating the prognostic power of the 70-genes in stage I and II patients (n = 295). Kaplan-Meier curves of the probability that patients would remain free of distant metastases (A) and the probability of overall survival (B) among all patients . (van de Vijver MJ, He YD, van't Veer LJ, et al. A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med. 2002;347:1999-2009. ©2002 Massachusetts Medical Society. All rights reserved. Adapted by permission, 2005.)
• Fifty fibroblast cultures derived from ten anatomic sites were cultured in 10% fetal bovine serum or in media containing only 0.1 % fetal bovine serum • Analysis of the global gene expression patterns, using human cDNA microarrays containing approximately 36,000 genes, revealed that although fibroblasts from different sites have distinctly different gene expression programs, they share a stereotype gene expression program in response to serum expo sure (Figure 9) • Based on the genes in the serum-response signature, two groups of breast carcinomas could be recognized . Tumors with an "activated" wound gene expression signature suggestive of active wounds and tumors with a "quiescent" gene expression signature (Figure 10) • In particular, Chang et al found the wound signature to be an extremely strong predictor of death and metastasis in the panel of 295 breast tumors. • Given the ability of the wound signature to accurately predict metastasis , the signature further predicted which breast cancer patients would have benefited from chemotherapy (Figure 11)
220
Predictive Genomic and Clinico-Genomic Decision Tree Models • Gene expression signatures can also be combined; an example is given as follows Example • Chang et aI. also developed a decision tree for combining the 70-gene signature and wound signature . • First, patients were classified according to the 70-gene prognosis profile into the good or poor prognosis group . Subsequently, the tumors from the poor prognostic group were classified according to the wound signature as wound activated or quiescent (Figure 12) • Those patients with a poor prognosis 70-gene profile, but a quiescent wound signature showed a risk similar to baseline , whereas those patients with both poor prognosis and wound-response signature showed a risk of metastatic disease 6.4 fold higher than baseline • This approach shows that combining different signatures with non-overlapping features can be used to strengthen the predictor
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Clinical Genomics in Oncology
APPLICATION IN DIAGNOSTICS OF CLINICAL ONCOLOGY
General • Microarrays have been used to study several tumor types, most notably breast, ovary, colon, gastric, pancreatic, prostate, lung, melanoma, leukemia, and malignant lymphoma • To understand the current status and relevance of gene expression profiles that have been developed we have highlighted a few important examples as follows
Clinical Genomics in Breast Cancer • Currently, in breast cancer research three relevant gene expression profiles associated with prognosis have been identified, a 70-gene classifier, a 21-gene signature, and a 76-gene expression profile. The 70-gene prognosis profile has been described in Statistical Analysis section, the other two gene expression signatures are described next - 2l-gene signature • In 2004, the company Genomic Health in cooperation with the American National Surgical Adjuvant Breast and Bowel Project identified a "recurrence-score" • This score of 21-genes quantifies the likelihood of distant metastasis in tamoxifen-treated patients with lymph node-negative, estrogen-positive breast cancer • Gene expression in fixed, paraffin-embedded tumor tissue was measured as described by Cronin et al. and has resulted in the Oncotype DX assay (Genomic Health [Redwood City, CAD • The list of 2 l -genes and the recurrence-score algorithm were generated by analyzing the results from three independent preliminary studies involving 447 patients and 250 candidate genes found in earlier studies (including microarray-based studies) • 16 cancer-related genes were selected primarily based on the correlation with outcome in three trials • To test the prognostic value of the recurrence score, real-time polymerase chain reaction was successfully tested in 668 paraffin-embedded tumor blocks out of a larger study population of tamoxifen-treated patients in the B-14 study of the National Surgical Adjuvant Breast and Bowel Project • Using this recurrence-score 338 patients (51%) had a low-risk, 22% an intermediate-risk and 27% a high-risk profile for distant metastasis - 76-gene expression profile
• In 2005, the Erasmus Medical Center (Rotterdam, the Netherlands) in cooperation with the American company Veridex identified a signature of 76-genes, which identifies lymph node-negative breast cancer patients at high risk of distant recurrence and eligible for adjuvant systemic therapy • An Affymetrix-chip U133a containing 22,000 genes, was used to measure the level of gene expression • Frozen samples of 286 untreated node-negative TlT3/4 breast cancer patients of all ages were included • This 76-genes prognostic signature was identified using a training series of 171 tumors and consists of two separate profiles, one for estrogen receptorpositive (60 genes) and one for estrogen receptornegative breast carcinomas (16 genes) • The gene expression levels were analyzed using log rank analysis and validated on an independent validation set of 115 tumors, without any overlap with the training set • The distant metastasis-free survival of a "poor" profile present in 65% of the patients was after 60 months 53% and after 80 months 49% . For a "good" profile present in 35% of the patients, the disease-free survival after 60 months was 93% and after 80 months 88% • The overall survival after 60 months is for a "poor" profile 70% and after 80 months 63% and for a "good" profile 97% and 95%, respectively • Although the 70-gene profile from the Amsterdam group and the 76-gene profile of the Rotterdam group have only three genes in common, most genes are involved in the same regulatory pathways • Another reason why the overlap between these two signatures may be small, is that different microarray platforms were used, Agilent (dual label experiment) and Affymetrics (single label experiment) • New additional signatures are also being developed for example
Clinical Genomics in Hematologic Malignancies • Acute lymphoblastic leukemia (ALL) - Yeoh et al. studied 360 cases of pediatric ALL - Using unsupervised hierarchical cluster analysis they were able to correctly identify the leukemia subtypes of prognostic significance, i.e., T-lineage leukemia (T-cell ALL), B-lineage leukemia's (E2A-PBXl, BCR-ABL, TEL-AML) and mixed-lineage leukemia (MLL), and hyperdiploid type
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- The same scientists could develop a gene expression profile with an accuracy of 97% - This gene expression profile holds great promise in clinical practice and efforts will be made to refine the signature for general application • Mixed-Lineage Leukemia - Gene expression profiling is not only aimed at better classification, but can also lead to the identification of novel targets for therapy - Mixed-lineage leukemia, MLL cells distinctly have elevated levels of the receptor tyrosine kinase fmsrelated tyrosine kinase 3 (FLT3) and thereby represent an opportunity for targeted therapy - Armstrong et al. studied the effect of the small molecule inhibitor of FLT3, and showed that the drug stopped tumor progression • Diffuse large B-cell Lymphoma - Gene expression profiling of diffuse large B-cell lymphoma has allowed its categorization into groups based on cellular origin - One subgroup has gene expression characteristics of germinal center B cells, i.e., "germinal" center B-like diffuse large B-celllymphoma (DLBCL), while the second group consists of genes normally induced during in vitro activation of peripheral blood B cells, i.e., "activated" B-like DLBCL - Patients with germinal center B-like DLBCL have significantly better overall survival than those with activated B-like DLBCL and this knowledge can allow stratification of patients for clinical management - To validate these results have shown that the BCL6 and HGAL genes that are specifically expressed in the germinal-center B cells predict overall survival in unrelated groups of patients, while other genes differentially expressed, such as CDlO
Clinical Genomics in Prostate Cancer • Prostate cancer - Yu et al. studied 152 prostate tissue specimens, amongst which were frozen tissue samples from tumor and adjacent non-tumor tissue Using an Affymetrix platform they analyzed 37,777 probe elements and using a combination of principal component analysis, supervised hierarchical clustering, and lO-fold cross validation they developed a 70-gene expression profile predictor of aggressiveness with an accuracy of 93% The results were validated on a small independent group of 23 patients The group of Lapointe et al. studied 121 frozen tissue samples, comprising 62 cases of prostate cancer, 41 normal tissue specimens, and 9 lymph node metastases They studied 39,711 gene probes and used unsupervised hierarchical clustering to analyze the group
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- Malignant and normal tissue could be distinguished and further the malignant tumors could be classified into groups based on risk of recurrence - Using the microarray data immunohistochemical analysis for mucin I (MUCl) on 225 independent prostate tumors was developed and elevated levels of MUC 1 were correlated with aggressiveness of the prostate tumors in the training set and concurrent high risk of recurrence in the independent validation set (p =0.003) - These data show that using gene expression profiling it is possible not only to identify those genes that discriminate indolent from aggressive tumors, but also highlight the power of microarrays to quickly screen for genes of interest that can be measured using for example immunohistochemistry - Although gene expression profiling in prostate cancer is possible, it is not yet ready for clinical use as validation is still lacking
Clinical Genomics in Gastrointestinal Tumors • Eschrich et al. studied 78 colon cancer specimens and developed a 43-gene prognostic classifier that could predict with 90% accuracy the likelihood of survival at 36 months . There are only very little additional data on gene expression profiling in colorectal cancer • Lymph node metastasis is a determinant of therapeutic strategy for patients with oesophageal carcinoma. However, current methods such as computed tomography scanning and endoscopic ultrasound do not provide accurate assessments of lymph node status . Kan et al. studied 28 primary oesophageal squamous cell carcinomas and applied a supervised classification technique called artificial neural networks to develop a gene expression signature that was 86% accurate in predicting lymph node metastasis. Tamoto et al. performed a similar type of analysis using 36 oesophageal tumor specimens. They developed a 44-gene signature that was predictive of lymph node metastasis. Some genes present in this signature are known to have a biologic role in metastasis • Like many current microarray experiments the obvious differences amongst similar types of studies are due to the small numbers of tumor samples and methodologic differences. For example, in the aforementioned studies on oesophageal cancer, one pertinent difference is that Kan et al. used normal oesophageal tissue as a reference RNA, whilst Tamoto et al. used tumor tissue
Clinical Genomics in Carcinoma of Unknown Primary (CUP) • Patients with metastatic carcinoma in which no primary site of malignancy can be identified, despite extensive and standardized investigation, constitute approximately 3-5% of all malignancies • For optimal treatment decisions it is of great benefit to identify the true nature of the process
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Clinical Genomics in Oncology
• Recently, several studies using gene expression microarrays have demonstrated that the expression levels of thousands of genes can be used as a "molecular phenotype" to classify a multitude of tumor types. Examples are given as follows - Examples • Using a support vector machine algorithm, Ramaswamy et al. demonstrated 78% classification accuracy in classifying 14 common tumor types • Bloom et al. extended the coverage of tumor types to 21 by combining multiple datasets and built a neural network classifier with 85% accuracy • While these studies have demonstrated that gene expression microarrays hold great promise as a powerful tool for cancer diagnosis, their survey of the human tumor universe has been rather limited (at most 21 types). They also require the use of frozen tumor biopsies, which are not readily available in the current clinical setting. To overcome these shortcomings Ma et al. have established a comprehensive microarray database
•
•
•
•
•
of human tumors (466 fresh frozen, 112 formalinfixed paraffin-embedded, and 39 tumor types). Ma et al. generated a 92-gene quantitative real-time polymerase chain reaction assay for classifying 32 tumor classes, which can use archival formalinfixed paraffin-embedded tissues MicroRNA gene expression profiling may be an even better method to improve diagnostic accuracy in CUP Lu et al. used a training set of 68 more differentiated tumors, representing II tumor types and for which both mRNA and microRNA profiles were available in order to generate a classifier This classifier was then used without modification to classify the 17 poorly differentiated tumor samples, representing CUP The microRNA-based classifier established the correct diagnosisof the poorly differentiated samples with far greater accuracy (12/17) than the mRNA-based classifier (1/17)
INTERPRETATION OF GENE LISTS • Getting insight in biologic processess from microarray data is one of the major goals • Examples of methods that have been developed to identify biologic themes are; DAVID, The Database for Annotation, Visualization and Integrated Discovery; CLENCH, a program for calculating Cluster Enrichment; eGON, a program to explore Gene Ontology terms; GOstat, computes GO statistics of a list of genes selected from a microarray experiment; OM, Onto-Miner, will return all known information about a given list of genes; IPA, Ingenuity Pathways Analysis
- Examples • Gene set enrichments Analysis developed by Subramanian • Oncogenic signatures can be detected within human cancers and associated with disease outcome (Bild 2006) • "Molecular modules" underlying human malignancies
POINTS OF ATTENTION IN THE DESIGN OF MICROARRAY EXPERIMENTS
The Sample
The Microarray
• Frozen tumor tissueis indispensable as the RNA quality remains optimal due to the rapid fixation method, but is not widely available as in most hospitals the tumorsamples are directly fixed in formalin and embedded in paraffin blocks • Problems related to making use of mRNA - mRNA is a very fragile molecule, which can degrade within minutes of surgical manipulation dramatically affecting the final result of microarray data - Likewise, subtle variations in tissue handling and method of RNA extraction from samples can result in different levels of gene expression
• Differences in platform design (single vs dual label experiments), microarray design, probe annotation, methods of RNA labeling, the process of hybridization, data acquisition, and normalization make direct comparison of cross platform comparisons of gene expression studies difficult
The Statistics • "Over fitting" is one of the limitations of clustering methods. This certainly explains often that the proposed association of a "Genetic Signature" with disease outcome is significantly stronger in preliminary studies
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than in subsequent research. It indicates that the number of parameters of a model is too largely relative to the cases or specimens studied • Studies with a small sample size «50) tend to have large variance and confidence intervals • It is essential to validate a predictive gene expression pattern in a sufficiently large independent series of tumors/patients
• A limitation of unsupervised cluster analyses is it provides qualitative and not statistically valid quantitative information on differences between genes or classes • Gene lists derived from a single data set have been found to be highly dependent on the composition of the dataset
CONCLUSIONS AND FUTURE DIRECTIONS • Although genomic technologies have been most successfully applied to characterize human cancers and have the ability to predict clinical outcomes, several challenges need to be overcome before these techniques will be implemented in patient management • Until a consensus is reached amongst all laboratories for standardization of probe hybridization, image quantification, normalization, and data interpretation, inter-experimental variability will remain commonplace inhibiting the transfer of microarrays from the bench to the bedside • Validating the results of microarray experiments is an important challenge for the future
• Lack of frozen tumor material from patients with adequate clinic al pathologic annotation, including outcome data, is a major limiting factor. An advance will be to incorporate frozen sample collection and gene expression profiling into prospective randomized clinical trials • The primary goal will be to generate signatures that are robust and reproducible • In the end genomic technologies, including gene expression profiling, will improve our ability to define more precisely disease subtypes and to come to more accurate patient tailored treatment
SUGGESTED READING Application in Diagnostics of Clinical Oncology Nevins JR, Huang ES, Dressman H, et al. Towards integrated clinicogenomic models for personalized medicine : comb ining gene expression signatures and clinical factors in breast cancer outcome s prediction . Hum Mol Genet. 2oo3;12 :RI53-RI57. Quackenbush J . Microarray analysis and tumor classification. N Engl J Med. 2006;354:2463-2472. Ramaswamy S, Golub TR. DNA microarrays in clinical oncology. J Clin Oncol. 2002 ;20:1932-1941. Rebbeck TR. Inherited genetic markers and cancer outcomes: personalized medicine in the postgenome era. J Clin Oncol. 2006;24:1972-1974.
Clinical Genomics in Breast Cancer Chang JC, Hilsenbeck SG, Fuqua SA. The promise of microarrays in the management and treatment of breast cancer. Breast Cancer Res. 2005;7:100-104. Cronin M, Pho M, Dutta D, et al. Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay. Am J Pathol. 2004 ;164:35-42. Lonning PE, Sorlie T, Borresen-Dale AL. Genomics in breast cancertherapeutic implication s. Nat Clin Pract Oncol. 2005 ;2:26--33. Paik S, Shak S, Tang G, et al. A muItigene assay to predict recurrence of tamoxifen-tre ated, node-negative breast cancer. N Engl J Med. 2004;351:2817- 2826.
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Pawitan Y, Bjohle J , Amler L, et al, Gene expression profiling spares early breast cancer patients from adjuvant therapy: derived and validated in two population -based cohorts. Breast Cancer Res. 2oo5;7:R953-R964. Rels-Fllho JS, Westbury C, Pierga JY. The impact of expression profiling on prognostic and predictive testing in breast cancer. J Clin Pathol. 2006;59:225-231 . Wang Y, Klijn JG, Zhang Y, et al. Gene-expres sion profiles to predict distant metastasis of lymph-node-neg ative primary breast cancer. Lancet 2005;365:671-679.
Clinical Genomics in Carcinoma of Unknown Primary Bloom G, Yang IV, Boulware D, et al, Multi-platform . multi-site. microarray-based human tumor classification. Am J Pathol. 2004;164:9-16. Buckhaults P, Zhang Z, Chen YC, et al, Identifying tumor origin using a gene expression-based classification map. Cancer Res. 2003;63:4144-4149 . Dennis J, Hvidsten T, Wit E, et al, Markers of adenocarcinom a characteristic of the site of origin: development of a diagnostic algorithm . Clin Cancer Res. 2005 ;11:3766--3772. Dennis JL, Vass JK, Wit EC, et al, Identification from public data of molecular markers of adenocarcinom a characteristic of the site of origin. Cancer Res. 2002 ;62:5999-6005. Lu J, Getz G, Miska E, et al, MicroRNA expression profiles classify human cancers. Nature 2005 ;435:834-838.
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Clinical Genomics in Oncology Ma XJ, Patel R, Wang X, et al, Molecular classification of human cancers using a 92-gene real-time quantitative polymerase chain reaction assay.
Arch PatholLab Med. 2006;130:465-473. Pavlidis N, Fizazi K. Cancer of unknown primary (CUP). Crit Rev Oneal
Decision Tree Models Chang HY, Nuyten DSA, Sneddon JB, et al, From the cover: robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival. PNAS 2005;102:3738-3743 .
Hematol. 2005;54:243-250. Ramaswamy S, Tamayo P, Rifkin R, et al. Multiclass cancer diagnosis using tumor gene expression signatures. Proc Natl Acad Sci USA 2001;98:15,149-15 ,154. Tothill RW, Kowalczyk A, Rischin D, et al. An expression-based site of origin diagnostic method designed for clinical application to cancer of unknown origin. Cancer Res. 2005;65:4031-4040. Varadhachary GR, Abbruzzese JL, Lenzi R. Diagnostic strategies for unknown primary cancer. Cancer 2004; I00:1776-1785 .
Clinical Genomics in Gastrointestinal Thmors Eschrich S, Yang I, Bloom G, et al, Molecular staging for survival prediction of colorectal cancer patients. J Clin Oncol. 2005;23:3526-3535. Kan T, Shimada Y, Sato F, et al. Prediction of lymph node metastasis with use of artificial neural networks based on gene expression profiles in esophageal squamous cell carcinoma . Ann Surg Oncol. 2004; II :1070-1078. Tamoto E, Tada M, Murakawa K, et al. Gene-expression profile changes correlated with tumor progression and lymph node metastasis in esophageal cancer. Clin Cancer Res. 2004;10:3629-3638.
Clinical Genomics in Haematologic Malignancies Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-ceillymphoma identified by gene expression profiling. Nature 2000;403:503-511.
"Functional" Gene Expression Signatures Chang HY, Sneddon JB, Alizadeh AA, et al. Gene expression signature of fibroblast serum response predicts human cancer progression: similarities between tumors and wounds. PLoS Biol. 2004;2:E7.
Hierarchical Unsupervised Classification Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumors. Nature2000;406:747-752. Sorlie T, Perou C, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. PNAS 2001;98:10,869-10,874. Sotiriou C, Neo SY, McShane LM, et al, Breast cancer classification and prognosis based on gene expression profiles from a population-based study. ProcNatl Acad Sci USA 2003; I00:10,393-10,398.
Interpretation of Gene Lists Bild AH, Yao G, Chang JT, et al. Oncogenic pathway signatures in human cancers as a guide to targeted therapies. Nature2006;439:353-357. Segal E, Shapira M, Regev A, et al, Module networks: identifying regulatory modules and their condition-specific regulators from gene expression data. Nat Genet. 2003;34: 166-176. Slonim DK. From patterns to pathways: gene expression data analysis comes of age. Nat Genet. 2002;Suppl 32:502-508. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. ProcNatl Acad Sci USA 2005;102:15,545-15,550.
Armstrong SA, Kung AL, Mabon ME, et al. Inhibition of FLT3 in MLL. Validation of a therapeutic target identified by gene expression based classification. Cancer Cell 2003;3:173-183 .
Points of Attention in the Design of Microarray Experiments
Ebert BL, Golub TR. Genomic approaches to hematologic malignancies.
Abdullah-Sayani A, Bueno-de-Mesquita JM, van de Vijver MJ. Technology Insight: tuning into the genetic orchestra using microarrayslimitations of DNA microarrays in clinical practice. Nat Clin Pract
Blood 2004;104:923-932. Lossos IS, Alizadeh AA, Rajapaksa R, et al. HGAL is a novel interleukin-4-inducible gene that strongly predicts survival in diffuse large B-ceillymphoma. Blood 2003; 101:433-440. Lossos IS, Jones CD, Warnke R, et al. Expression of a single gene, BCL-6, strongly predicts survival in patients with diffuse large B-cell lymphoma. Blood 200 1;98:945-951 . Ross ME, Zhou X, Song G, et al, Classification of pediatric acute lymphoblastic leukemia by gene expression profiling. Blood 2003;102:2951-2959. Yeoh EJ, Ross ME, Shurtleff SA, et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell 2002;1:133-143.
Oncol. 2006;3:501-516. Chuaqui RF, Bonner RF, Best CJ, et al. Post-analysis follow-up and validation of microarray experiments. Nat Genet. 2002;32 Suppl:509-514 . Tinker AV, Boussioutas A, Bowtell DD. The challenges of gene expression microarrays for the study of human cancer. CancerCell 2006;9:333-339.
Statistical Analysis Eisen M, Spellman P, Brown P, et al. Cluster analysis and display of genome-wide expression patterns. PNAS 1998;95:14,863-14,868 . Statnikov A, Aliferis CF, Tsamardinos I, et al. A comprehensive evaluation of multicategory classification methods for microarray gene expression cancer diagnosis. Bioinformatics 2005;21:63 1--643.
Clinical Genomics in Prostate Cancer
Supervised Classification (Knowledge Driven)
Lapointe J, Li C, Higgins JP, et al, Gene expression profiling identifies clinically relevant subtypes of prostate cancer. ProcNatl Acad Sci USA 2004;101:811-816.
van de Vijver MJ, He YD, van't Veer LJ, et al. A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med. 2002;347:1999-2009 .
Yu YP, Landsittel D, Jing L, et al, Gene expression alterations in prostate cancer predicting tumor aggression and preceding development of malignancy. J Clin Oncol. 2004;22:2790-2799.
van't Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002 ;415:530-536.
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9 Clinical Proteomics David H. Geho, MD, PhD, Virginia Espina, MS, Lance A. Liotta, MD, PhD, Emanuel F. Petricoin, PhD, and Julia D. Wulfkuhle, PhD
CONTENTS
I. Introduction to Clinical Proteomics
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Protein-Building Blocks Protein Structure Tools Used for Protein Studies: An Overview Physical Detection Systems Mass Spectrometry Surface Plasmon Resonance UV Spectroscopy Circular Dichroism Electrophoresis Capillary Electrophores is Chromatography Specific Affinity Techn iques for Protein Detection Hybrid Technologies Limitations of Classical Protein Detection Tools as Clinical Tools
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II. Protein Microarray-Based Clinical Proteomics Clinica l Proteomic Diagnostics: Protein Microarrays Specimen Procural Laser Capture Microdi ssectio n Protein Microarrays The Architect ure of a Protein Microarray Forward Phase Arrays Reverse Phase Microarrays
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Array Surfaces Arrayer Technologies Contact Printers Non-Contact Printers Additional Consideration s Antibodies Microarray Reporter Technologies Chromogenic Reporter Technologies Nanoparticle Reporter Technologies Image Acquisition Data Analysis Analysis Software Downstream Ana lysis Protein Microarray Conclusions
III. Mass Spectrometry Based Proteomics Methods of Ionization Solid Tissue Mass Spectrometry Body Fluid Mass Spectrometry Working Model for the Genesis of the Serum Peptidome High-Abundance vs Low-Abundance Blood Proteome Mass Spectrometry-Based Proteomics Work Flow MS-Based Diagnostics Specimen Procural and Preservation
IV. Suggested Reading
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Molecular Genetic Pathology
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INTRODUCTION TO CLINICAL PROTEOMICS • Clinical proteomics is a rapidly maturing research discipline • The fundamental question s of clinical proteomics are the following: - What proteins or protein isoform s are present in a disease process? - How do those proteins interact? - What are the relative abundance and activation states of disease-related proteins? • This chapter reviews technologies and experimental procedures that enable clinical proteomics research
Protein-Building Blocks • Proteins are polymers of amino acids, linked together by peptide bonds. A peptide bond is the amide linkage between a carboxyl group in one amino acid and amino group in another amino acid
structures. The aggregation of sickle hemoglobin resulting in the formation of rigid fibers causes red blood cell sickling • Amino acid side chains within proteins are sites for the covalent addition of molecules such as phosphates, sugars, and lipids. These modifications occur after the protein has been translated from mRNA and are termed post-translational modifications (i.e., phosphorylation, glycosylation, and lipidation) • The study of post-translational modified proteins represents a vast and important area of disease pathophysiology research. Certain proteins are phosphorylated or dephosphorylated on specific residues in response to cellular signals. These signaling cascades play an important role in orchestrating cellular growth, migration, and apoptosis, among other functions. In general, post-translational modifications are not detected using genomic approaches
• Vast numbers of potential amino acid sequences are possible
Tools Used for Protein Studies: An Overview
• However, the human genome is made of about 3-4 x 104 genes. Therefore, the proteins produced in the human system are a small subset of the theoretical protein complement
• Broadly speaking, two classes of protein characteristics have been used to study protein functions
• Clinical proteomics focuses on the relatively limited population of clinically relevant proteins transcribed from the genome
Protein Structure • The amino acid sequence of a protein is called its primary structure. Out of this sequence arise the intrinsic properties of the protein, such as surface shape, size, and charge. These are important characteristics of a protein, which determine the ultimate function(s) of a protein • Based on the primary structure, a linear chain of amino acids coalesce and fold to form a series of secondary structural elements such as a-helices, p-pleated sheets , and random coils • Tertiary structure is comprised of higher order arrangements of secondary structural motifs. The secondary structural elements fold in such a manner so as to assume a thermodynamically stable conformation • Cysteines can be linked via disulfide bonds , which provide further structural stabil ity • Quaternary structure of a protein refers to the higher order arrangements of tertiary structures • Historically, structural changes in proteins have been linked to diseases. As one example, sickle cell anemia results from of a single amino acid change in an otherwise unaltered primary amino acid sequence. This substitution gives rise to a protein with a different shape than normal hemoglobin and altered higher order protein
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- Intrinsic physical properties. These are broadly applicable across a range of protein types and provide information about the protein's mass, charge, or structure. Examples of this type of technology are mass spectrometry, surface plasmon resonance, electrophoresis, ultraviolet (UV) spectroscopy, and chromatography Protein-specific properties. These techniques are protein-specific and provide information about posttranslational modifications as well as presence or absence of specific proteins in complex mixtures. They are usually derived from previously wellcharacterized individual proteins, such as antibodybased detection systems . Examples of approaches used to study protein-specific properties include flow cytometry, immunohistochemistry, enzyme-linked immunosorbent assay, and Western blots
Physical Detection Systems • Within a protein , the overall sum of amino acids, the intrinsic qualities and relative abundance of the amino acids provide physical elements suitable for detection by a number of complimentary technologies, including mass spectrometry, chromatography, electrophoresis, surface plasmon resonance , and circular dichroism
Mass Spectrometry • Mass spectrometry takes advantage of the behavior of a charged molecule in magnetic fields in order to classify proteins based on mass:charge ratios . This is discussed in further detail later
9-3
Clinical Proteomics
Surface Plasmon Resonance • The biomolecular interactions of unlabeled proteins can be studied using surface plasmon resonance. Proteins are immobilized onto a thin metal film. If ligand binding occurs, the refractive index changes and these changes are detected by an optical sensor. Analyte association! dissociation rate constants may be calculated
UV Spectroscopy • By measuring the absorbance of UV light by aromatic side chains of constituent amino acids within proteins, UV spectroscopy enables protein detection and quantitation
Circular Dichroism
high-resolution protein separations when voltage is applied to the system
Chromatography • Proteins can be separated by passing them over a resin that partitions the molecules between the liquid or gas phase and the bound resin. Types of chromatography include size exclusion, ion exchange, and high-pressure liquid chromatography
Specific Affinity Techniques for Protein Detection • A molecule or macromolecular structure that selectively interacts with a protein can be utilized as an affinity system for specific protein detection. Affinity reagents that can be used to isolate specific proteins include metals, carbohydrates, proteins, and nucleic acids
• Circular dichroism spectroscopy uses circularly polarized light of one direction as a quick, low-resolution method for determining protein structure. Within a protein, the relative abundance of secondary structural elements such as a-helices, and B-pleated sheets can be measured
• A commonly used type of affinity reagent is an antibody that binds to a specific protein. Validation of a reagent's sensitivity and specificity must be performed for each antibody
Electrophoresis
• Other uses of antibody reagents for detection of specific proteins within clinical samples include immunohistochemistry, immuno -flow cytometry, Western blots, and enzyme-linked immunosorbent assay
• Standard one-dimensional (lD) gel electrophoresis provides a means for protein separation. The movement of proteins through a solution in a polymeric matrix based on the application of an electrical field represents the primary technology. Differences in migration are based primarily on size of the protein • In two-dimensional electrophoresis, another level of separation, isoelectric potential , is used to further separate protein species in addition to size-based separation. In the first step, a pH gradient permits movement of the constituent proteins until they reach their isoelectric point. In the second step, the separation occurs at right angles to the first dimension and is based on protein size as in ID electrophoresis
Capillary Electrophoresis • Electrophoresis can also be performed on protein samples within a capillary tube, which provides
Hybrid Technologies • Many times, a system for protein characterization and detection represents the fusion of several technologic approaches. A physical detection method , such as electrophoresis, can be paired with an antibody probing step, as is the case with a Western blot. Affinity chromatography using antibodies immobilized to a chromatography resin represents another example
Limitations of Classical Protein Detection Tools as Clinical Tools • Proteomic tests in a clinical setting must be rapidly performed using very limited clinical material. Classical proteomic tools have had limited applicability due to the need for relatively large sample volume and time constraints
PROTEIN MICROARRAY-BASED CLINICAL PROTEOMICS • Clinical proteomics is a new field that combines components of classical protein detection technologies with new technologies to create high throughput assays that effectively utilize the proteomic information available in limited sample volumes • Patient tissue specimens contain a wealth of potential diagnostic molecular descriptors
• Of particular interest are proteins that are involved in cell signaling, metabolism, migration, cell division, immunity, and epigenetic processes within diseased cells • Many of the protein s of interest undergo posttranslational modifications, such as phosphorylation and cleavage. For example, many cell signaling pathways involve cascades of phosphorylated signaling proteins
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Molecular Genetic Pathology
9-4
Clinical proteomics Reverse phaseprotein microarray Lesion biopsied Tissuefrozen Frozen section Lasercapture microdissection
• Protease and phosphatase inhibitors may be added to the samples
Tumor cells isolated
Laser Capture Microdissection
Extract proteins
• Many distinct cell types are present within a tissue specimen, including cells of epithelial, endothelial, hematopoietic, or mesenchymal derivation
Spot proteins on array Process arrayusing validated antibodies Acquire imageof arrays Bioinformatic analysis
Fig. 1. Clinical proteomics work flow for reverse phase protein microarrays. Immediately after harvesting, the tissue specimen must be frozen in order to preserve labile molecular end points. A frozen section is later prepared and stained. A targeted cell population, such as tumor cells, is isolated using laser capture microdissection. Proteins are extracted from the isolated cells and arrayed onto an array substrate using a high-throughput protein microarrayer. The array is further developed by applying validated antibodies onto the array surface as well as a reporter detection system (such as streptavidin-HRP). The array is then imaged and bioinformatics analysis and clinical correlation is performed.
• A genomics-only approach will not detect this posttranslationally defined molecular information. Tools with high sensitivity are required, as protein does not have an intrinsic amplification system as is found with DNA
Clinical Proteomic Diagnostics: Protein Microarrays • Critical phases in a protein microarray evaluation include the following : specimen procurement, specimen preservation, specimen processing, molecular characterization, data analysis, and clinical correlation (see Figure 1) • Key technologies for protein microarray analysis: Laser capture microdissection, a reliable arraying device, arrays, an image acquisition system , and bioinformatics analysis
Specimen Procural • Integration of a clinical proteomics assay into pathology practice requires the development of a comprehensive program that involves nurses, physicians, tissue procurement specialists, tissue processing facilities, and sufficient storage space
234
• For clinical proteomics assessments, the tissues must be rapidly processed shortly after procurement. Typically tissue is frozen at -80 DC or placed in liquid nitrogen. Rapid processing limits protein degradation and phosphatase activity
• For this reason, it is important to isolate specific cell populations, such as tumor cells, from a biopsy for clinical proteomic assessments • Laser capture microdissection enables pure cell populations, such as tumor cells, to be isolated from the complex tissue microenvironment
Protein Microarrays • Protein microarrays provide a means for measuring the levels of disease-related proteins extracted from patient tissues
The Architecture of a Protein Microarray • Certain technologies are required for protein microarrays as an assay class: an arraying device, a substrate that functions as the array surface, antibodies, or some other detection probe , a reporter system , a detection device , and bioinformatics analysis • Varying microarray architectures have been employed. There are two fundamental types of arrays: forward phase arrays and reverse phase arrays (see Figure 2). The difference lies in how the analytes, or proteins of interest, are captured for study • Once captured, the analytes are detected using a specific probe, such as an antibody
Forward Phase Arrays • An array surface is coated with bait molecules, such as an antibody, to capture a particular analyte • Capture moiety (bait molecules) can be arrayed in dilution curves on the same array • Multiple bait molecules can be spotted on the same array • The array is then incubated with a mixture of analytes • Analytes bound to the bait molecule are detected using a second distinct antibody. Alternatively, the analytes may be directly labeled • For the antibody detection system, two distinct epitopes or antibody-binding regions, must be available on the same analyte, which may be difficult for small analytes
9-5
Clinical Proteomics
• Denatured or non-denatured analytes may be arrayed Protein microar rays
• Analyte s may be arrayed in a dilution curve that enables the linear component of the antibody-analyte interaction to be evaluated
Biotinylated secondary antibOdY!
Streptavidin -+- conjugate
Forward phase array
+--
Peptide - -
Detection antibody
Capture +-- antibody
Array Surfaces • An array surface must be capable of binding proteins . One example is nitrocellulose-coated glass slides
!
Array substrate
• The substrate must have a high surface area and low intrin sic background signal
Arrayer Technologies
Reverse phase array
Biotinylated secon dary antibOdY!
• A reliable arrayer is an important technology for clinical proteomics microarray s. These devices must have reliable, reproducible delivery (printing) of proteins onto the array surface -+- St re~tavi d i n
conjugate Detection antibody
~
Peptide _ _
r:
• Two primary forms of printing are available commercially: contact and non-contact devices
Array
Fig. 2. Compari son of forward phase arrays and reverse phase arrays . For forward phase arrays, an array surface is coated with bait molecule s, such as an antibody, to capture a particular protein analyte. The remainder of the array surface is blocked to decrease non-specific binding interactions. The array is then incubated with a mixture of analytes. Analytes bound to the bait molecule are detected using a second distinct antibody. Alternatively, the analytes may be directly labeled . For the antibody detection system, two distinct epitope s, or antibody binding regions, must be available on the same analyte, which may be difficult for small analytes. The binding of the second antibod y is detected using a biotinylated detection antibody. Streptavidin linked to HRP is then incubated on the array. The array is then treated with a chromogenic substrate such as DAB (Diaminobenzidine). For reverse phase arrays , the cellular proteins are directly arrayed onto a substrate. The remainder of the array surface is blocked to decrease non-specific binding interaction s. A detection antibody is then incubated on the array surface. The presence of bound detection antibody is in tum probed using a biotinylated secondary antibody. Streptavidin linked to HRP is then incubated on the array. The array is then treated with a chromogeni c substrate such as DAB.
Reverse Phase Microarrays • In the reverse phase architecture, the cellular protein s are directly arrayed onto a substrate • MUltiple samples are immobilized on the array substrate
Contact Printers • Contact printers depo sit samples via direct contact of the print head with the substrate and utilize solid pins or split pins with a built-in microreservoir to transfer sample to the substratum • Pin diameter and the fluid propertie s of the sample dictate the volume deposited and the spot size • Solid pin printers require repeated dipping into a sample reservoir to print replicate spots onto the array • Variations such as the pin and ring ass embly, ink jet technology, and split pin configurations allow multiple spots to be printed from each sample without repeated repleni shment from the main sample reservoir • Volumes deposited are generally in the nanoliter range
Non-Contact Printers • Non-contact printing devices utilize a sensor for depositing sample from above the substratum • These arrayer s may involve a piezoelectric crystal sensor or employ syringe- solenoid technology • Piezoelectric printing technology utilizes a piezoelectric crystal closely appo sed to a fluid reservoir with a capillary tip. Changes in electrical voltage control crystal deformation such that sample is ejected through the glass capillary • Syringe-solenoid technology utilizes a syringe pump coupled to a solenoid valve to as pirate sa mple into a reser voir, which is then dispen sed at high pressure upon opening of the solenoid valve • Volumes dispensed from these devices are in the picoliter to nanoliter range
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9-6
Additional Considerations
Molecular Genetic Pathology
• These particle-based fluorophores resist photobleaching, unlike organic fluorophores, which makes them suitable for array retesting at a later date
• Control of temperature and humidity conditions are critical to printing quality and reproducibility in that they affect sample evaporation from the reservoir and/or water condensation on the instrument or the substratum
Image Acquisition
• Pin washing between samples is critical for reproducibility and prevents sample carry-over
• For chromogenic arrays, a flatbed scanner can be used to acquire an image of an array
• Pin calibration for the degree of contact with the substrate is an important consideration with contact printing devices affecting spot size and quality
• For fluorescent arrays, a CCD (e.g., Novaray Imager, Alpha Innotech, San Leandro, CA) camera or a hyperspectral imaging system can be used to detect signals from arrays
• Dust and debris on the printing substratum can lead to printing irregularities, damage to the substrate, or cause interference with spot detection
Data Analysis
Antibodies
• Programs capable of quantifying pixel intensity are essential for the analysis of protein microarray data
• For reverse phase arrays, after analyte spotting, the array surface can be probed with molecules, such as antibodies that specifically recognize proteins of interest within the lysate • The specificity of an antibody must be validated using a Western blot assay • For forward phase arrays, antibodies are used to both capture and detect the analytes of interest • A limitation of proteomic arrays is the availability of sensitive and specific antibodies that detect posttranslational modifications such as phosphorylation • A widespread effort, including academic and industry laboratories is underway to produce and characterize antibody libraries to meet this critical need
Microarray Reporter Technologies • The detection of an analyte by an antibody probe must be transformed into a detectable signal. Reporter technologies enable this transformation either through chromagen deposition, fluorophore deposition, or a radioactive tag
Chromogenic Reporter Technologies • Chromogenic detection systems provide reproducible, sensitive signals • As in immunohistochemistry, a biotinylated antibody is bound by streptavidin linked to an enzyme such as horseradish peroxidase (HRP) • Enzymatic signal amplification strategies can be used to increase the sensitivity of the assay, such as biotinyl tyramide amplification
Nanoparticle Reporter Technologies • Semiconductor nanoparticles, such as quantum dots, provide intense fluorescent signals for protein microarrays • For example, streptavidin-linked quantum dots can be used as reporter agents for biotinylated antibodies
236
• Additionally, statistical software packages for downstream analysis of data and correlations with clinical parameters are essential for individualized therapy
Analysis Software • In reverse phase microarray analysis, utilization of the printed sample dilution curve to identify and analyze the linear range of detection for the antibody-analyte interaction in each sample can provide more accurate and reproducible results than single data point analysis • Array analysis software that provides automated spot detection and local area background subtraction can greatly increase efficiency and reduce operator bias during analysis • Subtraction of negative control spot (secondary antibody alone) intensities may also be necessary when working with samples that have unusually high levels of endogenous peroxidases, avidin, or biotin • For data comparisons across samples, normalization of the data to total protein values or to a housekeeping protein is necessary
Downstream Analysis • Final data output can be used to compare samples or groups with a variety of univariate and multivariate statistical methods • Unsupervised hierarchical clustering of the entire data set can identify differences/similarities in protein(s) levels/activity that lead to formation of subgroups within the dataset • Additional tests, such as principal component analysis can help to identify key analytes underlying differences between groups of interest
Protein Microarray Conclusions • Numerous protein array studies have used patient specimens to define a specific protein's contribution to disease processes such as ovarian cancer, prostate cancer, breast cancer, and lymphoma
Clinical Proteomics
9-7
• In order for a proteomic tool to be applied to clinical proteomics research, it must meet high thresholds for sensitivity and reproducibility, as the molecular information that is queried is often of low abundance
• Challenges for the clinical application of protein microarray technologies include standardization of the procedure across laboratories and technologic improvements in arrayers, substrates, reporter technologies, and imaging systems
MASS SPECTROMETRY BASED PROTEOMICS • Mass spectrometry, a highly sensitive proteomics tool, is becoming widely used as a tool to discover and catalog disease-related proteins in solid tissues and body fluids
• Direct tissue mass spectrometry is another method, wherein a frozen tissue section is applied directly onto a mass spectrometry substrate
• Mass spectrometry has been used as a method for physical analysis of molecules for many years
Body Fluid Mass Spectrometry
• The basis for the technique is the behavior of charged particles in magnetic fields • The molecule to be studied can be charged either through the addition or removal of protons or electrons • The charged molecule is introduced into a chamber, which is under a vacuum, and hurtled through a highelectromagnetic field • MS analysis utilizes some type of mass analyzer, including ion trap, time of flight, quadrapole, or Fourier transform ion cyclotron resonance. These analyzers enable a mass/charge (m/z) value to be assigned to the molecules undergoing analysis • Using protein digestion methods (i.e., trypsin-based fragmentation), the identity of proteins can be determined by coupling the fragment patterns with computer bioinformatics algorithms
Methods of Ionization • With electrospray ionization, molecules in a solution are sprayed through an electric field. The field introduces a charged state within the fluid. The solvent is evaporated and the charges become associated with the molecules • Matrix-assisted laser desorption ionization (MALDI) , is another type of ionization wherein a matrix containing molecules of interest is broken up in the presence of laser light. This process ionizes the molecules for further analysis • Surface-enhanced laser desorption and ionization is a type of MALDI , wherein the matrix has surface properties that enable binding of a subset of proteins (i.e., weak cations, strong anions, and so on)
Solid Tissue Mass Spectrometry • Solid tissues can be evaluated in a variety of ways • One method utilizes protein lysates derived from laser capture microdissected cells. The lysates are then studied using mass spectrometry
• The presence of disease-related proteins in body fluids, including plasma, serum , and cerebrospinal fluid, has been examined using mass spectrometry
Working Model for the Genesis of the Serum Peptidome • Many cell types contribute to the formation of a disease microenvironment • For a cancer, the cells that coalesce to form a disease microenvironment include the neoplastic cells and surrounding stromal cells • All of these cells produce proteins that are released into the interstitium • Proteins may also be released as a cell dies and degrades. Resident enzymatic proteins cleave the proteins, resulting in an array of proteolytic fragments • The fragments enter the vascular compartment • These shed, fragmented molecules provide a molecular portrait of ongoing processes within a tissue • The peptide fragments are protected by association with larger, high-abundance proteins in the blood, such as albumin • Further degradation of the proteins in blood may occur once the blood specimen is collected, contributing to the array of protein fragments available for study
High-Abundance vs Low-Abundance Blood Proteome • Most of the proteins in blood are high-abundance, highmolecular weight molecules (99% of the mass) such as albumin, transferrin, and immunoglobulins • It is in the low-molecular weight fraction of the blood where significant attention is currently focused for discovery of disease-specific proteomic information • Experimental evidence indicates that the low-molecular weight molecules (LMW) in blood are non-covalently bound to high-abundance proteins like albumin
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Molecular Genetic Pathology
• A current challenge for blood-based proteomics is the development of technologies that release the peptide fragments from proteins such as albumin, so they can be measured using mass spectrometry • One approach that has been used is to isolate albumin, strip it of its non-covalently bound molecular cargo, and study the dissociated cargo molecules. This type of approach has been applied to the blood of patients with ovarian cancer
• The resulting polypeptide fragments are separated using a device such as high-performance liquid chromatography • The fragments are then ionized and detected using mass spectrometry systems such as electrospray ionization or MALDI • Bioinformatics programs enable the polypeptide fragments to be identified
• A current goal is to find strategies that fully utilize this LMW information for the purposes of biomarker discovery and detection. One potential strategy for achieving this is to generate harvesting platforms out of nanoscale particles that bind and preserve low-abundance, LMW biomarkers
MS-Based Diagnostics
Mass Spectrometry-Based Proteomics Work Flow
Specimen Procural and Preservation
• Tissue specimens are highly complex . Detection and identification of low-abundance proteins can be enhanced using affinity chromatography, gel electrophoresis, or some other method of fractionation
• Tissues must be processed according to a standardized protocol
• Often, tryptic digestion of the fractionated proteins occurs during proteomic analysis
• Samples should be processed promptly after collection and serum/plasma should be stored at -80°C
• Bioinformatic interrogation of MS data can also be used to identify sets of peptides/proteins that are uniquely found in disease or healthy patients
• Specimens should be handled in the same manner for all of the patients in a particular study
SUGGESTED READING Body Fluid Proteomics
Direct Tissue Mass Spectrometry
Anderson NL, Anderson NG . The human plasma proteome: history character, and diagnostic prospects. Mol Cell Proteornics 2002; I :845-867.
Chaurand P, Caprioli RM. Direct profiling and imaging of peptides and proteins from mammalian cells and tissue sections by mass spectrometry. Electrophoresis 2002;23;3125-3135.
Deutsch EW, Eng JK, Zhang H, et aI. Human Plasma PeptideAtias . Proteornics 2005;5:3497-3500.
Stoeckli M, Chaurand P, Hallahan DE, Caprioli RM. Imaging mass spectrometry : a new technology for the analysis of protein expression in mammalian tissues. Nat Med. 200 1;7:493--496.
Lowenthal MS, Mehta AI, Frogale K, et aI. Analysis of albuminassociated peptides and proteins from ovarian cancer patients . CUn Chern. 2005;51:1933-1945.
Laser Capture Microdissection
Mehta AI, Ross S, Lowenthal MS, et al, Biomarker amplification by serum carrier protein binding. Dis Markers 2003;19:1-10.
Emmert-Buck MR, Bonner RF, Smith PD, et al. Laser capture microdissection. Science 1996;274:998-1001.
Rosenblatt KP, Bryant-Greenwood P, Killian JK, et aI. Serum proteomics in cancer diagnosis and management. Annu Rev Med. 2004;55 ;97-112.
Mass Spectrometry
Clinical Proteomics
Hillenkamp F, Karas M. Mass spectrometry of peptides and proteins by matrix-assisted ultraviolet laser desorption/ionization. Methods Enzymol. 1990;193:280-295.
Celis JE, Gromov P. Proteomics in translational cancer research; toward an integrated approach. Cancer Cell 2003;3:9-15. Liotta LA, Kohn EC, Petricoin EF. Clinical proteomics ; personalized molecular medicine. lAMA 2001;286 ;2211-2214. Petricoin EF, Zoon KC, Kohn EC, Barrett JC, Liotta LA. Clinical proteomics : translating benchside promise into bedside reality. Nat Rev Drug Discov. 2002; I:683-695. Wultkuhle JD, McLean KC, Paweletz CP, et al. New approaches to proteomic analysis of breast cancer. Proteornics 2001; I;1205-1215.
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Gygi SP, Aebersold R. Mass spectrometry and proteomics. Curr Opin Chern Bioi. 2000;4:489--494.
Protein Microarrays Grubb RL, Calvert VS, Wulkuhle JD, et aI. Signal pathway profiling of prostate cancer using reverse phase protein arrays. Proteornics 2003;3:2142-2146. Gulmann C, Espina V, Petricoin E, et aI. Proteomic analysis of apoptotic pathways reveals prognostic factors in follicular lymphoma . CUn Cancer Res. 2005;11:5847-5855.
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Clinical Proteom ics
Herrm ann PC, Gillespie J W, Cha r bo nea u L, et al, Mitochondrial proteome: altered cytochrome c oxidase subunit levels in prostate cancer.
Proteomics 2003;3:1801- 1810. Knezevic V, Leethanakul C, Bich sel VE, et at. Proteomic profiling of the cancer microenvironment by antibody arrays. Proteomics
2001;I:1271- 1278.
Pawel et z CP, Charboneau L, Bichsel VE, et at. Reverse phase protein microarrays which capture disease progression show activatio n of prosurvival pathways at the cancer invasion front. Oncogene
2001;20:1981-1989 .
Thmor Microenvironment
Liotta LA, Espina V, Mehta AI, et al, Protein microarrays: meeting analytical challenges for clinical applications. Cancer CeI/2003;3:317-325.
Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature
Mac Beath G. Protein microarrays and proteom ics. Nat Genet. Suppl
Liotta LA, Kohn EC. The microenvironment of the tumour-host interface.
2002;32:526--532.
2001;411:355- 365.
Nature 2001;411:375-379.
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10
Clinical Pharmacogenomics Catalina Lopez-Correa,
MD, PhD
and Lawrence M. Gelbert,
PhD
CONTENTS I. Introduction
l 0-2
II. Pharmacogenetic/Pharmacogenomic Patient Stratification 10-2 III. The Evolution of Pharmacogenetic and PharmacogenomicTests
10-3
IV. Glossary
10-4
V. Pharmacokinetics vs Pharmacodynamics VI. Genetic Variants Single Nucleotide Polymorphisms Microsatellites Variable Number of Tandem Repeats Copy Number Variants (CNV s)
VII. Genetic Markers for Association and Linkage Studies
XIII. PGx of Asthma Treatment B2-Agonists Leukotrienes
XIV. PGx of Cardiovascular Disorders 10-4 10-4 10-5 10-5 10-5 10-5
Hypertension Angioten sin-Converting Enzyme (ACE) Angioten sinogen (AGT) Gene ~-Blockers
PGx of Lipid-Lowering Agents Emerging Trend s in Cardiovascular Pharmacogenetics
XV. PGx of Neuropsychiatric Diseases 10-5
VIII. Pharmacogenomic Association Studies
XII. Warfarin (Coumarin): An Example of DME and Target Variation Affecting Safety and Efficacy (Therapeutic Index)
10-6
Alzheimer's Disease Parkinson's Disease Major Depression Schizophrenia
10-11 10-12 10-12 10-12
10-13 10-13 10-13 10-14 10-14 10-14 10-14
10-14 10-15 10-15 10-15 10-15
10-6 10-6 10-7
XVI. Cancer PGx ••••••••••••••••••.•.•••••••.•.....•••10-16
10-7
Monogenic Cancer Pharmacogenetics; TPMT Multi-Gene Cancer PGx-Microarray Expression Profiling
10-16
X. Regulatory Issues in PGx
10-7
XVII. Web Resources ••••••••••••••••••••••.•.•••••..10-18
XI. Pharmacogenetics: DMEs
10-8
XVIII. Suggested Reading
Candidate Gene Search Genome Wide Association (GWA) Candidate Pathway Approach
IX. Genetic Testing
I0-16
10-18
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Molecular Genetic Pathology
10-2
INTRODUCTION • -OME is from Latin -oma meaning "mass ." A genome is defined as the complete DNA sequence of an organism, and genomic s (also known as functional genomics) is the comprehensive analysis of genomic structure and function. The development of genomics was greatly facilitated by the Human Genome Project and the publication of the draft sequence of the human genome in 2001. This and the development of several enabling technologies such as automated high-throughput DNA sequencing and genotyping, microarray technology, and bioinformatics now allow for complete views of human biological systems including drug activity. These developments have shifted research from gene discovery to the association of specific genes and genetic variants with disease susceptibility, response to therapy, and other phenotypes of importance in healthcare • The role of genetic variability in response to drug therapy was first described in the 1950s. Pharmacogenetics is
defined as the analysis of genetic factors influencing response to drug treatment. The term pharmacogenomics (PGx) is used to describe the expanded analysis of multiple genetic factors affecting drug efficacy and toxicity, and disease susceptibility. Although pharmacogenetics and PGx are commonly used interchangeably, more recently, pharmacogenetics has been used to define the genetic factors specifically associated with drug metabolizing enzymes (DMEs) . The goal of both these fields is to identify genetic biomarkers to predict disease susceptibility/progression (disease biomarker) and response to therapy (drug activity biomarkers) • In this chapter, we will provide a historical background to the development of pharmacogeneticslPGx, and emerging trends and examples of the current state of PGx in several disease areas
PHARMACOGENETIC/PHARMACOGENOMIC PATIENT STRATIFICATION • Drug therapy for many diseases has significantly reduced mortality, improved quality of life, and has a significant positive economic impact. However, because of genetic and environmental heterogeneity (e.g., diet and exercise), the response rate for most commonly prescribed drugs ranges from 25% for cancer treatments to 80% for analgesic COX-2 inhibitors. Similarly, environmental and genetic factors impact drug safety, contributing to approximately 6.5% of the patient population having an adverse drug reaction (ADR) resulting in >100,000 deaths annually
• Figure 1 summarizes the use of PGx to stratify patient populations. Patients are first diagnosed with a disease (such as hypertension, Alzheimer's, cancer, and asthma) for which there is a standard drug therapy. The goal of PGx is to enrich patient populations for responders (PGx efficacy tests), or to identify patients genetically predisposed for an ADR (PGx safety test) • Complex diseases result from multiple genetic and environmental factors whose individual effects are small and overlap. Therefore, pharmacogenomic tests are not absolutely predictive, but allow for the enrichment of patient populations to improve safety and/or efficacy • Factors driving the adoption of PGx include : - Drug safety : the recent recall of several marketed drugs has increased safety concerns and regulatory
242
expectations for new and existing drugs. Pharmacogenomic safety assays are viewed as a way to address these safety concerns - Drug cost: with the cost of healthcare rising, there is increasing pressure to contain costs, including those of prescription medicines. This is especially true for newer targeted cancer therapies and biologics, which tend to be more expensive. Targeting patients who respond is also of importance in developing countries, which have fewer financial resources for healthcare and will rely on genomic tests to maximize the benefit of prescription medic ines and other forms of disease management - Decreasing cost of drug discovery and development: the cost of developing a new drug now exceeds 800 million USD. The high cost of developing drugs results in great part from the attrition observed in clinical drug development. The overall success rate in clinical development is II %, and reducing the cost of clinical drug development is recognized as a primary factor in controlling the cost of new drugs. The early development of pharmacogenomic markers will improve patient selection for clinical trials, reducing their size and complexity, and allowing for a focus on efficacy testing; thus, resulting in reduced attrition and lower costs
10-3
Clinical Pharmacogenomics
Patient Stratification Using Genetic Biomarkers
All patients with same diagnos is 17/45 (38%) response rate
I "I'I'llt Il it
.u...,J.)\\: III
Population enriched for toxic responders PGx safety assay ~
t ltlt l tt Treat with alternative
II
I -Responders 17
I t
t-
Non-responder 23
t -Toxic responder 5
drug or dose PGx efficacy assay
. - - - - - -- - - - - , Population of non -responders, non-toxic 1/16 (6%)
Population enriched for responders 16/21 (76%)
tltlill III llill IIIII tTreat with conventional
1\\\1111\11 Treat with alternative druq or dose
drug or dose
Fig. 1. Strategy to enrich patient populations to improve efficacy and reduce adverse drug responses using pharmacogenomic biomarkers. Safety assays include those measuring drug metabolism or on-target toxicities . Efficacy assays identify those patients who will respond to a treatment. Efficacy markers also include drug activity biomarkers that are used to determine the biologically optimum dose in clinical drug development. Adapted and reprinted, with permission, from the Annual Review of Genomics and Human Genetics, Volume 2 ©2001 by Annual Reviews, www.annualreviews.org.
THE EVOLUTION OF PHARMACOGENETIC AND PHARMACOGENOMIC TESTS • Historically, pharmacogenetics has been used to describe single gene mutations or variants assayed with low content/low-throughput techniques such as DNA restriction fragment length polymorphisms (RFLPs), DNA sequencing, or differential electrophoresis methods such as single-stranded conformation polymorphism analysis . Such single gene/low content pharmacogenetic assays have been successful for diagnosing diseases or predicting drug response wherein a single gene with a large effect contributes to the phenotype (such as autosomal-recessive familial disorders, or genes regulating a rate limiting step in drug metabolism). In the common complex diseases multiple genes contribute to the phenotype, each with a small effect. To identify these
i
Gene discovery
genetic factors pharmacogenomic approaches now include microarrays, high-throughput automated DNA sequencing and genotyping, informatics, and mass spectrometry. These new approaches are both more efficient and sensitive allowing for multiplexed analysis of mutations/variants in several genes and thus are more predictive for complex diseases wherein multiple genetic factors are involved • Figure 2 summarizes the pharmacogenomic biomarker discovery and development process . The process has several steps : - Whole genome experiments (microarray gene expression or whole genome single nucleotide
Assay development and validation
Biomarker evaluation
Increasing number of genes
Increasing number of samples
~
Fig. 2. Development flow scheme for pharmacogenomic assays.
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polymorphism [SNP] analysis) drive the discovery phase when it is necessary to analyze a large number of genes in a small to moderate number of samples (patient specimens or in pre-clinical models) - Assay development and validation involves development of specific assays to measure a subset of genetic changes that are predictiveof a specificclinical phenotype. This can include measurement of mRNA changes or genotyping using microarrays or by quantitative real time polymerase chain reaction
(PI-PCR) (TaqMan® Applied Biosystems, Foster City CA), and also includes approaches that measure proteins (e.g., Western blot, ELISA, and immunohistochemistry). Assay validation should be performed in both the original sample set as well as an expanded set of samples - Biomarker evaluation tests that the assay is robust enough to work in the appropriate biologic sample (fluid and tissue) under a variety of conditions observed in clinical applications (sample shipping, temperature variation, and so on)
GLOSSARY • Haplotype: a set of closely linked alleles (genes or DNA polymorphisms) inherited as a unit along a chromosome
• Variable numberof tandem repeat (VNTR) locus: a region of DNA that is hypervariable because of tandemly repeated DNA sequences. Presumably variability is generated by unequal crossing over or slippage during replication. The repetitive sequence is present in different numbers in different individuals of a population or in the two different chromosome homologues in one diploid individual
• SNP: a SNP is a specific position in a stretch of DNA wherein there is a single nucleotide substitution. Each alternate nucleotide is called an allele
• Pharmacokinetic (PK): biologic properties related to altered drug uptake, distribution, metabolism, or excretion of the agent administered • Pharmacodynamic (PD): biologic properties related to drug target modulation, or in its pathway, leading to altered drug efficacy • Linkage disequilibrium (LD): LD is often termed "allelic association." When alleles at two distinctive loci occur in gametes more frequently than expected given the known allele frequencies and recombination fraction between the two loci, the alleles are said to be in LD. Evidence for LD can be helpful in mapping disease genes because it suggests that the two may be very close to one another
PHARMACOKI NETICS VS PHARMACODYNAMICS • Genetic factors can affect PK and PD activities . Polymorphisms in drug metabolizing enzymes (DMEs) and transporters have their primary effect on drug PK properties, whereas those in a drug target or the target pathway primarily influence PD activities . Although these are separate biologic activities, it is ultimately the combination of PKlPD properties
that are responsible for a drug to be both efficacious and safe • The first pharmacogenetic studies were focused on PK changes and safety (polymorphisms in the metabolizing enzymes and transporters) but this has been shifting toward an increasing focus on PD variability (polymorphisms in the drug target and related pathways)
GENETIC VARIANTS • Genetic variation in the human genome takes many forms, ranging from large, cytogenetic rearrangements (including chromosomal deletions, inversions, and duplications) to single-nucleotide changes (mutations, SNPs)
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• Genetic variation can occur in the coding region of genes affecting the activity of the resulting protein or in regulatory regions of drug targets that can affect transcription, RNA splicing or stability, thereby increasing or decreasing the amount of protein present in a tissue
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Single Nucleotide Polymorphisms SNPs are the most abundant form of polymorphism in the human genome and have several advantages over previous genetic markers. The large number of SNPs and distribution across the genome provides a higher level of resolution for genetic studies than was previously possible; their power can be further enhanced by analyzing clusters of closely spaced SNPs called haplotypes, and because they are bi-allelic their genotyping and analysis can be automated.
• Synonymous SNP: generates a codon that encodes the same amino acid
handful of VNTRs have been reported to have functional consequences in gene expression or function • Genes containing functional VNTRs that affect gene expression and the encoded protein product include: insulin-like growth factor 2, the dopamine transporter solute carrier family 6, member 3, the dopamine transporter 1, dopamine receptor D4, and arachidonate 5-lipoxygenase (ALOX5) • Technologies used for VNTR genotyping are: - Southern blotting - DNA sequencing - PCR fragment sizing (gel electrophoresis)
• Non-synonymous SNP: generates an amino acid change • Nonsense SNP: generates a premature stop codon
- Denaturing high-performance liquid chromatography
Copy Number Variants (CNVs) Microsatellites • Microsatellites are also called simple sequence repeats or short tandem repeats. The usual repeat size is 1-5 bp • In contrast to SNPs, microsatellites can have multiple alleles and are hypervariable • Shorter overall length than VNTRs, and are more amenable to PCR-based analysis • Microsatellites have been largely used for family-based linkage studies
Variable Number of Tandem Repeats • In humans, different types of repetitive sequences account for approximately 50% of the genome. Subsets of these are the tandem repeats, which are consecutively perfect or slightly imperfect repeats of DNA motifs • VNTRs are defined as tandem repeats with a unit size of 6 bp or longer. Although extensively used as genetic markers in genetic linkage and forensic studies, only a
• Recent studies have discovered an abundance of submicroscopic copy number variation of DNA segments ranging from kilobases (kb) to megabases (Mb) in size • Deletions, insertions, duplications, and complex multisite variants, collectively termed CNVs or copy number polymorphisms, are found in healthy and unrelated individuals • Most of these CNV sequences may be functionally significant but this has yet to be fully ascertained • Different comparative genomic hybridization methods have been used to study copy number variations: - Comparative genomic hybridization (CGH) - SNP genotyping microarrays - Fiber fluorescent in situ hybridization (FiberFISH) • The chemokine (C-C motif) ligand 3 like-l (CCL3Ll) gene is an example of a CNV associated with a particular phenotype . Low CCL3Ll copy number was found to be associated with enhanced susceptibility to HIV infection and AIDS progression
GENETIC MARKERS FOR ASSOCIATION AND LINKAGE STUDIES • Until recently, microsatellites have been the predominant marker for family-based linkage analyses. They are abundant, well dispersed throughout the genome, and highly polymorphiclhighly informative. The increased availability of SNPs now provides an opportunity for alternative automated approaches. SNPs are more abundant than microsatellites and are also well dispersed throughout the genome, but they are less
informative than microsatellites (because they are bi-allelic). To overcome this last feature, a considerably larger number of SNP are required to achieve an information content equal to a small number of microsatellites • Each of those markers has advantages and disadvantages that should be carefully reviewed when designing genetic studies (Table 1)
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Table 1. Features of SNP and Microsatellite Genetic Markers Microsatellites
SNPs
Multi-allelic markers
Hi-allelic markers Genetically more stable Lower mutation rate
More unstable markers Higher mutation rate (_10-3)
(_10-9)
Low degree of heterozygosity «20%)
High degree of heterozygosity (-70%)
Shorter range of LD «20 kb)
Larger range of LD (-100 kb)
Need to analyze millions of SNPs to perform GWA studies
Few thousand for GWA
Capture more ancient events
Capture more recent events
More rapid and highly automated genotyping, lower overall cost and sample requirements
Expensive and time consuming genotyping and analysis, requires more DNA to analyze
Generally used for case and controls association studies
Generally used for familial based linkage studies
PHARMACOGENOMIC ASSOCIATION STUDIES • Genetic factors other than those associated with known drug metabolism and molecular drug targets affect drug safety and efficacy. Genetic association studies are used to identify such genetic factors
Candidate Gene Search • This approach directly tests the association of selected genes with particular phenotypes, drug response or ADRs • The main source of candidate genes is through hypothesis generation and literature surveys. This can include analysis of cDNA and expressed sequence tags databases that help identify new candidate genes by using sequences that are similar to genes known to be associated with a drug's activity. Whole genome expression profiling using microarrays is now routinely used to identify novel candidates that are differentially expressed in responder/non-responder cell lines or individuals • Genetic variants (e.g., SNPs and microsatellites) in candidate genes are analyzed in populations of patients treated with the drug of interest and tested for statistical association with drug response • This approach tests only known genes and therefore misses genes for which no previous involvement in drug response or particular phenotypes is unknown
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Genome Wide Association (GWA) • The candidate gene approach has considerable intuitive appeal, but is limited to the analysis of genes with known function, and excludes those whose function is not yet been determined • Increasingly robust and sensitive techniques that have been developed for the analysis of large sets of SNPs, spanning the entire genome, have now made GWA studies feasible • Genome wide analysis of random SNPs is based on LD mapping or statistical association between SNPs in proximity to each other. The number of SNPs used for a GWA study is determined by patterns and strength of LD in a given population. GWA studies rely on the assumption that LD enables one SNP to act as a marker for association to other sequence variants in that region • Large sets of affected individuals (cases) and controls are needed to perform these studies. The typical outcome is presented as one or more SNPs associated with the cases (or with drug responders) but not with the controls. Those SNPs can be used as markers to predict a certain phenotype. These studies are in general laborious and costly but are increasingly being used as they are more relevant for the analysis of complex diseases
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Candidate Pathway Approach • An appealing intermediate between the candidate gene and the genome wide approach is the analysis of wellcharacterized biologic networks/pathways including dozens or hundreds of interacting genes. Genetic markers located in those genes are used to test the large sets of patients and controls in order to identify genes that might be associated with particular disorders or phenotypes
• Increasingly sophisticated pathway analysis software and computational tools are helping to analyze how the behavior of a complex biologic system changes in response to the variation of an individual component of that network • As for other areas of genomics, pathway and biologic network analysis is expected to positively impact PGx through the association of novel genes and their function with drug response
GENETIC TESTING
• An increasing number of genetic association studies are performed to discover new genetic variants that could be translated into tests of drug response and/or disease susceptibility
- DGTlAI variants and toxicity to the anti-colon cancer drug irinotecan • Genetic tests can evaluate different types of genetic changes:
• Confirming initial pharmacogenomic associations is a challenging endeavor. Ideally different researchers, in different settings and working with different populations should obtain similar results in order to validate the association of a genetic variant with a certain phenotype
- Heritable change s (constitutional) such us the genetic variants in the cytochrome P450 DMEs
• A multitude of pharmacogenomic testing services are available directly from diagno stic labs but most of them remain at the exploratory level (sometimes called Home Brew assays) • Only few pharmacogenomic associations have been extensively validated to date. Well known and often cited example s include : - Thiopurine S-methyltransferase (TPMT) variants and toxicity to the anti-cancer drug 6-mercaptopurine (6-MP) in children with acute lymphoblastic leukemia
- CYP2C9 and VKORCI variants and dose requirements of the blood thinner drug warfarin in patient s with clotting disorders
- Non-heritable changes (somatic) such us the genetic variants observed in cancer tissues and detected by gene expression profiling or gene amplification analysis • Currently there are only a handful of marketed pharmacogenomic test kits - Herceptin f (trastuzumab therapy) for breast cancer treatment - Amplif.hip'" (Roche Molecular Systems Inc, Branchburg, NJ) test for two of the cytochrome P450 DMEs (CYP2D6 and CYP2CJ9) - Oncotype Dx" (Genomic Health Inc, Redwood City, CA) and MammaPrint Dx® (Agendia, Amsterdam, The Netherlands) gene expres sion assays in adjuvant breast cancer chemotherapy
REGULATORY ISSUES IN PGx
• As part of the Food and Drug Administration's (FDA) strategic plan, the FDA is developing standards to apply emerging technologies (e.g., PGx and other biomarkers) to provide effecti ve translation of new scientific discoveries into safe and effective medical product s • The FDA published initial guidelines for genomic data submi ssion in 2004. This guidance was intended to
encourage voluntary genomic data submission by sponsors using PGx in exploratory research during drug development • The FDA is encouraging the application of phannacogenomic approaches and data into the evaluation of patient variability during clinical drug development. This data can now be submitted to the FDA
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PHARMACOGENETICS: DMEs • DMEis a term used to define genes whose activities are involved in the metabolic activation and/orinactivation of xenobiotics (environmental chemicals) including drugs, and were the initial focus of clinical pharmacogenetics. DMEs are functionally divided into phases I and II
DMEs. Phase I DMEs convert xenobiotics to reactive intermediates through oxidation, reduction, or hydrolysis reactions, whereas phase II DMEs conjugate the reactive intermediate to a small functional group, which makes the compound more easily excreted (Figure 3A)
A Phase I DMEs (predominantly cytochrome P450)
.
.
Phase II DMEs . (including glutathione transferases, N-acetyltransferases. sulfotransferases, and UDP-glucuronosyltransferases)
Conjugated metabolite
Reactive intermediate: oxidation, reduction or hy&olysis
B UGT2B7
Morphine (10%)
Morphine-6-glucuronide (M6G) Morphine-3-glucuronide (M3G) Analgesia
Morphine (0%) No clinical effect (7-S % Caucasians)
Morphine (2o-S0X intermediate)
M6G (1o-1000X intermediate) .
Toxicity (1-2% Caucasians)
Fig. 3. Pharmacogenetics of drugmetabolism. (A)Overview of phases I andII biotransformation of drugs. Phase I DMEs generate a polar functional group through oxidation, reduction or hydrolysis that may sometimes be reactive. In drug metabolism, these newly revealed functional groups constitute an intermediate metabolite, which is subsequently conjugated by a phase II enzyme to a polarendogenous compound likea sugarthatfacilitates excretion. The balance of phases I and II activity is important to prevent theoverproduction of intermediates andendproducts thatcause oxidative stress, may be toxins, or carcinogens. Naturally occurring variants in the genes encoding DMEs result in imbalance of the system, as do environmental factors (drug-drug and drug-diet interactions). (B)Example of drugmetabolism andimpact of pharmacogenetics. Codeine is a prodrug thatpossesses littleanalgesic activity. Green text indicates metabolites with analgesic activities and red inactive metabolites. The majority of codeine is biotransformed by CYP3A4 to norcodeine. Biotransformation by CYP2D6 to morphine, and subsequently by the phase II enzyme glucuronosyltransferase UGT2B7 to M6G and morphine-3-glucuronide (M3G). Morphine and M6G have analgesic activity, whereas norcodeine and M3G are inactive. Genetic variation in CYP2D6, UGT2B7, and CYP3A4 results in different ratios of the active metabolites of codeine, resulting in different clinical outcomes including no analgesia (CYP2D6 null) and codiene toxicity (CYP2D6 ultra-metabolizers). Relative levels of morphine and M6G estimated from data reported by Gasche et al.
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• The impact of genetic factors on drug response was first noted by Arno Multulsky in a 1957 report. In the 1970s, Robert L Smith and colleagues studied the metabolism and PKs of debrisoquine (an antihypertensive drug) by administering the drug to themselves and monitoring its metabolism. In response to a standard dose, Smith soon became dizzy and hypotensive for 2 days. Drug metabolite analysis showed that Smith eliminated debrisoquine almost completely as the parent compound (poor metabolizer), rather than the 4-hydroxy metabolite observed in individuals who did not exhibit any adverse effects. Subsequent studies in larger populations showed that approximately 6-10% of Caucasians were poor metabolizers of debrisoquine, which was later shown to result from genetic variants in the CYP2D6 gene . Genotype-phenotype studies of CYP2D6 variants are now routinely performed using debrisoquine or dextromethorphan as surrogate substrates • The large number of DME genes and variants in the human genome are believed to result from the selective advantage of these variants provided during the coevolution of plants and animals. The molecular mechanisms for the functional variation of DME genes include: - Splice site mutations resulting in exon skipping (e.g., DPD, CYP2C19, and CYP3A5) - Microsatellite repeats (e.g., CYP2D6) - Gene duplications (e.g., CYP2D6) - Point mutations resulting in early stop codons (e.g., CYP2D6) - Enhanced proteinolysis (e.g., TPMT) - Altered promoter functions (e.g., CYP2A5 and UGTIAl) - SNPs causing amino acid substitutions (e.g., NAT2, CYP2D6, CYP2C9, and CYP2C19) - Large gene deletions (e.g., GSTTl, GSTMl, and CYP2D6) • There are over 70 alleles of CYP2D6, many of which alter the enzymatic activity of the encoded protein. The level of enzymatic activity varies from no activity (null), poor, intermediate (average), extensive metabolizers, and ultra-metabolizers. CYP2D6 is responsible metabolism of approximately 25% of all drugs (Table 2), and genetic variation in this gene is responsible for a significant amount of ADRs or a lack of efficacy • Although CYP2D6 poor metabolizers are responsible for most of the variability in drug efficacy and ADRs, variants that increase enzymatic activity and result in an ultra-rapid metabolism phenotype are also clinically important. Codeine is an example of a drug wherein clinical efficacy and safety is affected by rapid drug metabolism. Codeine is a prodrug that is metabolically activated to morphine and morphine-6-glucuronide (M6G) by the CYP2D6 and UGT2B7 . Codeine clearance is modulated by another phase I DME,
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Table 2. Common Drugs That are Metabolized by CYP2D6, Pro-Drugs Activated by CYP2D6 are Indicated byTamoxifen and Codiene
Class
Drug S-metaoprodol
~-blocker
Propafenone
~-blocker
Timolol
~-blocker
Dextromethorphan
Anti-tussive
Paroxetine (Paxil)
Anti-depressant
Haloperidol
Anti-psychotic
Resperidone
Anti-psychotic
Thioridzine
Anti-psychotic
Tamoxifen
Anti-estrogen-breast cancer prevention
Codiene
Analgesic
Table 3. Prevalence of CYP2D6 and CYP2C19 Poor Metabolizers in Different Ethnic Groups Population
PM phenotype (%) Median
CYP2D6 African Asian Caucasian CYP2C19 African (Sub-Saharan) Asian Caucasian Middle EastJNorth Africa
3.4 0.5 7.2 4 15.7 2.9 2
CYP3A4. Genetic variation in CYP2D6 has been shown to be associated with lack of efficacy and toxicity (Figure 3B) • The frequency of phenotypic variation in DMEs varies in different populations, as shown for CYP2D6 and CYP2C19 poor metabolizers in Table 3 . The variation in
metabolic frequencies between different ethnic populations, and the large number of variants that affect metabolism highlights the essential need for a costeffective and accurate genotyping platform to make clinical pharmacogenetics a reality
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• Initial DME genotyping assays utilized RFLPor PCR technology, but were technologically cumbersome, time consuming, and error prone. Recent advances in microarray technology now allowfor the genotyping of a largenumber of different alleles on a singleassay platform. The AmpliChip CYP450 is a microarray for genotyping clinically relevant CYP2D6 and CYP2CJ9 variants (Figures 4 and 5). The use of microarray technology allows for a single standardized platform, reduced cost, and more robustdata, which will speed the transition of DME genotyping from the laboratory to the patient
• The extensive knowledge on genetic variation in DMEs has led to rational adjustment of dose or dosinginterval, and to appropriate warnings or precautions. For example, the labeling of atomoxetine (Strattera, Eli Lilly and Company, Indianapolis, IN), thioridazine (Mellaril, Novartis Pharmaceuticals, Basel, Switzerland), voriconazole (Vfend, Pfizer, NewYork, NY), and irinotecan (Comptosar, Pfizer, NewYork, NY, Pharmacia and Upjohn) now contain information about the genetics of metabolizing enzymes
Amplichip CYP450 Microarray for CYP2D6 and CYP2C19 Genotyping
Each 20 llm 2 cell on the array can contain 107 DNA fragments . or "probes"
Fig. 4. Microarray assay platform for the comprehensive analysis of DME polymorphisms. The Amplichip CYP450 is an oligonucleotide array that can detect alleles of CYP2D6 and CYP2CJ9 that affect drug metabolism. Each array contains over 15,000 different olionucleotide probes, approximately 240 for each polymorphism. Genomic DNA is amplified in two multiplexed PCR reactions, and hybridizedto the array. The hybridization to specificsets of oligonucleotides representingspecific alleles is detected by fluorescent staining of a biotin label incorporated into the sample. System software is then used to detect and call the genotype. The microarray format allows for the incorporation of a large number of controls and a standardize assay system which increases the accuracy of the data.
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Fig . 5. Genotyping results from Amplichip CYP450 array for CYP2D6 gene. Allele number indicates change nucleotide sequence of the gene (http ://www.cypalleles.ki.se/cyp2d6.htm) and color indicates predicted phenotype. The use of a single assay platform improves the accuracy of gentoyping. Full detail s can be found in the package insert for the product (http ://www.amplichip.us/presslounge/).
WARFARIN (COUMARIN): AN EXAMPLE OF DME AND TARGET VARIATION AFFECTING SAFETY AND EFFICACY (THERAPEUTIC INDEX) • Warfarin is a commonly prescribed anti-coagulant drug for the prevention and treatment of venous and arterial thromboembolic disorders. However, warfarin therapy is difficult to manage because of the drug's narrow therapeutic index and the wide inter-individual variability in both drug metabolism and efficacy (e.g., resistance) • Bleeding is a severe side effect of warfarin, both gastrointestinal and cerebral bleeds are of particular concern; therefore the usual protocol is to start on a low dose and "titrate" to achieve the desired degree of anticoagulation. However, dose titration has been proven ineffective in many cases and carries inherent risk for adverse effects • CYP2C9 is associated with warfarin toxicity as a result of drug metabolism. This gene encodes the enzyme responsible for metabolizing more potent S-enantiomer form of warfarin to inactive metabolites • Since the initial cloning of CYP2C9, a large number of allelic variants of the gene have been associated with drug response to warfarin. The two commonest are CYP2C9*2 , in which cysteine substitutes for arginine at amino acid 144 (RI44L), and CYP2C9 *3, in which leucine substitutes for isoleucine at residue 359 (I359L) .
Both are well known to result in decreased S-warfarin metabolism. Patients with these variant alleles require significantly lower doses of warfarin than patients with the wild-type gene. The frequency of carriers for one or more of these variants is roughly 30% of the general population • In 2004, coding-region mutations in vitamin K epoxide reductase complex, subunit 1 (VKORC1, warfarin 's pharmacologic target) were found to cause a rare syndrome of warfarin resistance. This discovery led to the investigation of the association of other VKORC I genetic variants with drug response to warfarin . This is an example of variable drug response related to variation of the target gene • Common variants in VKORCI have now been found to account for a much greater fraction of variability in warfarin response (21%) than do variants in CYP2C9 (6%). VKORC1 haplotypes can be used to stratify patients into low, intermediate, and high dose warfarin groups • This example illustrates the importance of making a comprehensive pharmacogenetics analysis including PD and PK aspects. Recently clinical trials have been initiated to assess the extent to which pharmacogenomic
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testing will reduce adverse effects with warfarin treatment (www.fda.gov/OHRMSIDOCKETS/AC/OS/ minutes/200S-41 94M l.pdt). Translation of this
knowledge into clinical guidelines for warfarin use will be likely to have a major impact on the safety and efficacy of warfarin
PGx OF ASTHMA TREATMENT
• Asthma affects an estimated 300 million individuals worldwide, resulting in substantial morbidity, mortality, and healthcare utilization . Asthma is a complex respiratory disease characterized by airway inflammation and reversible airflow obstruction. The prevalence of the disease has increased dramatically over the past two decades, accentuating the need for effective pharmacologic treatments. Over the past few years, substantial effort has been made to explore how a patient's genotype determines asthma drug efficacy and ADRs • Response to the three major classes of asthma therapy, p-agonists, leukotriene antagonists, and inhaled corticosteroids demonstrates wide inter-individual variability, including a significant number of nonresponders. Available data suggests that genetics contributes as much as 60-80% to the inter-individual variability in treatment response. Here we present two different examples of genetic variants that have been show to affect drug response and could eventually help in asthma patient stratification to improve drug response
B2-Agonists • B2-adrenergic agonists (e.g., albuterol) are the first line therapy for bronchodilatation in asthma patients. They act through binding to B2-adrenergic receptors (B 2AR) , a cell surface G protein-coupled receptor. The B,~R gene contains numerous SNPs and some of these polymorphisms may act as disease modifiers in asthma and may be responsible for part of the known interindividual variation in the bronchodilating response to B2-agonists • The B,~R variants Arg16Gly, Gln27Glu, and Thr164I1e appear to be functionally relevant based on data generated using cell gene transfection systems, from ex vivo studies from individuals with known genotype, and from population studies (Table 4). These "nonsynonymous" variants result in amino acid substitutions altering receptor structure and therefore appear to be the most clinically relevant • These polymorphisms are common, with allele frequencies as high as SO% in some cases , and have been associated with secondary effects related to the chronic use of long-acting p-agonists and also with general response to B2-agonists in asthmatic patients • In addition to studies of single base pair changes, there have also been studies focused on the analysis of
2S2
haplotypes and their association with drug response in asthma patients. One example is a haplotype of 13 SNPs in the promoter and coding region of B,~R that suggest altered receptor function correlated with clinical phenotype • Despite these developments for B2-agonist PGx, significant gaps in understanding the genetics remain and need to be answered before clinical pharmacogenomic assays can be developed . It is still unknown if other polymorphisms that occur in the non-coding and flanking regions of the B-zAR gene may be important in regulating B-zAR expression, thereby affecting individual drug responses. Little is known about other B-zAR related genes (same pathway or transduction cascade) that may playa role in asthma drug response • Owing to the complexity of the action of B 2-agonists and its therapeutic response, a broader genomic/genetic analysis (including other genes in the same pathway and other-non-coding regions-of the B-zAR gene) will help in optimizing therapy for the individual patient
Leukotrienes • Leukotrienes are important mediators of asthma as they induce bronchoconstriction, tissue edema, and airway secretion. Therefore, inhibition of leukotriene activity has therapeutic benefit in asthma patients . Leukotrienes are synthesized from arachidonic acid by 5-lipoxygenase (ALOX5) and several pharmacologic studies for leukotriene inhibition have focused on genes from the ALOX5 pathway, mainly 5-lipoxygenase (ALOX5) and S-lipoxygenase activating protein (ALOX5AP) • Studies have associated clinical response for a selective inhibitor of ALOX5 with VNTR variations in the promoter region of the ALOX5 gene, which naturally varies in the general population. The number of repeat copies is associated with changes in gene expression and promoter activity. Patients carrying the common length VNTR (5 copies) have normal gene expression levels whereas patients carrying the less common or mutant variants of the VNTR (3,4, 6, and 7 copies) show diminished gene transcription with decrease ALOX5 production and a diminished clinical response to treatments with drugs targeting this pathway. Individuals homozygous for any of the mutant alleles (3, 4, 6, or 7) demonstrated significantly decreased response as measured by lung function tests when compared with individuals heterozygous or homozygous for the common
Clinical Pharmacogenomics
10-13
Table 4. Summary of Polymorphisms, in Two Different Genes That Affect Asthma Drug Treatment Pathway
Gene
Location
P2-adrenoceptor
B~R
5q31.32
16 (Arg -> Gly) 27 (GIn -> Glu) 164 (Thr- >Ile)
Decreased response to P2-agonists Reduced bronchial responsiveness Potentiall y decreased response to P2-agonists
ALOX5
lOql1.12
Number of tandem (VNTR) Sp-l and Egr-l binding sites in promoter region
Decreased response to the ALOX5 inhibitors
Leukotriene synthesis
Polymorphism
Associated phenotype
These examples must be consideredin the light that until recently PGx studies were limited in scope and considered only a verysmall subset of variations in a few genes. These examples show the wide range of genomic variants that might influence drug response
or wild-type allele. However, there is some discrepan cy in the data published so far with some articles indicating that the mutant VNTR is associated with increase gene expression and increase leukotriene B4 production and inflammation • The distribution of the VNTR rare allele s varies; it is present in 6% of the North American population, and has been shown to vary across different ethnic group s with higher prevalence among Asians (19.4%), blacks (24%) than among Hispanic (3.6%), and non-Hispanic whites (3.1%). This variability in allele distribution indicate s that response to ALOX5 inhibitors will not be the same in all the human populations
• This example highlights the importance of the variation in the regulatory (promoter) region of genes , in addition to the coding regions of the gene itself, and of the differences in human populations • The complexity in understand ing drug response to leukotriene inhibitors is illu strated by the fact that onl y 6% of asthma patients carry the rare VNTR alleles at the ALOX5 promoter locu s, but >6 % of asthmatic patients do not respond to leukotriene inhibitors. Thu s, there are likely other genetic variant s in this pathway, yet to be identified, that playa role in leukotriene regulation
PGx OF CARDIOVASCULAR DISORDERS
• Cardio vascular disease is one of the leading causes of morbidity and mortality, and drug therapy is a major modality to attenuate its burden. At present, conditions such as hypertension, lipid disorder s, and heart failure are pharmacologically managed with an empirical trial-and-error approach. However, this approach fails to adequately addre ss the therapeutic needs of many patients, and pharmacogenetics has been explored as a tool to enhance patient-specific drug therapy • There are a number of studies in the published literature that provide proof of concept that genetic variation contributes to the variable respon se that is observed on administration of cardiovascular drugs. Some of these examples are discussed here, mainly focusing on the PD aspects (genetic variants in drug targets)
Hypertension • Hypertension is the most common chronic disease in the western world. Even with the large number of drugs from which to choose therapy, only 34% of North American s respond to these treatments • The renin-angiotensin system plays an important role in blood pressure (BP) regulation , and previous studie s have reported that response to anti-hypertensive medications is influenced by genetic variation in the renin-angiotensin-aldosterone system
Angiotensin-Converting Enzyme (ACE) • ACE is essential for the production of angiotensin II and for the degradation of bradykinin, two peptides involved in vascular physiology and regulating BP. ACE inhibitors reduce peripheral vascular resistance and therefore reduce BP
253
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Molecular Genetic Pathology
• Several studies have associated an insertion/deletion (lnJDel) polymorphism of the ACE gene with the occurrence of cardiovascular diseases, myocardial infarction and drug response to ACE inhibitors. The ACE lID polymorphism results from the presence or absence of a 287-bp DNA fragment in intron 16 of the ACE gene on chromosome 17. This intronic variation shows tight LD with the clinical phenotype, but the causative (functional) variant has not yet been determined • The D allele has a frequency of approximately 0.53 in Caucasian populations and has been associated with higher levels of ACE activity. Some studies have shown that individuals homozygous deletion (Del/Del) appear to have a highest response to ACE inhibitors whereas individuals homozygous for the insertion (InJIn) genotype show a lower response. Differences in plasma ACE activity associated with the ACE genotype that affect the therapeutic response of ACE inhibitors explain in the inter-individual variability in cardiovascular or renal response to equivalent doses of ACE inhibitors • However, clear patterns of association with the ACE InJDel polymorphism have failed to emerge, with as many studies pointing to the increased response of 1allele carriers as studies pointing to D-allele carriers. Other studies have found no pharmacogenetic effect of the InJDel polymorphism and ACE inhibitors on BP and related outcomes. These divergent results suggest other genetic and environmental factors are associated with response to ACE inhibitors
Angiotensinogen (AGT) Gene • A polymorphism in the AGT gene, M235T, wherein the T allele is associated with higher plasma AGT has been linked to elevated blood pressure (BP) and myocardial infarction . However, neither the M235T nor the Tl74M polymorphisms, both in exon 2, seem to affect function, secretion, or metabolism of AGT ~-Blockers
• ~-Blockers (e.g., atenolol and bisoprolol fumarate) are a first-line treatment for hypertension . Several studies have found significant pharmacogenetic effects with ~-blockers, including studies that reported an association
in the ~-l-adrenergic receptor Arg389 variant and response to ~-blockers. Other polymorphisms have been described but most of them have not been confirmed in independent populations • Pharmacogenetic studies of ~-blockers have been complicated by relatively small sample size and had variable durations of treatment. However, in aggregate they suggest that variants in the ~-l-adrenergic receptor gene may play a future role in determining response to ~-blockers
PGx of Lipid-Lowering Agents • The prevention of cardiovascular disease has been greatly facilitated by lipid-lowering therapy including 3-hydroxymethyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statin drugs, including pravastatin and atorvastatin) and cholesterol absorption inhibitors. These drugs are generally well tolerated but severe adverse effects occurs in a small number of patients and there is also a subset of patients that do not respond • Studies have shown that SNPs in HMG-CoA are significantly associated with reduced efficacy of pravastatin . Individuals heterozygous for variants in the HMG-CoA reductase gene may experience significantly smaller reductions of cholesterol (22% lower reduction of total cholesterol and 19% lower reduction of low-density lipoprotein) when treated with pravastatin . However, these initial results have not yet been confirmed in other populations
Emerging Trends in Cardiovascular Pharmacogenetics • Although numerous polymorphisms in several genes have been associated with drug response to ~-blockers, ACEinhibitors , and statins, additional studies are required to translate the PGx of cardiovascular disorders into clinical practice . Future studies are required that use adequately sized patient cohorts, reduce variability in methodology, and assess complexity of the larger biologic organization (e.g., gene networks and pathways) Additional challenges include the applicability of pharmacogenetic findings across specific population groups as a number of report show differences in cardiovascular disorders and severity among different racial categories
PGx OF NEUROPSYCHIATRIC DISEASES
• Impressive advances have been made in the genetics of neuropsychiatric diseases . Synergies between genetic studies, elaboration of intermediate phenotypes, and novel applications in neuroimaging are revealing the effects of positively associated disease alleles on aspects of neurologic function
254
• Genetic and pharmacogenomic studies suggest that the sub-categorization of individuals based on various sets of susceptibility alleles, could make the treatment of neuropsychiatric more predictable and effective • Much of the pharmacogenomic and pharmacogenetic data available today has come from psychiatric disorders as
10-15
Clinical Pharmacogenomics
well as from neurologic conditions often resulting in psychiatric sequelae (e.g., Alzheimer's and Parkinson's disease), and these areas will be the focus of this chapter
Alzheimer's Disease • Genetic polymorphi sms in the apolipoprotein E (APOE) gene are associated with predicting response to therapy for Alzheimer 's disease as well as for lipid-lowering drugs • There are numerous allelic variants of the human APOE gene (e.g., APOEe2, APOE£3, APOEe4, and so on), which contain one or more SNPs that alter the amino acid sequence of the encoded protein • Studies with tacrine (a cholinesterase inhibitor) to treat Alzheimer's disease have shown that 83% of patients lacking the APOEe4 allele had a positive response to tacrine as compared with 40% of the patients with at least one APOEe4 allele . However, the greate st improvement was observed in a patient with a single APOEe4 allele , the unfavorable genotype, illustrating that a single gene will not always predict the respon se to a given treatment • Follow-up studies indicate that the prognostic value of APOE genotype for tacrine treatment was stronge st in female patient s, suggesting that additional genes and other factors may be involved in the response to tacrine treatment • Other studies have suggested that patient s not carrying the APOEe4 allele respond to PPARyagonist rosiglitazone (cognitive and functional improvement), whereas APOEe4 allele carriers showed no improvement • Analysis of APOE has shown association between APOE genotype and susceptibility to Alzheimer's and response to treatment. However, prospective clinical evaluation with robust clinical end point s and sufficient sample size are needed to define better the usefulne ss of the clinical implementation of an APOE pharmacogenetics test
Parkinson's Disease • More than 50% of Parkinson's disease (PD) patients treated with L-DOPA develop L-DOPA-induceddyskinesias (LIDs). Some patients exhibit severe dyskinesia soon after starting low doses of L-DOPA, whereas other patients remain free of this disabling complication despite long-term treatment. Avoiding or delaying the appearance of LIDs is a major issue in the management of PD • Some studies have associated several genetic polymorphisms with the risk of developing LIDs, including variation in the dopamine receptors 2, 3, and 5 • Genetic predisposition to LIDs is likely to involve several distinct genes (or multiple allele s), each producing a small effect that might increase the risk of developin g LIDs by two- to threefold
Major Depression • Serotonergic activity is thought to play an important role in the regulation of mood, motor activity, and sleep
patterns. Serotonin re-uptake is controlled by the serotonin transporter gene (SERT or SLC6A4)
• SERT displays an lID polymorphi sm in its promoter region (5-HTILPR), which is the presence or absence of a 44-bp insertion. This variant results in a bi-allelic polymorphism designated long (I) and short (s). The shorter variant of the promoter is associated with reduction in the transcriptional efficiency of the gene resulting in decreased gene expression • Anti-depressant efficacy of selective serotonin re-uptake inhibitors (SSRls) has been shown to depend on this functional promoter polymorphism. It has been reported that carriers of the short allele have a poor outcome after treatment with SSRls and a higher rate of adverse effects whereas individuals homozygous for the long variant have two times more efficient response to SSRls and might have a better long-term outcome when treated with anti-depressants • However, contradictory results that might be explained by interethnic difference s, or differences in haplotypes, have been reported. Therefore, sufficiently large and wellplanned, controlled randomized studies are needed to finally prove that anti-depressant therapy might benefit from SERT genotype diagno sis
Schizophrenia • There is substantial unexplained inter-individual variability in the drug treatment of schizophrenia with an important proportion of patient s that respond inadequately to anti-psychotic drugs, and many experience limiting side effects. Converging data suggest that the identification of the molecul ar variants that influence anti-psychotic drug response and adverse effects may soon be feasible • For the most part , the pharmacogenomic studies in schizophrenia have focu sed on the secondary effects of the new atypical anti-psychotic agents . One of the most studied of such secondary effects is weight gain associated with those agents. Weight gain appears to be a serious side effect encountered during treatment with many anti-psychotic drugs. Although the propensitie s of induc ing weight gain vary con siderably between agents, this adverse effect is mostly obser ved with administration of atypical anti-psychotic drugs • A specific SNP in the 5-hydroxytryptam ine 2C (5-HT2c) receptor has been associated with weight gain across diverse samples. Several recent reports have linked a -759Crr polymorphi sm of the 5HT2C receptor gene with chlorpromazine, risperidone, and clozapineinduced weight gain • Aside from adverse effects some studies have suggested that variation in the gene that codes for the dopamine D2 receptor may significantly influence the clinical efficacy of a number of typical and atypical anti-psychotic drugs. Some of these polymorphi sms are the -141C In/Del
255
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Molecular Genetic Pathology
polymorphism and the TaqI polymorphism. Although several advances have been made, particularly in understanding the pharmacogenetics of some limiting
side effects, genetic prediction of drug response remains elusive and more studies with larger and better-defined populations are needed
CANCER PGx
• Tumorigenesis is a multi-step process whereincells acquire somatic genetic alterations followed by clonal expansion of those carrying alterations providinga growth advantage. This results in a tumor that has a genotype unique from the normal tissue which it was derived. Thus, cancer is a genetic disease, which makes it ideally suited for the use of genetic assays for tumor classification/outcomes and POx • The use of POx in cancer is further driven by the narrow margin of safety (MOS-the ratio the toxic dose/ therapeutic doses)for cancer cytotoxic drugs, and the emergence of targeted therapies such as trastuzumab (Herceptin"), imatinib (Gleevec"), and gefitinib (Iressa'") that have efficacy only in tumor harboring specific genetic alterations. The first widespread use of a pharmacogenomic companion diagnostic to assess drug targetstatus was for trastuzumab • Detailed descriptions of cancer POx are provided in other chapters. Here, we will provide a brief historical overview and frame the current status of pharmacogenomic applications
Monogenic Cancer Pharmacogenetics; TPMT • Thiopurine drugs are used to treat leukemia (acute myelogenous leukemia and acute lymphoblastic leukemia), autoimmune diseases (inflammatory bowel disease, systemic lupus erythematosus, and rheumatoid arthritis), and organ transplantpatients. This class of drugs includes 6-MP, 6-thioguanine, and azathioprine (6-MP prodrug). Myelosuppression is the primary toxicity observed with this class of drugs • TPMT is a cytosolic enzyme that catalyzes the S-methylation of these drugs using S-adenosyl-L-methionine (SAM) as a methyl donor (Figure 6). Inherited variation in TPMT was first reported in 1980 by Weinshilboum and Sladek. Measuring TPMT enzymatic activity in red blood cells of unrelated adults and in families, they showed a distribution of TPMT that conformedto Hardy-Weinberg prediction for codominant autosomal alleles for high and low activity, and suggested inherited variation TPMT was responsible in part for the clinical response/adverse events of thiopurines. Approximately 89% Caucasian and AfricanAmericans have high activity, II % intermediate, and
256
known to be because of sequence variation. Genomic characterization of the TPMT gene has shown that the gene is located on chromosome 6p22.3 and consists of 10 exons. Twenty one TPMT polymorphisms have been identified, 18 of which are non-synonymous SNPs (alleles *2, *3A, *3B, *3C, *5- 14, and *16-*19), two of which alter mRNA splice sites (alleles *4 and *15), one premature stop codon (*3D), and one changing the translational start codon (*14) • The most common alleles for low activity in Caucasians are *2, *3A, and *3C all which result in a protein with essentially no enzymatic activity. The mechanism by which this mutation enhanced protein ubiquitin proteasomemediated degradation as a result of protein misfolding and aggresome formation • The pharmacogenetic analysis of TPMT allows for strategies for eliminating thiopurine toxicity and improving efficacy by adjustingdose based on TMPT genotype. Several genotyping and phenotyping methods are used including RFLP-PCR, direct DNA sequencing, single-stranded conformation polymorphism, and denaturing high-performance liquid chromatography analysis, and TPMT enzymatic/metabolite measurement in erythrocytes • Significant dose adjustment (6-10% standard dose) is recommended for patients with low TMPT activity. Although more controversial, studies suggest that dose adjustment (-65 % standard dose) reduces ADRs in intermediate patients
Multi-Gene Cancer PGx-Microarray Expression Profiling • The first whole genome analysis of genomic changes in human cancers showedthe number and complexity of genomic changes was significantly different from what was predictedfrom previous studies. The first large-scale cancer genome re-sequencing projects focused on the analysis of genes that were known to be mutated in cancer (i.e., kinases). Stephenset al. re-sequenced essentially all human kinases (n =518) in a small set of 25 breast cancers. This was followed with larger studies in differenttumor types and with a larger set of genes. In general, although these studies found mutations in genes known to be altered in cancer, the number of novel alterations was larger and the distribution and frequency of known cancer genes more complex than previously
(I inical Pharmacogenomics
10-17
S-adenosyl-Lmethionine
A
adenosyl-.L-
(SA~ 2~:te,"e
6-Mercaptopurine (6-MP)
6-Methylmercaptopurine (6-MMP)
...
v~ ~~~.+~.~
B
Allele: Nucleotide (Amino acid):
c
t
TPMT*2 G238C (Ala80Pro)
'\
/""-
TPMT*3A G240A (Ala154Thr) A719G (Tyr240Cys)
Genotype
Caucasian frequency
WTNIT
89.8
High
WT I*2
0.5
Intermediate
TPMT*3C A719G (Tyr240Cys)
TPMT phenotype
WTI*3A
8.0
Intermediate
WT I*3C
0.6
Intermediate
' 3N'3A
0.4
Low
'3N'3C
0.2%
Low
Fig. 6. Polymorphisms in the TPMT gene. (A) illustrates the enzymatic activity of TPMT. Using SAM as a methyl donor, TPMT catalyzes the S-methylation of 6-MP to 6-methylmercaptopurine. Yellow indicate sulfur atom, blue-nitrogen, gray--carbon, and cyan-hydrogen. (B) Structure of the TPMT gene . There are a total of 10 exons, eight of which encode the protein . Coding regions are indicated by green , and non-coding by red. Location of the three most common genetic variants is shown below. Allele *3A contains two non-synonymous SNPs resulting in amino acid changes in exons 7 and 10. A VNTR located in the promoter region has been shown to be polymorphic, but the significance of this VNTR on TPMT activity is not yet been determined. (C) Genotype-phenotype correlation in Caucasians for the three most common alleles (*2, *3A, and *3C). Phenotyping is performed in erythrocytes measuring enzymatic conversion of 6-MP to 6-MMP using radio-labeled SAM .
thought. The results of these and other studies have led to the development of the National Institute s of Health (NIH) funded project, The Cancer Genome Anatomy (http://cancergenome.nih.gov). Currently The Cancer Genome Anatomy is funding a 100 million USD pilot to globall y assess genomic changes in cancer, including somatic mutations, transcriptional change s, and epigenetic alterations such as DNA methylation and histone modifications • One genomic approach that has already been exploited to analyze clinical PGx of cancer is whole genome expression profiling using microarrays. Microarrays are
manufactured and automated tools that allow for the analysis of a large number of nucleic acid sequences on a small solid support. Current microarray technology is based on technology first described by Ed Southern in 1975 for the immobili zation of nucleic acids on a solid support. Currently, there are two predominant microarray technologies, cDNA and oligonucleotide arrays, and since the first description of cDNA microarrays in 1995 and oligonucleotide arrays in 1996, their use in cancer research has rapidly expanded and is now an establi shed method of classifying tumors and to predict clinical outcome (survival, response to treatment)
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Molecular Genetic Pathology
10-18
• The rapid uptake of mircoarray technology in cancer research has resulted in the development of the first disease-specific multi-gene pharmacogenomic assays ; the Mammaprint for breast cancer patient stratification (risk of recurrence), and oncotype Dx which is used to assess the need for adjuvant
tomoxifen treatment in estrogen receptor positivel node-negative breast cancer. These assays are a significant advancement in clinical PGx, and are described in greater detail in Chapter 8
WEB RESOURCES
• Professional education for genetic assessment and screening-http://www.pegasus.nhs.ukl
• NCBI-http://www.ncbLnlm.nih.gov/ • PharrnGKB-http://www.pharrngkb.orgl • Genetests-http://www.genetests.org/ • NIH biomarkers-http://ospp.od.nih.gov/biomarkersl • FDA-http://www.fda.gov/cder/genomics/default.htm
• PGx for every nation-http://pgenLunc.edul • National coalition for health professional education in genetics-http://www.nchpeg.org/
• Comprehensive research on expressed alleles in the therapeutic evaluationhttp://pharmacogenetics.wustI.edu/ • Personalized medicine coalitionhttp://www.personalizedmedicinecoalition.org/ • Drug interaction websitehttp://medicine.iupui.edulflockhartl
SUGGESTED READING Basile VS, Masellis M, De Luea V, et al, 759Crr genetic variation of 5HT(2C) receptor and clozapine-induced weight gain. Lancet 2002;360:1790-1791 . Cheok MH, Evans WE . Acute lymphobl astic leukaemia: a model for the pharmacogenomics of cancer therapy. Nat Rev Cancer 2006;6:117. Davies H, Hunter C, Smith R, et al, Somatic mutations of the protein kinase gene family in human lung cancer. Cancer Res. 2005;65:7591-7595. Drysdale CM, McGraw DW, Stack CB, et aI. Complex promoter and coding region beta 2-adrenerg ic receptor haplotypes alter receptor expression and predict in vivo responsivene ss. Proc Natl Acad Sci USA 2000;97:10,483-10,488. Dwyer JH, Allayee H, Dwyer KM, et al, Arachidonate 5-lipoxygena se promoter genotype, dietary arachidonic acid, and atherosclerosis. N Engl J Med. 2004;350:29-37. Evans WE, Hon YY, Bomgaars L, et aI. Preponderance of thiopurine Smethyltransferase deficiency and heterozygosity among patients intolerant to mercaptopurine or azathioprine. J Clin Oncol. 2001;19:2293-2301. Farlow MR, Lahiri DK, Poirier J, et al. Apolipoprotein E genotype and gender influence response to tacrine therapy. Ann NY Acad Sci. 1996;802:10I-I 10. Gasche Y, Daali Y, Fathi M, et al. Codeine intoxication associated with ultrarapid CYP2D6 metabolism . N Engl J Med. 2004;351:2827- 2831. Heils A, Teufel A, Petri S, et aI. Allelic variation of human serotonin transporter gene express ion. J Neurochem. 1996;66:2621-2624.
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Israel E, Chinchilli VM, Ford JG, et al, Use of regularly scheduled albuterol treatment in asthma : genotype-stratifi ed, randomised, placebo -controlled cross-over trial. Lancet 2004 ;364 : 1505-1512. Kalayci 0, Birben E, Sackesen C, et al. ALOX5 promoter genotype , asthma severity and LTC production by eosinophils. Allergy 2006;61:97- 103. Kirchheiner J, Fuhr U, Brockmoller J. Pharmacogeneti cs-based therapeut ic recommend ations-ready for clinical practice ? Nat Rev Drug Discov.2oo5 ;4:639-647 . Krynetski EY, Tai HL, Yates CR, et aI. Genetic polymorphi sm of thiopurine S-methyltransferase: clinical importance and molecular mechanisms. Pharmacogenetics 1996;6:279-290 . Liggett SB, Mlalet-Perez J, Thaneemlt-Chen S, et aI. A polymorphism within a conserved beta(l )-adrenergic receptor motif alters cardiac function and beta-blocker response in human heart failure. Proc Natl Acad Sci USA 2006;103:11.288-11,293. Linazasoro G. New ideas on the origin of L-dopa-induced dyskinesias: age, genes, and neural plasticity. Trends Pharmacol Sci. 2005;26(8):391-397. Marshall E. Preventing toxicity with a gene test. Science 2003;302:588-590. Motulsky AG. Drug reactions enzymes, and biochemical genetics. JAm MedA ssoc.1957;165:835-837 . Nebert DW. Polymorphisms in drug-metaboli zing enzyme s: what is their clinical relevance and why do they exist? Am J Hum Genet. 1997;60:265-271.
Cl ini cal Pharmaco genomi cs
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Palm er CN, Lipw orth BJ , Lee S, et aI. Arginine-16 beta2 adrenoceptor genotype predisposes to exacerbation s in young asthmatics taking regular salmeterol. Thorax. 2006;6 1:940-944.
Smith RL. The Paton Prize Award. The discovery of the debrisoquine hydroxylation polymorphism: scientific and clinical impact and consequences. Toxicology 200 I; 168(1):11- 19.
Poirier J , Delisle MC, Quirion R, et aI. Apolipoprotein E4 allele as a predi ctor of choline rgic defi cit s and treatment outcome in Alzheimer disease. Proc Natl Acad Sci USA 1995;92: 12,260-12,264 .
Southern, EM . Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Bioi. 1975;98(3):503-517 .
Ried er MJ, Rein er AP, Gage BF, et aI. Effect of VKORC I haplotypes on transcriptio nal regulation and warfarin dose. N Engl J Med. 2005;352:2285-2293. Risner ME, Saunders AM, Altma n JF, et aI. Efficacy of rosiglitazone in a genetically defined population with mild-to-moderate Alzheimer's disease. Pharmacogenomics J. 2006;6:246-254. Schafer M, Rujescu D, Giegling I, et aI. Association of short-term response to haloperidol treatment with a polymorphism in the dopamine 0 (2) receptor gene. Am J Psychiatry 200 I;158:802- 804. Sjoblom T, Jones S, Wood LD, et aI. The consensus coding sequences of human breast and colorectal cancers. Science 2006;314:268-2 74.
Stephens P, Edkins S, Davies H, et aI. A screen of the complete protein kinase gene family identifies diverse patterns of somatic mutations in human breast cancer. Nat Genet. 2005;37:590-592. Taylor DR. Pharmacogenetics of beta2-agonist drugs in asthma. Clin Rev Allergy lmmunol. 2006;31:247- 258. Wang L, Weinshilboum R. Thiopurine S-metbyltransferase pharmacogenetics: insights, challenges and future directions. Oncogene 2006;25:1629. Weinshilboum RM, Sladek SL. Mercaptopurine pharmacogenetics: monogenic inheritance of erythrocyte thiopurine methyltransferase activity. Am J Hum Genet. 1980;32:651-{)62. Wilrrert B, Zaal R, Brouwers JR. Pharmacogenetics as a tool in the therapy of schizophrenia. Pharm World Sci. 2005;27:20-30. Winkelmann BR, Russ AP, Nauck M, et al. Angiotensinogen M235T polymorphism is associated with plasma angiotensinogen and cardiovascular disease. Am Heart 1. 1999;137:698-7 05.
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11 Clonality Analysis in Modern Oncology and Surgical Pathology Liang Cheng,
Shaobo Zhang, MD, Timothy D. Jones, and Deborah E. Blue, MD
MD,
MD,
CONTENTS
I. Clonal Expansion is the Hallmark of Neoplasia
11-3
Overview Tumorigenesis Models Monoclonal Tumor Model Multi-Step Carcinogenesis Polyclonal Tumor Model Patch Phenomenon
11-3 11-3 11-3 11-4 11-5 11-6
II. X Chromosome-Linked Clonality Analysis
11-7
Principle and Implication of X Chromosome Inactivation 11-7 X Chromosome Inactivation Control Mechanisms 11-9 Human Androgen-Receptor Gene X Chromosome Inactivation Analysis 11-9 Advantages and Limitations 11-12 Technical Considerations 11-12 Other X Chromosome-Linked Clonality Analyses 11-15 Selected Applications 11-16
III. Loss of Heterozygosity (LOH) as a Clonal Marker Overview Evaluation and Interpretation Advantages and Limitations Technical Considerations
11-1 7 11-17 11-18 11-19 11-19
Allele Drop-Off Loss of Sensiti vity LOH in Normal Cells Methods of LOH Analysis Radioisotope PCR Incorporation-Gel Electrophoresis High-Resolution Fluorescent Microsatellite Analysis High-Performance Liquid Chromatography (HPLC) High-Density Oligonucleotide Single Nucleotide Polymorphism Array Selected Applications
IV. Other Methods of Clonality Analysis Somatic Mutation Gene Rearrangement Analysis Cytogenetic Analysis and FISH DNA Methylation as a Clonal Marker Micro satellite Instability Viral Integration Analysis Comparative Genomic Hybridization (CGH) Gene Expres sion Profiling/Array-Based Clonality Analy sis MicroRNA Signatures Protein-Based Clonality Analysis
11-19 11-19 11-19 11-19 11-19 11-20 11-20
11-20 11-20
11-21 11-21 11-23 11-23 11-25 11-25 11-27 11-27 11-28 11-28 .11-28
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Molecular Genetic Pathology
11-2 V. Tissue Contamination and Patient Identity Mismatch Testing Overview Technical Approaches Caveats
VI. Identification of Donor Origin in Transplantation Patients Overview Technical Approaches Caveats
VII.
Bone Marrow Engraftment Testing Overview Technical Approaches Caveats
262
11-29
VIII. Molecular Diagnosis of Hydatidiform Mole
11-29 11-29 11-31
11-32
Overview Technical Approaches Caveats
IX. Cancer of Unknown Primary Origin (CUP)
11-32 11-33 11-33
Overview Technical Approaches Clinical Pathology Molecular
11-34 11-34 11-34 11-35
x.
Summary
XI. Suggested Reading
11-35 11-35 11-35 11-36
11-36 11-36 11-37 11-37 11-37 11-38 11-39
11-39
Clonality Analysis in Modern Oncology and Surgical Pathology
11-3
CLONAL EXPANSION IS THE HALLMARK OF NEOPLASIA Overview Normal stem cells
• Clonality refers to the principle that a cell or group of cells are descended from and geneticall y identical to a single common ancestor, a stem cell • Clonal proliferation is a fundamental characteristic of all human cancers - Tumors arise as a result of a series of mutations occurring in a stem cell and in its progeny
~ Progenitor cells
- One cancer stem cell gives rise to daughter cell s, which exhibit the same genetic changes that initially provided a growth advantage to the stem cell Further genetic alterations in subsequent daughter cells provide additional growth advantages
,, ,, , ,, ,, ,
1
.
Genetic mutation
• Recent revolutionary progress in human genomics is reshaping the approach to cancer therapy and diagnosis • The major principle for clonality analysis was proposed in the late 1970s, and the basi s for which was the discovery of differential X chromosomal inactivation patterns in female patients and of a variety of somatic mutations • The principle of clonality is evolving over time and so are the methods for clonality analysis - Traditional cancer models suggest that tumors are monoclonal - All cells can form tumors and are, therefore, equally tumorigenic • Unregulated growth is due to a series of acqui sitions of genetic alterations, which lead to the expression of genes that promote cell proliferation and the concomitant silencing of growth inhibitory genes and/or the blunting of cell death genes • Any cell type could be the target of carcinogenesis - Modern cancer models propose that only a minority of tumor stem cells can form new tumors
Differentiated cells
Clonal cancer
Cancer stem cell seu" O wal .
~
.>
Fig . I. Clonal proliferation is the hallm ark of cancer. Normal stem cell s renew themselves through asymmetric division to maintain the stem cell population and generate more differentiated cell populations. Carcinogenetic factors can affect stem cell s in its self-renewal process, causing epigenetic and geneti c alterations and transform the stem cell s into cancer stem cells. Cancer stem cells are the cellular source of cancer. These cells divide to expand the cancer cell population, forming a clonal cancer. The modern carcinogenesis theory suggests that cancer develops from a cancer stem cell; whereas, the traditional carcinogenesis theory suggests that cancer can develop from any cell type , even differentiated cell s as indicated by the dashed line.
(Figure 1) • Unregulated cell growth in tumors results from disruption of the regulatory mechanism of stem cell self-renewal • Thus, cancer is a regulatory disorder in stem cells and not simply an augmentation of proliferation signals • Only stem cells or progenitor cells are targets of carcinogenesis and possess the capacity for selfrenewal (see also Chapter 7) • The genetic change is inheritable and shared by the entire population of cancer cells that are derived from the same stem cell origin
Thmorigenesis Models Monoclonal Tumor Model • Genetic damage or epigenetic alterations play critical roles in carcinogenesis • Carcinogenesis is a multi-step process at both the phenotypic and genetic levels • Many tumors have been reported to result from the clonal expansion of a single stem cell founder (cancer stem cell); thus, the tumor is composed of a clonal population of tumor cells
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Physiological Condition Regulatory element
-
C£roteiV
Abnormal Condition Regulatory element
Non-related gene
- ~
- ~ -
Fig. 2. Proto-oncogenes are functional genes in physiologic conditions. Normal proto-oncogenes are expressed in a wellcontrolled way and produce normal amounts of protein product (aqua coded oval). An oncogene is a modified proto-oncogene, which produces either an increased amount of normal protein (aqua) or a mutated form of the protein (orange), which is involved in cancer development. Upregulation of oncogene expression can be induced by mutation of the regulatory fragment or by incorporation of viral DNA, which may provide a stronger promoter and increased expression of a normal form of oncoprotein (aqua). Also if the coding sequence is linked to another gene sequence by a chromosomal translocation, a fusion protein may result (blue and green) .
• The monoclonal cancer theory is appealing in that it agrees with many known molecular events in tumorigenesis • Somatic mutations and/or epigenetic alterations acquired by cancer stem cells can accumulate and be transferred from the cancer stem cell into daughter cells to form a tumor clone with the same genotype and phenotype among all cells • Because the mutations acquired by the cancer stem cell are stably passed to its progeny, the presence of these mutations could be used as clonal markers • The clonal tumor cells exhibit a concordant pattern of somatic mutations and/or epigenetic alterations • Activation of an oncogene or deletion/inactivation of a tumor suppressor gene in a cancer stem cell and its progeny are common pathways in carcinogenesis (Figure 2) • Activation of anti-apoptosis genes or inactivation/loss of apoptosis genes are also characteristics shared by clonal cancers - Simultaneous upregulation of anti-apoptosis genes and downregulation of apoptosis genes results in a selective kinetic advantage and the expansion of a dominant clone
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• Epigenetic disorders alter the expression levels of related genes during carcinogenesis - The epigenetic status is often consistent among a clonal population of cells and can, thus, be used in clonality analysis - The most important epigenetic process is mediated through DNA methylation, which silences genes
Multi-Step Carcinogenesis • In 1997, Kinzler and Vogelstein proposed the concept of two different types of carcinogenetic genes, gatekeepers and caretakers (Figure 3) - Gatekeepers are genes that directly regulate the growth of tumors by inhibiting cell growth or promoting cell death. Known gatekeeper genes include APe, ~-catenin, Rb, NF J, VHL, and others • A specific cell type has only a few gatekeepers. Inactivation of specific gatekeeper leads to a specific cancer • In the gatekeeper pathway only one additional mutation, which inactivates the remaining allele, is needed to initiate a cancer
Clonality Analysis in Modern Oncology and Surgical Pathology
Normal cells
Epigenetic alterations
Loss of tumor suppressor gene allele
Genetic mutations of gatekeeper and carekeeper genes
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Tumor progression
Fig. 3. Multi-step carcinogenesis theory involves several epigenetic and genetic alterations. Epigenetic alterations and loss of tumor suppressor gene alleles are thought to be important initial steps in carcinogenesis. The accumulation of genetic mutation s involving gatekeeper and caretaker genes will eventually initiate cancer development and eventually lead to a more advanced malignancy.
- Caretakers are genes that maintain the integrity of the genome. Mutation of these genes can promote tumor initiation indirectly • Three subsequent somatic mutations are required to initiate a cancer: mutation of the normal caretaker allele, and mutations of both gatekeeper alleles • Caretakers include nucleotide excision repair genes , mismatch repair genes , ATM genes, BRCAJ and BRCA2 genes - Cancer incidence increases with age, supporting the theory of multi-step carcinogenesis • Multi- step carcinogenesis is a progressive event in which the sequential accumulation of somatic mutations transforms the stem cell into a cancer stem cell, which clonally expands into a cancer • Along with the accumulations of mutations, the affected cells also experience a morphologic transition from normal histology to hyperplastic/dysplastic lesions , and finally to cancer • The genetic changes are inheritable and shared by the entire population of cancer cells that are derived from the same stem cell origin
Polyclonal Tumor Model Field Cancerization Theory • The concept of field cancerization was first introduced by Slaughter et al. in 1953 - Slaughter proposed that the multi-centric origin and high probability of cancer recurrence in the oral mucosa suggests a microscopically invisible "field" where genetically altered pre-cancerous cells exist
Distinct X chromosomal inactivation patterns and discordant patterns of genomic mutations in the same tumor (or in separate tumors in the same patient) provides supporting evidence for the hypothesis • The theory explains the development of multiple primary tumors and of locally recurrent tumors Field cancerization generates multi-focal areas of cancer development from multiple genetically distinct clones due to carcinogenic events (Table 1) - Each cancer clone originates from a different cancer stem cell and bears different genetic alterations - The cancer clone can form tumors synchronously or metachronously within the field • Many organ systems have been studied including oral cavity, lung, esophagus, breast, skin, urinary bladder, vulva, and colon - Molecular findings have supported this field cancerization model in that genetic alterations can be detected in many field s in which the cells acquire genetic alterations and grow to form a patch (see Patch Phenomenon section) - External carcinogens cause independent genetic alterations at different sites leading to the development of multiple , genetically unrelated tumors • The important implication is that some effects of the carcinogens in the field remain after the primary cancer is removed and may lead to new cancers • Field carcinogenesis assumes a multi-step process in which neoplastic changes evolve over a period of time due to the accumulation of somatic mutations in a single cell lineage
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Table 1. Monoclonal vs Polyclonal Origin Theories of Tumorigenesis
Monoclonal origin
Polyclonal origin
Founder
Single transformed cancer stem cell
Multiple transformed cells in the field
Tumor clone
Single
Multiple
Somatic mutations
Similar mutations in all cancer cells
Different mutations
Multifocal development
Implantation, migration
Locally transformed
Mechanism of carcinogenesis
Monoclonal proliferation
Field cancerization
- One of the important effects of field cancerization is that genetic alterations are often found within the morphologically normal cells surrounding the tumor in the same field - These morphologically normal cells adjacent to the tumor occupy an intermediate step in the transformation from biologically normal to a dysplastic epithelium • Separate origination of multiple tumors suggests a concept of generalized carcinogenesis, through which a larger area or even the whole organ is affected by the carcinogens • Although multiple tumor clones may exist during the carcinogenetic process, one clone may develop an additional growth advantage, becoming dominant and resulting in a pseudomonoclonal appearance on clonality analyses • The genetic alterations may be found long before histologic evidence of cancer development, and the cells bearing the alterations mayor may not themselves be precursors of cancer
Random Collision Theory • Collision tumors have been reported in various organs and they represent a co-existence of two adjacent but histologically distinct tumors (without histologic admixture) in an organ • Random collision theory proposes the possibility that two distinct tumor types initiated in a close proximity can result in a polyclonal neoplasm that may be recognized clinically as a single tumor • Collision tumors have been reported in the stomach, lung, esophagus, ovary, and intestine • Collision tumors are polyclonal and display different genetic alterations in each component
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Patch Phenomenon • Patch phenomenon is a concept introduced by Schmidt and Mead in the 1990s through the use of X chromosome inactivation analysis A patch is generally regarded as a group of cells which are derived from a common stem cell, sharing common genetic characteristics and having inactivation of the same X chromosome in female individuals (Figure 4) - Patch size varies. The patch size in bladder and stomach is up to 1 ern? and in hair follicle is 0.2-1.0 cm-. In other tissues, the patch size is about 0.2-0.3 ern? - Some patches correspond to anatomic boundaries such as an intestinal crypt or breast duct-lobular unit; however, some do not follow anatomic boundaries, such as patches in the liver - Because of potentially large patch sizes, cells isolated for X chromosome inactivation analysis may come from a single patch. A multiple site cell isolation technique, in which much larger areas can be sampled, may be helpful in achieving more accurate results in these clonality analyses • The terminal lobuloductular unit of breast often lies entirely within one patch; therefore, a polyclonal origin of breast tumors may be difficult to demonstrate unless a proper normal control is used • Because of the large patch sizes in colon epithelium, X chromosome inactivation studies are heavily biased toward monoclonal results • Patch phenomenon in field carcinogenesis - In field carcinogenesis, many stem cells in the field acquire somatic mutations and divide to form genetically altered cell clusters as patches
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Fig. 4. A patch is a cluster of cells derived from the same founder cell. Cells in female tissues demonstrate a mosaic pattern of methylated X chromosomes (from either maternal or paternal origin). In this illustration, the red and blue cells represent cells with inactivated X chromosomes of maternal and paternal origin , respectively. Sometimes the cells can grow in clusters, remaining adjacent to one another as a result of clonal growth in a discrete territory referred to as a patch. The cells in a patch share identical genetic characteristics. The cluster of blue cells in the left lower comer constitutes a patch, all of which show inactivation of the paternally derived X chromosome.
- These altered cells in the patch mayor may not be morphologically recognizable but all have identical genomic changes • These patches can be recognized on the basis of genetic mutations such as TP53 mutations or abnormal epigenetic processes such as non-random X chromosome inactivation
- A single patch may be formed from the progeny of one or several stem cells that show the same genetic alteration pattern • The cells in these patches are monophenotypic but mayor may not be monoclonal • The patch proliferation-malignant transformation model is one basis for multi-focal, multi-clonal carcinogenesis (i.e., more than one genetically unrelated primary tumor)
• The cells in a patch may be more susceptible to carcinogens and to undergoing malignant transformation as somatic mutations accumulate
• To truly demonstrate monoclonality, postulated somatic genetic changes have to be directly demonstrated rather than inferred on the basis of apparent X chromosome inactivation data
X CHROMOSOME-LINKED CLONALITY ANALYSIS
Principle and Implication of X Chromosome Inactivation • X chromosome inactivation (also called lyonization) is a process proposed by Mary Lyon in 1961. It is a process
of chromosome-wide epigenetic gene silencing by which one of the two copies of the X chromosome present in female mammals is randomly and permanently inactivated (for purposes of dosage compensation)
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Fig. 5. X chromosomes of female cells are of paternal (P, green) and maternal (M, red) origin. In early embryogenesis, the paternally and maternally derived X chromosomes are both in an activated state. One of the X chromosomes in each cell becomes randomly inactivated after early embryogenesis (inactivated X chromosomes are gray). Normal female tissues are composed of a mosaic of cells having either the maternal or paternal X chromosome inactivated (red and green cells in lower box represent cells having an active maternal or paternal X chromosome, respectively). Theoretically, 50% of cells will have an inactivated paternal X chromosome and 50% of cells will have an inactivated maternal X chromosome. The X chromosome that is inactivated in a cell will be stably passed to its progeny, allowing this feature to be used as a clonal marker.
• The inactive X chromosome is methylated, followed by histone deacetylation, resulting in compaction of the chromatin into repressive heterochromatin, forming a Barr body (also called sex chromatin) • X chromosome inactivation occurs in early embryogenesis (blastocyst stage) and is permanent (Figure 5). The process is random, and either the maternally or paternally derived X chromosome is inactivated
268
• Once established, the same inactivated X chromosome is stably maintained and passed to daughter cells through all subsequent cell divisions • Analysis of the differential methylat ion (inactivation) of X chromosomes forms the basis of clonality analysis in women • Cells derived from a common progenitor share the same inactivated X chromosome in all progeny
Clonality Analysis in Modern Oncology and Surgical Pathology
A
B
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•• • •••• : N
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- The process is initiated by transcription and cis-localization of the non-coding Xist RNA, which then recruits much of the epigenetic machinery associated with maintenance of constitutive heterochromatin and silencing of genes on the inactivated X chromosome (e.g., histone modifications and DNA methylation) - The Xist gene transcribes a large RNA, which coats the inactivated chromosome. The non-inactivated X chromosome is not RNA coated - X chromosomes which lack the Xist gene cannot be inactivated. Artificially placing and expressing the Xist gene on another chromosome leads to silencing of that chromosome - The Tsix RNA is transcribed antisense to Xist
Fig. 6. Tumor cells are derived from a common progenitor cell and, therefore, share the same X chromosome inactivation pattern. Panel A is a schematic illustration showing random (normal) and non-random (tumor) X chromosome inactivation. Panel B shows a gel picture of X chromosome inactivation analysis. N designates normal control and T designates tumor. + and - indicate with or without HhaI methylation sensitive restriction enzyme digestion. The upper allele of tumor could not be amplified after HhaI digestion, indicating a non-random X chromosome inactivation pattern. Two bands are seen in normal tissue after HhaI digestion, consistent with randomly inactivated X chromosomes in the constituent cells.
- The Tsix gene overlaps the Xist gene and is transcribed on the opposite strand of DNA from the Xist gene. Thus, Tsix is a negative regulator of Xist • Inactivated X chromosomes have high levels of DNA methylation, low levels of acetylated isoforms of histone H4, low levels of histone H3 lysine-4 methylation, and high levels of histone H3 lysine-9 methylation
Human Androgen-Receptor Gene X Chromosome Inactivation Analysis • HUMARA X chromosome inactivation analysis is based on methylation sensitive restriction enzyme can only cut non-methylated restriction site (Figure 7) - The HUMARA is located at Xq11.2-12
• X chromosome inactivation is normally a random process with approximately equal numbers of maternally and paternally derived X chromosomes being inactivated in the female • As a consequence, normal tissues are composed of cellular mosaics with random X chromosome inactivation patterns. In contrast, a clonal expansion of cells, such as that present in tumors , exhibits a non-random pattern of X chromosome inactivation in all cells. Tumors in a female arising from a single progenitor cell have the same inactive X chromosomes (Figure 6)
X Chromosome Inactivation Control Mechanisms • The X-inactivation center (XIC) is located at Xq12-13 on the X chromosome and it is necessary and sufficient to cause X chromosome inactivation • The XIC contains two non-translated RNA genes, X inactivation-specific transcript (Xist) and Tsix (full-length RNA product, which is complementary to Xist RNA), which are involved in X chromosome inactivation. The XIC also contains binding sites for both known and unknown regulatory proteins
- A highly variable region of CAG trinucleotide repeats is located within the first exon of the gene . The repeats are in close proximity to differential methylation sites and to methylation-sensitive restriction endonuclease (HhaI or HpaII) cutting sites • Brief procedures: - Genomic DNA is extracted from microdissected normal and cancer tissues - Polymerase chain reaction (PCR) amplification of HUMARA locus on methylation-sensitive restriction endonuclease-digested genomic DNA to generate the allele fragments - The methylated (inactivated) X chromosome cannot be digested by methylation-sensitive restriction enzymes HhaI or Hpall - The digested (active) X chromosome cannot be amplified - The PCR products are subjected to electrophoresis • Interpretation of the X chromosome inactivation analysis results - Clonality analysis of a cell population can be interpreted correctly only in relation to the clonality of surrounding normal tissue of the same embryologic origin
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Methylated CAG polymorphic site
/\ Hhal
PCR primer
Hhal
Allele 1
Unmethylated
/\
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Hhal
Allele 2
+--- PCR primer
1 -/+ Hhal digestion
PCR amplif ication
Electrophresis
1 Normal
+
Allele 1 Allele 2
--
Tumor
+
--
Fig. 7. Schematic illustration showing the HUMARA X chromo some inactivation assay. Genomic DNA is isolated from normal tissue and from tumor and is subjected to methylation-sensitive restriction enzyme HhaI digestion. Allele I (blue) and allele 2 (purple) are from different parental origins. Allele I is methylated and cannot be digested; allele 2 is non-methylated and, thus, can be digested. Digested (+) and non-digested (-) DNA is PCR-amplified and separated by gel electrophore sis. The digested normal tissue shows a double band pattern since its X chromosomes are randomly inactivated. The digested tumor DNA shows only one band since its X chromo somes are non-randoml y inactivated. The X chromo some inactivation pattern is shared among the cells in the clonal population . - Informative case: two allelic bands were present after PCR amplification without methylationsensitive restriction enzyme digestion in normal control samples
270
- Non-informative case: only one allelic band was present after PCR amplification without enzyme digestion in normal control samples
Clonality Analysis in Modern Oncology and Surgical Pathology
Tumor 1
11-11
Tumor 2
DNA extraction
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-
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T2
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Fig. 8. X chromosome inactivation analysis can be used to analyze clonal relation ships of separate tumors . Genom ic DNAs from a normal control and from two separate tumors are isolated and subjected to a methylation sensitive restriction enzyme digestion followed by PCR amplification and gel electrophoresis. In pattern A the two tumors (TJ and T2) display an opposite pattern s of non-random X chromosome inactivation, indicating different clonal origin s. Pattern B shows identical pattern s of non-random X chromosome inactivation in each tumor, indicating a commo n clonal origin . As expected , normal control tissue (N) displays a random pattern of X chromosome inactivation with two bands present on gel electrophoresis before and after methylation sensitive enzyme digestion. - In informative cases, the PCR product s from digested normal control tissue show a double band on electrophoresis (random X chromo some inactivation), while digested clonal tumor DNA shows only one band (non-random X chromosome inactivation ) - A non-random X chromosome inactivation pattern indicates clonal proliferation; a random
X chromo some inactivation pattern indicate s a polyclonal process (mosaic cell populations) - Identical X-inactivation pattern s in two distinct tumors (with either upper or lower bands dimini shed) suggests a possible monoclonal origin (Figure 8)
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Molecular Genetic Pathology
- Opposite X-inactivation patterns, such as loss of upper band in one tumor and loss of lower band in another tumor support an independent origin for each tumor
Advantages and Limitations of HUMARA X Chromosome Inactivation Clonality Analysis • Advantages: - X chromosome inactivation is the most consistently informative marker of clonal proliferation in women - 90% of female s are heterozygous and suitable for X chromosome inactivation analysis - A highly variable region of CAG trinucleotide repeats is located within the first exon of the HUMARA gene - The proximity of differentially methylated restriction sites to the loci of genes allows for the use of these techniques on fragmented DNA (the size of PCR products is about 200-280 bp) - The method is simple and the result is stable • Limitations: - X chromosome inactivation is only applicable to women - Inherited or acquired unbalanced methylation (skewed, non-random pattern of X chromosome inactivation) and abnormal patterns of DNA methylation in malignancies can complicate the interpretation of results - Strict dosage compensation may not be necessary for all genes in the X chromosome • 15% of X-linked genes escape inactivation and an additional 10% of X-linked genes show variable patterns of inactivation - X-linked clonal ity analysis only distinguishes random and non-random X chromosome inactivation - Not all clonal proliferations are neoplastic. Clonal processes are not equivalent to neoplastic processes
Table 2. Mechanisms of Skewed X-Chromosome Inactivation in Normal Tissue Inherited Dysregulation, mutation, or deletion of XIC at Xq12-13 Xq28 deletion Xist mutation
XCE human equivalent of mutant murine Xce Decreasedprecursorpool size • Monozygotic twinning • Confinedplacental mosaicism Carrier state of X-linkeddiseases • Agammaglobulinemia • Severecombinedimmunodeficiency • Wiskott-Aldrich syndrome • Adrenoleukodystrophy • Incontinentia pigmenti type II
• u-Thalassemia with mental retardation syndrome • Duchenemuscular dystrophy • HPRTdeficiency • Facial dermal hypoplasia • Dyskeratosis congenital
Acquired Selection and growth advantage Aging process Tissue-specific (e.g., hematopoietic cells) Artifact
Technical Considerations Skewed DNA Methylation • The process of X chromosome inactivation is usually random , producing tissues with equal mixtures of cells having active X chromosomes of either maternal or paternal origin • However, skewed X-inactivation patterns can occur and this skewing can be inherited or acquired and can result from complex mechanisms of action during the early phase of embryonal life (Table 2) • Skewed methylation is an asymmetric distribution of inactivated maternally or paternally derived X chromosomes (Figure 9) • X chromosome inactivation with a 3: I ratio of inactivated Xp:Xm or vice versa, is accepted as skewed X chromosome inactivation
272
- Artificial allelic dropout • Reduction of DNA template quantity • Damagedor salt-contaminated DNA - Different laboratory criteria for skewing
• Primary skewed methylation patterns are related to the limited number of stem cells present at the time of random X chromosome inactivation during embryogenesis - Asymmetric division of stem cells is another cause of skewed methylation due to loss of some stem cells through terminal differentiation - Decreased precursor pool size contributes to skewed X chromosome inactivation
Clonality Analysis in Modern Oncology and Surgical Pathology
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Fig. 9. Schematic illustration of a mechanism for a skewed methylation pattern . The random inactivation of an X chromosome in each cell of a female occurs during early embryogenesis. If equal splitting of cells with an inactivated maternal or paternal X chromosome occurs during the early X chromosome inactivation process, the tissues will be composed of a cellular mosaic . If there is unequal splitting of stem cells at this time, the stem cells with inactivated paternal (green) and maternal (red) X chromosomes will be in unequal number, resulting in a skewed pattern of X chromosome inactivation .
- Monozygotic twins have an excessive skewing rate which may be related to the twinning process . The twinning process reduces the number of cells contributing to the embryo • Skewed methylation patterns can be influenced by hereditary factors and can be genetically transmitted. Family concentrations of highly skewed methylation patterns with preferential activation of one parental X chromosome has been reported • Mutation or abnormal imprinting of the Xist gene can result in skewed inactivation • Dysregulation of XIC or physical deletion of Xq28 (pseudoautosomal region) could cause skewing of X chromosome inactivation • Xist mutations are also directly related to skewing of X chromosome inactivation • Several X-linked disorders are known to cause skewing of X chromosome inactivation (Table 2) • Acquired skewing of X chromosome inactivation may also be related to selection and the aging processing - Age has been shown to have a direct influence on inactivation. A much higher incidence of extremely unbalanced X-inactivation patterns are seen in elderly women. There is depletion of stem cells through the aging process - Somatic selection occurs in the aging process . A small growth advantage may preferentially affect certain cell populations or clones. Such selection is variable among different tissues • X chromosome inactivation patterns in different tissues in the same female may vary, a phenomenon which is referred to as tissue-specific X-inactivation pattern
- Skewed methylation is low in gastrointestinal mucosa and thyroid but significantly higher in blood cells - Hematopoietic cells are particularly subject to the selection process due to short life spans and high turnover. Depending upon the definition and the quantitative accuracy of the measurement, up to 20% of such specimens may have skewed X chromosome inactivation - Before a clonal population of cells can be demonstrated with X chromosome inactivation analysis, the pattern of X chromosome inactivation observed in a tissue sample must be interpreted with reference to that seen in normal tissue of the same lineage. If a hematologic malignancy is being studied, an alternative method of clonality analysis should be considered because of the high incidence of skewed methylation in blood cells • Methodology divergence may also cause skewed results on X chromosome inactivation analysis. For example , artificial allelic dropout due to insufficient PCR amplification, reduction of DNA template quantity due to tissue preservation and processing, damaged or saltcontaminated DNA, and different laboratory criteria for skewing
Patch Phenomenon • Patch size can be large or small in normal tissues (see previous discussion). A patch can contain 200 or more cells and have a diameter of 2-3 mm or greater in some tissues - For example, there have been reports that the patch size in bladder epithelium could be about 120 mm? and composed of 2 x 106 cells
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- Monoclonal patch size of normal human thyroid tissue is between 48 and 128 mm'', containing 4 x 105 cells. In fact, when 20 normal thyroids were microdissected and subjected to X chromosome inactivation analysis, 70% demonstrated monoclonality, a likely reflection of large patch size • X chromosome inactivation analysis cannot discriminate between a monoclonal proliferation and a patch • Without considering the patch factor, X chromosome inactivation studies in human tissue, especially when applied to epithelial neoplasms, cannot readily answer questions about clonality A microdissection area of >2 mm? or multiple site cell harvesting could be helpful in differentiating between a normal patch and an abnormal clonal proliferation
res Bias • PCR bias is the phenomenon in which PCR can preferentially amplify one of two heterozygous alleles • Differential methylation of androgen receptor gene, HUMARA, permits identification of non-random X-inactivation in a monoclonal tumor • Co-amplification of two alleles in a heterozygote generates PCR products in different sizes • Under optimized conditions the amplification efficiency of two alleles is equivalent yielding equal band intensities • Highly imbalanced PCR products of heterozygous alleles may be present with preferential amplification of lower molecular weight alleles - PCR bias can be caused by different factors - Biased amplification consistently favors the lower allele - Regional secondary structure of DNA is another factor leading to PCR bias. Titrating the melting temperature is necessary to solve this issue - Adequate genomic DNA quantities are essential for a consistent allelic amplification. Five nanogram or more of genomic DNA can generate consistent amplifications - The quality of genomic DNA is critical when DNA is extracted from paraffin-embedded tissues. A report suggests that using 7-deaza-2'-dGTP could adjust the upper band coefficient by fourfold
Persistence of Biallelic Bands in Tumor Samples (Table 3) • Up to 40% of cancers may have a random X chromosome inactivation patterns • The loss of X chromosome inactivation may be related to loss of the XIC located at Xq13 • Contamination with normal tissue is one of the major causes of loss of sensitivity. Precise microdissection may be required for accurate interpretation
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Table 3. Persistence of Biallelic Bands in Tumor Samples Possible explanations • Deletion of XIC • Contamination with normal tissue • Incomplete DNA digestion • X chromosome aneuploidy • Co-existence of multiple tumor subclones of independent origin • Variable methylation patterns at the HUMARA locus • Reactivation of inactive X chromosome • Others
• Incomplete DNA digestion leads to the amplification of non-methylated alleles, which will greatly reduce the sensitivityby showing a pseudorandom inactivation • Methodology divergence, including different laboratory criteria for allelic loss, PCR conditions, and assay method selection, are also among reasons for persistence of biallelic patterns • X chromosomeaneuploidy - X chromosome inactivation mechanisms result in only one active X chromosome. The other is subject to inactivation even in the setting of X chromosome aneuploidy such as XXX, XXY, or XXXX Multiple X chromosomes from paternal and maternal origin may be inactivated and show falsely random inactivation in clonality analysis. Pseudorandom X chromosome inactivation should be excluded through other techniques, such as fluorescence in situ hybridization (FISH) • Co-existence of multiple tumor clones of independent origin may show false random X chromosome inactivation representing more than one clonal tumor. Precise small area microdissection of tumor cells may be helpful in solving this problem • Variable methylation patterns at the HUMARA locus can be seen in neoplastic and non-neoplastic cells - About 15% of X-linked genes escape inactivation to some degree, and the proportion of genes escaping inactivation differs dramatically between different regions of the X chromosome - The incidence of variable X chromosome inactivation in healthy females varies from 4 to 33%, which may be related to tissue-specific X chromosome inactivation patterns
Clonality Analysis in Modern Oncology and Surgical Pathology
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Table 4. Commonly Used Clonalitv Analysis Techniques X chromosome-linked methods • DNA-based : - DNA methylation
• Human androgen receptor locus (HUMARA) • M27~ probe for DXS255 locus - Restriction fragment length polymorphism (RFLP) • Glucose-6-phosphate dehydrogenase (G6PD) locus • Hypoxanthine phosphoribosyl transferase (HPRT) • Phosphoglycerate kinase (PGK) locus • RNA-based : - Palmitoylated membrane protein p55 gene locus - Iduronate-2-sulfatase (IDS) gene locus • Protein-based: - Glucose-6-phosphate dehydrogenase (G6PD) isoenzyme
Non-X chromosome-linked method • Loss of hetero zygosity • Somatic mutation (e.g., p53) • Gene rearrangement (e.g., T cell receptor and immunoglobin rearrangement for lymphom a work-up) • Restriction fragment length polymorphism (RFLP) • Cytogenetics and fluorescen ce in situ hybridi zation (e.g., FISH for il2p) • DNA methylation • Microsatellite instability • Viral integration analysis (e.g., EBV, HBV, HCV, HPV) • Comparative genomic hybridization (CGH) • Microarray-based cionality analysis • MicroRNA fingerprint • Protein based analysis (e.g., OCT4, TfFI) EBY, Epstein-Barr virus; HBY, Hepatiti s B virus; HCY, Hepatit is C virus ; HPY, human papillomavirus
• Age-related reactivation of inactivated X chromosomes. This may be related to the loss of a critical methylation sites on the X-linked genes
Other X Chromosome-Linked Clonality Analyses (Table 4) • Various methods have been used in the past, essentially all of which have been gradually replaced by HUMARA X chromosome inactivation analysis . The reader may refer to specific articles for more detailed discussions • M27~ probe for DXS255 locus - Locus DSX255 on the X chromosome is consistently differentially methylated and can be analyzed directly to assess X chromosome inactivation status
- The M27~ probe is hybridized to electrophoretically separated DNA - The polyclonal tissue shows two equal bands and the monoclonal tumor shows a pattern of unequal bands • Gluco se-6-phosphate dehydrogenase (G6PD) , hypoxanthine phosphoribosyl transferase (HPRT), and phosphoglycerate kinase gene s - These are restriction fragment length polymorphism (RFLP)-based analyses - DNA from tumor and control are extracted and purified and then peR amplified - The amplified DNA is cut into restriction fragments using suitable endonucleases, which only cut the DNA molecule at a specific recognition sequence
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- The restriction fragments are then separated by agarose gel electrophoresis - Clonal and non-clonal populations show different band patterns accordingly • Palmitoylated membrane protein p55 gene locus - Human p55, located at Xq28, encodes an abundantly palmitoylated phosphoprotein of the erythroid membrane - The allele on the inactivated X chromosome is silenced - mRNA is extracted and reverse transcribed using random primers . cDNAs are amplified with specific primer sets for two round s - Two bands are seen when X chromosome inactivation is random but cells with non-random X chromosome inactivation show a single band • Iduronate -2-sulfatase (IDS) gene locus - IDS clonality analysis is an mRNA analysis for a functional silencing of the gene - mRNA is extracted and reverse transcribed by random primers . PCR and primer extension analysis are carried out for IDS - Two bands are seen when X chromosome inactivation is random • G6PD isoenzyme analysis - This is a classic tool that has be used to study X chromosome inactivation status and clonality since the 1970s - Protein is extracted from cells and the G6PD fraction is collected and then separated by electrophoresis - The protein bands are compared with known heterozygous blood cells - A clonal cell population will show only one isoenzyme band
Selected Applications Defines the Monoclonal Nature of the Lesion • Renal angiomyolipoma is a benign neoplasm composed of blood vessels, smooth muscle, and adipose tissue. Whether renal angiomyolipoma is a hamartoma or a neoplastic process has long been debated. It is also uncertain which components of angiomyolipoma represents clonal growths and if various components share the same clonal origin. Cheng et al. (2001) found the smooth muscle cells and the adipose tissue to have differing pattern s of non-random X chromosome inactivation, indicating that both are monoclonal and probably originate from independent clones (Figure 10)
Defines the Clonal Relationship of Separate Tumors • Two proliferative cell populations (e.g., two separate tumors) that share the same non-random X chromosome
276
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+
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BV
+
Fig. 10. A representative gel picture of X chromosome inactivation analysis of the various components of a renal angiomyolipoma. N, normal; SM, smooth muscle; AT, adipose tissue; and BV, blood vessel. - and + indicate that DNA is non-digested or digested with methylation sensitive restriction enzyme . In SM and AT, opposite patterns of nonrandom X chromo some inactivation indicate that the lesions are clonal proliferations with each component having a different clonal origin. BV shows a random X chromosome inactivation pattern (two allelic bands).
inactivation patterns have a 50% probability of being derived from a common progenitor cell (monoclonal) since the chance of either paternal or maternal origin of the inactivated X chromosome is 50% • Different patterns of non-random X chromosome inactivation in separate tumors support independent origin • With a larger number of cell populations analyzed, results of clonality assessments become more meaningful. The probability of different cell populations with the same pattern of X chromosome inactivation representing a polyclonal process (i.e., unique genetic origin) decreases as the sample number (n) of cell populations increases [probability =(0.5)n] • For example, patients with ovarian papillary serous tumor of low malignant potential (LMP) may have peritoneal "implants" of histologically identical tumors. Gu et al. (2001) studied a group of women with advanced-stage ovarian papillary serous tumors of LMP using X chromosome inactivation. Most of the patients with peritoneal and ovarian tumors showed different X-inactivation patterns, suggesting that peritoneal tumors asso ciated with ovarian LMP tumors may arise independently from their own primary tumor clones rather than through an "implantation" process. Some patients with bilateral ovarian tumors of LMP showed different X chromosome inactiv ation patterns in tumors of each ovary, indicating that the patients had bilateral primary tumors instead of one ovarian tumor with metastasis to the oppo site ovary (Figure 11)
Defines the Clonal Relationship of Different Components of the Same Tumor • The identification of components of different biologic aggressivenes s within a single neoplasm is a common finding in pathology. These variable components are
Clonalitv Analysis in Modern Oncology and Surgical Pathology
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Fig. 11. X chromosome inactivation analysis of multi-focal ovarian papillary serous tumors of LMP. Tumor locations are as follows : La, left ovary; OM, right ovary; SBS, peritoneum; and PN, pelvic lymph node. N represents normal. - and + indicates that DNA is non-digested or digested with methylation sensitive restriction enzyme . The tumor from the LO and SBS share the same X chromosome inactivation pattern, both showing loss of the upper alleles after digestion . This is suggestive of a common clonal origin. However, tumor from the PN shows a different clonal origin . The allele from the right ovary shows a reduced upper allele but does not reach the cutoff value and thus is interpreted as negative.
thought to result from tumor cell dedifferentiation or transformation, with the subsequent evolution of different sub-populations of tumor cells, a concept that is exemplified by the co-existence of small cell and urothelial carcinoma of the bladder. Cheng et al. (2005) found a concordant pattern of non-random X chromosome inactivation between small cell cancer and co-existing urothelial carcinoma, suggesting that both tumor components originate from the same progenitor cells in the urothelium. These findings may provide new insights into the treatment of small cell carcinoma of the urinary bladder
Defines the Clonal Relationship Between Precursors (Such as Intra-Epithelial Neoplasia) and Cancer • Guo et al. (2000) studied cervical intra-epithelial neoplasia with co-existing invasive cancers using X chromosome inactivation. The authors found that
pre-cancerous lesions and co-existing cervical cancers were clonally related. Carcinogenesis of cervical cancers involves selection of sub-clones from originally polyclonal precursors
Defines the Clonal RelationshipsBetween Primary and Metastatic Tumors • Evidence of genetic heterogeneity and tumor sub-clones within urothelial carcinoma of the bladder has raised questions about the clonal origin of urothelial carcinoma and its metastases . Jones et al. (2005) investigated female patients who underwent radical cystectomy for urothelial carcinoma. The X chromosome inactivation analysis showed identical non-random inactivation patterns in primary bladder cancer and pelvic lymph node metastases, suggesting that the capacity for metastasis arises in only a single clonal population in the primary tumor
LOSS OF HETEROZYGOSITY (LOH) AS A CLONAL MARKER Overview • Microsatellites are polymorphic loci that consist of repeating units of 2-6 bp that repeat 10-100 times without interruption. There are approximately 200,000 rnicrosatellite loci in the human genome • The polymorphism of microsatellites is the basis for rnicrosatellite analysis • Slipped strand rnispairing is the primary mechanism for polymorphism, which leads to deletion or insertion of the microsatellite repeat unit - Slipping is caused by regional non-pairing, which forms a "bubble" containing one or more repeat units.
If the slippage involves a newly synthesized strand it is called "backward slippage;" if it involves the parental strand it is called "forward slippage" - 5'-3' slipping (backward slippage) causes insertion of a repeat unit and 3'-5'-slipping (forward slippage) causes deletion of a repeat unit • In a heterozygote, peR amplification using rnicrosatellite locus-specific primers will result in two distinct bands, representing maternally and paternally derived alleles • LOH represents the loss of one parent's contribution to the cellular genome . (Figure 12)
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A Fig. 12. LOH in a cell represents the loss of one parent's contribution of microsatellite DNA to a cell's genome . It often indicates the presence of tumor suppressor gene loss adjacent to the microsatellite locus. An informative locus is the one that the maternal and paternal alleles are different in repeat numbers . Specific primers (green) are used to amplify the alleles . For normal (N) cells, both alleles are intact and, thus, can be amplified. When the tumor cells (T) have lost a segment of its DNA, the lost allele (boxed) cannot be amplified . Two allelic bands are seen in normal control DNA; whereas, tumor DNA shows loss of the lower allele (LOH). • LOH often indicates the presence of a tumor suppressor gene in the lost region
- If LOH occurs at a selected polymorphic region that is related to a known tumor suppressor gene, it is highly suggestive of deletion of the corresponding gene as this also explains the growth advantage of the tumor cells carrying that genetic alteration . LOH can result in haploinsufficiency of a specific allele - LOH has been considered an early event during carcinogenesis. Often, the remaining copy of the tumor suppressor gene will be inactivated by mutations during cancer progression - LOH is inheritable and can be passed to the cellular derivatives. The presence of a uniform and nonrandom alteration of a tumor suppressor gene as demon strated by LOH analysis in all cells of a tumor confirms a clonal origin - LOH is related to chromosomal instability. A cell with chromosomal instability carries mutations that result in an increased rate of LOH • A number of mechani sms can lead to LOH, including local deletion, non-disjunction of chromo somes, mitotic recombination, gene conversion , double-strand break resulting in loss of a chromosome arm, or whole chromosome loss • The principle of LOH clonality analysis is that the clonal expanding cells share a common set of allelic losses
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Fig. 13. An LOH-based clonality test compares the allelic banding patterns between tumors and normal tissue . Pattern X shows an opposite allelic loss pattern in two separate tumors (A and B), suggesting a different clonal origin. Pattern Y shows the same allelic loss pattern in each tumor, suggesting a common clonal origin. Normal tissue shows double alleles indicating that the patient is heterozygous (informative) at the locus .
Evaluation and Interpretation of the LOH Analysis Results • LOH can be identified by detecting the presence of heterozygosity in germline DNA and the absence of heterozygosity at the same locus in the tumor DNA • LOH can be used as a clonal marker. Tumor cells derived from the same progenitor cells will share the same LOH patterns at multiple microsatellite loci • Brief procedures (Figure 13): - Genomic DNA is extracted from microdissected normal and cancer tissues - PCR amplification of microsatellite loci from tumors and normal control DNA is performed - The PCR products are subjected to electrophoresis. Microsatellite patterns of normal and tumor samples are compared • Informative case: - Two alleles are present after PCR amplification in normal control samples - Informative is a synonym for the heterozygous state - Microsatellite loci with a relatively high heterozygous rate should be selected for the study • Non-informative case: - Only one allelic band is present in normal control DNA after PCR amplification
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- This is due to the identical size of two alleles which could not be distinguished, as seen in the homozygous state • LOH clonality analysis results are based on the comparison of band patterns in tumor and normal control DNA. A non-informative case cannot be analyzed by this method • Identical allelic loss patterns shared among separate tumors suggests a common clonal origin; different or opposite allelic loss patterns in two separate tumors suggests a different clonal origin (Figure 13)
Advantages and Limitations of LOH Analysis • Advantages: - Unlike X chromosome inactivation, this technique is applicable to both men and women - LOH is a sensitive marker for morphologically normal pre-cancerous cells - Many microsatellite loci have high LOH rates in different malignancies - In analysis of multiple tumors, LOH analysis generally uses multiple microsatellite markers ; thus, decreasing the likelihood of random matches (see discussion below) - The clonal population of cells may be recognizable even during sub-clone evolution in tumorigenesis • Limitations: - Accurate interpretation of results requires a pure population of target cells . Tissue microdisssection is often required to avoid contamination - Selection of appropriate microsatellite loci for specific applications may be difficult - For formalin-fixed, paraffin-embedded tissue, a long PCR microsatellite fragment is difficult to amplify (PCR product of <200 bp is preferred) - LOH may occur in normal tissue (see discussion below) - The occurrence of homozygous deletion and microsatellite instability (MSI) may complicate interpretation of results
Technical Considerations Allele Drop-Off • Allele drop-off refers to PCR bias against one allele, especially the upper one, resulting in preferential amplification of the lower allele • Allele drop off should not be misinterpreted as noninformative or LOH • To avoid this artifact, appropriate DNA extraction methods should be used. PCR conditions, especially the cycle number, need to be precisely controlled
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Loss of Sensitivity • Loss of sensitivity is a common reason for failure of the test - Most commonly due to amplifying the locus for too many cycles . When genomic DNA is in low concentration or in lower quality, there is a tendency to amplify for more cycles, which equalizes the alleles. The PCR amplification cycle number should be controlled within the range of log phase The genomic DNA from formalin-fixed and paraffinembedded tissue is fragmented . Selection of microsatellite markers with PCR products <200 bp may enhance the sensitivity A different method (such as FISH) should be used to exclude homozygous deletion of a specific allele
LOH in Normal Cells • The background level of LOH in normal tissues has been reported to be from 4 to 20%, regardless of the detection system used • LOH could also be randomly acquired and irrelevant to tumor development - The existence of genomic alterations in morphologically normal cells is not an indication of malignancy Random genomic alteration in morphologically normal cells has less tendency to involve multiple loci - Incidences of LOH are significantly higher in precursor and cancer cells • Histologically normal epithelium and/or simple hyperplasia associated with cancer is often characterized by genetic alterations - Genetic alterations , such as LOH leading to tumor development, occur earlier than morphologic alterations in pre-cancerous cells - The genetic alteration may represent generalized carcinogenesis and might be the basis for the development of multi-focal tumors or recurrence of tumors (see field cancerization above) - These genetic alterations may represent an intermediate step in carcinogenesis • Careful selection of normal controls is critical for accurate interpretation of results
Methods for LOH Analysis Radioisotope PCR Incorporation-Gel Electrophoresis • Genomic DNA is prepared from the pure cell population, generally from microdissected paraffin sections • Radioisotope PCR incorporation-gel electrophoresis uses locus specific primer pairs to amplify the heterozygous loci from the normal and tumor genomic DNA in the presence of radioisotope-labeled dNTPs
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• The PCR products are separated by electrophoresis and visualized by autoradiography • The intensity of gel bands from normal control and tumor DNA are compared to decide if there is a diminished allele at the locus • LOH is present when one band from either allele has disappeared or is greatly decreased when compared with the other allele • The method is stable, simple, and inexpensive but radioisotope is needed
High-Resolution Fluorescent Microsatellite Analysis • Genomic DNAs are extracted from tissue sections and amplified using primers labeled with fluorescent compounds • The PCR products are denatured and loaded onto a sequencer. The mobility of each sample is processed using computer software to convert amplified DNA to fluorescence band peaks • The height of each allele peak is compared with the normal control to decide if LOH is present • This method is sensitive and standard ized but needs special equipment and software such as ABI Genetic Analyzers (Applied Biosystem, Foster City, CA)
High-Performance Liquid Chromatography (HPLC) • Genomic DNAs are amplified using primers labeled with fluorescent compounds • Denatured amplicon is then gradually reannealed by decreasing sample temperature and injecting it into the HPLC analyzer • DNA is eluted from the column • DNA fragments of different sizes can be separated by elution over time • The method is sensitive and highly repeatable but a specific analyzer is needed (e.g., WAVER Fragment Analysis System [Transgenomic , Omaha, NED
High-Density Oligonucleotide Single Nucleotide Polymorphism Array • Simultaneously analyze thousands of Single nucleotide polymorphism markers, thus allowing identification of LOH in the absence of heterozygous loci • Whole genome screening for LOH could provide allele typing for each tumor
Selected Applications (Table 4) Defining Clonal Relationships of Separate Tumors • Concordant LOH patterns involving multiple loci in separate tumors support a common clonal origin; discordant LOH patterns over multiple loci suggest an independent clonal origin (Figure 13)
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• For example, the prostate gland usually contains two or more widely separate tumors. A critical issue is whether the multiple tumors are independent in origin. Cheng et al. (1998) found a discordant pattern of allelic deletion in the majority of multi-focal prostate cancers . The data support that multiple precursor lesions of the prostate arise from a field effect and independent expansion of these premalignant lesions can lead to the development of multiple tumors within the same gland
Defining Clonal Relationships of Different Histologic Components of the Same Tumor • Teratomas of the testis in post-pubertal patients are histologically diverse tumors that often co-exist with other types of germ cell tumors. In an effort to assess the clonality of mature teratoma and its relationship to other components of malignant mixed germ cell tumors , Kernek et al. (2003) investigated these issues using LOH analysis. Complete concordant allelic loss patterns between mature teratoma and other germ cell tumor components were seen in over 70% of patients in which mature teratoma demonstrated LOH. The study provides insight into the histogenetic relationships of various germ cell tumor components and testicular tumorigenesis
Defining the Precursor Nature of Lesions • Atypical adenomatous hyperplasia (AAH) of the prostate has been proposed as a possible precursor lesion of prostate cancer. Cheng et al. (1998) identified a high frequency of LOH, similar to that commonly seen in high grade prostatic intra-epithelial neoplasia (PIN) and prostate cancer, in AAH. This result provides evidence of a genetic link between some cases of AAH and prostate carcinoma
Defining Clonal Relationships Between Precursor Lesions and Cancer • Prostate carcinoma usually is heterogeneous and multifocal, with diverse clinical and morphologic manifestations. Understanding the molecular basis for this heterogeneity is limited, particularly for the putative precursor, high grade PIN. Bostwick et al. (1998) analyzed LOH in multiple foci of PIN and matched foci of carcinoma. The strong genetic similarities between PIN and prostate carcinoma suggest that evolution and clonal expansion of PIN may account for the multi-focal etiology of prostate cancer
Defining Clonal Relationships Between Primary and Metastatic Tumors • Epidermotropic metastases of melanoma are unique in their varied histopathologic appearances, which can mimic a primary lesion . Bahrami et al. (2007) found that primary lesions demonstrated a concordant LOH pattern with corresponding epidermotropic metastases.
Clonality Analysis in Modern Oncology and Surgical Pathology
The data suggest that these metastases are clonally related to the primary lesion and that some cases diagnosed as epidermotropic metastatic melanoma might be divergent clones or new primaries rather than metastatic disease
Defining the Genetic Basis of Tumorigenesis and Cancer Progression • Boland et al. (1995) analyzed the temporal sequence of allelic losses on 5q, l7p, and 18q during neoplastic progres sion of colorectal cancer. No allelic losses were found in normal tissues surrounding colorectal neoplasms, but losses occurred abruptly on 5q at the transition from normal colonic epithelium to the benign adenoma, and on 17p at the transition from adenoma to carcinoma. The authors conclude that allelic losses on 5q and 17p are associated with abrupt waves of clonal neoplastic expansion
Defining Stromal-Epithelial Interaction During Tumorigenesis • The stromal cells in post-chemotherapy surgical specimens of lymph nodes from testicular cancer patients have generally been considered "fibrosis" secondary to chemotherapy and the necrosis it causes ; however, the frequent cytologic atypia of the stromal cells suggests that they may be neoplastic. Brandli et al. (2003) found that the stromal cells adjacent to metastatic mature teratoma in postchemotherapy lymph node specimens frequently have genetic abnormalities similar to the metastatic teratoma. The highly concordant genetic alterations (LOH) observed in co-existing teratoma and stroma suggest that both are derived from the same element of the original germ cell tumor or the same progenitor cell
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Defining the Evolution of Neoplastic Cell Lineages in Carcinogenesis • Cancer progression develops as a consequence of an acquired genetic instability and the subsequent evolution of clonal populations with accumulated genetic errors. Barrett's esophagus is a precursor of esophageal cancer. Barrett et al. (1999) found that the clonal evolution of neoplastic progeny frequently involved LOH at 5q, 13q, and 18q that occurred in no obligate order relative to each other. These findings suggest a progenitor cell may undergo clonal expansion. With increasing genetic instability, these lesions will enter a phase of clonal evolution that begins in premalignant cells and continues after the development of invasive cancer
Defining Clonal Divergence and Genetic Heterogeneity in Cancer Progression • Cancer progresses through increasing genetic instability, which creates cellular diversity. It is uncertain whether genetic homogenization of the neoplasm from a clonal expansion or whether the accumulation of clonal diversity is more predictive of cancer progression. Maley et al. (2006) found that clonal diversity based on LOH can predict tumor progression from precursor lesions such as Barrett's esophagus • Some clear cell renal cell carcinomas contain components with sarcomatoid differentiation. It has been suggested that the sarcomatoid elements arise from the clear cell tumors as a consequence of clonal expansions of neoplastic cells with increasing genetic instability. Jones et al. (2005) found different patterns of allelic loss in multiple chromosomal regions in clear cell and sarcomatoid components from the same patient. The observed genetic heterogeneity indicates genetic divergence during the clonal evolution of renal cell carcinoma
OTHER METHODS OF CLONALITY ANALYSIS
Somatic Mutation
- DNA sequencing • DNA sequencing identifies a point mutation directly from the tumor DNA sequence
• Somatic mutations have been found in many human malignancies. They are acquired by the tumor founder cell and can be stably passed to all of the clonally-related daughter cell populations. Thus, they could be used as clonal markers
• DNA sequencing is mostly used to analyze positive cases after a qualitative test such as single-strand conformation polymorphism (SSCP)
• Somatic mutations are a part of the carcinogenesis process and are related to the biologic behavior of tumors . Somatic mutations most often affect oncogenes and tumor suppressor genes through different pathways (see Multi-Step Carcinogenesis section) • Methods for somatic mutation -related clonality analysis
• A similar mutation in key genes, such as an identical p53 mutation in the same codon in multiple samples from separate tumors, suggests a common clonal origin. Different p53 mutations, either different codon changes or different locations , are compatible with independent clonal origins
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- Single strand conformation polymorphism (SSCP) • The basic principle of SSCP is that the DNA sequence change will alter the three-dimensional conformation of single-stranded DNA, which alters the migration properties of DNA on gel electrophoresis. Differing band patterns indicate base changes • SSCP is a widely used PCR-based method to detect mutations in a large number of samples for clinical diagnostics, for population genetics, and for molecular epidemiologic study
Marker
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• A single nucleotide change in a sequence of double-stranded DNA cannot be distinguished by electrophoresis since the physical properties of the double strands are almost identical for both alleles • SSCP detects a conformational difference from single-stranded nucleotide sequences of identical length . The detected abnormal conformation needs to be verified by sequencing • DNA from tumor and from normal donor is included in each test • The PCR products are denatured and subjected to electrophoresis. After denaturation, the DNA becomes single-stranded. The strands undergo three-dimensional folding and may form different conformational states based on their DNA sequences
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• DNA strands may migrate at a different speed on a gel depending on various folded structures, even though the number of nucleotides is the same • The conformational states are subject to many experimental conditions and sequence differences mayor may not be detectable - Restriction fragment length polymorphism (RFLP) • The basic principle of RFLP is that mutation will destroy or create a restriction cutting site, leading to an altered restriction fragment banding profile
(Figure 14) • Regions of some genes encompass restriction enzyme cutting sites in their coding region, which will yield a specific restriction fragment pattern
Fig. 14. RFLP analysis. DNA from normal tissue and tumor are extracted followed by PCR amplification. The amplified DNA is digested into restriction fragments using a suitable endonuclease, which only cuts the DNA molecule at a specific recognition sequence. The restriction fragments are then separated by gel electrophoresis. Normal DNA shows a specific restriction fragment panel. The restriction site of tumor DNA was destroyed by a mutation (boxed). Therefore, the tumor DNA cannot be cut and shows a changed band pattern (arrow) .
• Genomic DNA is extracted and purified, followed by PCR amplification for the target fragment • The amplified DNA fragment is then cut with selected restriction endonucleases, which only cut the DNA sequence where specific restriction sites exist • A gene fragment with a single base mutation may destroy or create a restriction enzyme cutting site and therefore alter the restriction cutting pattern • The method is of low cost but only suitable for certain specific mutations • Tumors with the same altered RFLP pattern suggested a common clonal origin
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- Denaturing high performance liquid chromatography (HPLC) • Denaturing HPLC is a method of separating one chemical from another based upon their absorption of ultraviolet light and group interaction with the analytic column substrate • A gene fragment is amplified from genomic DNA extracted from microdissected tumor tissues • The PCR product is denatured and injected into the HPLC instrument to be column separated, and the fractions are analyzed
Clonality Analysis in Modern Oncology and Surgical Pathology
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Fig. 15. Arm painting of chromosomes. The fluorescence-labeled DNA probes are hybridized to specific chromosome arms, "painting" the arms into different colors. Chromosome arm painting is a useful tool in the identification of chromosomal translocations, isochromosomes, and arm losses.
• The method is excellent for the systemic detection of all the mutation sites in a gene fragment but it requires expensive instrumentation
Gene Rearrangement Analysis • Clonality analysis using T cell receptor and B cell immunoglobin gene rearrangement has been routinely employed in lymphoma work-ups (see Chapter 25 for details)
Cytogenetic Analysis and FISH • Cytogenetic analysis is an important tool for clonality analysis . The accumulated genetic instability leads to cancer evolution and progression • These genetic alterations are inherited in subsequent cell populations during clonal expansion, and can serve as clonal markers
• Karyotyping determines numerical and structural changes of chromosomes of a eukaryotic organism. More recently, chromosomal painting techniques have been widely used in cellular karyotyping . Chromosome painting can be used to label the whole chromosome or an arm. It is also useful in identifying chromosomal translocations (Figure 15) • FISH has been routinely employed for aiding in diagnosis and identification of tumor origin - FISH uses fluorescent-labeled probes to hybridize to part of a chromosome with a high degree of sequence similarity. It can be used to identify numerical and structural anomalies of chromosomes from interphase or metaphase cell nuclei (Figure 16A) (see Chapter 12 for more details) - FISH can be used to map sequences to a specific position on a chromosome, e.g., centromeres, arms, and telomeres showing loss and/or gain of DNA
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A Denaturization
Probe hybridization
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- Isochromosome 12p is characteristic of tumors of germ cell origin (Figure 16B) • Cheng et al. (2007) found a high incidence of chromosome 12p anomalies in "fibrosis" from residual retroperitoneal fibrous masses after chemotherapy for testicular germ cell tumors. These findings suggest that the stromal cells in the residual mass are derived from the same tumor progenitor cells as the pre-existing metastatic germ cell tumor - Many tumors are characterized by specific types of chromosomal translocations, such as the t(9;22)(q34;qll) translocation (bcr-abl) in chronic myelogenous leukemia, which can be demonstrated by FISH using a specific probe set (Figure 16C)
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• Epigenetic alterations, such as DNA methylation, can be inherited or acquired. Patterns of DNA methylation have been consistent findings in some cancer cells. Thus , epigenetic characteristics of tumor cells may provide highly specific and sensitive molecular markers for tumor clonality analyses . For example, Catto et al. (2006) analyzed DNA methylation of 7 gene promoters in bladder urothelial carcinoma and found that these epigenetic events are useful in defining the clonal origin of the tumor • Brief procedures for methylation analysis (Figure 17): - The genomic DNAs are extracted from microdissected normal and cancer tissues - The genomic DNAs are chemically modified by sodium bisulfite
- Consistent chromosomal changes suggest a clonal origin such as 1p36/19q 13 deletion in oligodendroglioma (Figure 16D)
• The unmethylated cytosine in the CpG site is converted into uracil but the methylated cytosine could not be converted
- FISH can also be used for chromosome painting to demonstrate entire chromosomes or chromosomal arm anomalies (Figure 16E)
• The primers designed according to methylated sequence (non-convertible C) or unmethylated sequence (convertible C) could only recognize and amplify the methylated or unmethylated sequences, respectively
- Multi-probe FISH, such as Urovysion' by Vysis, uses probes labeled with different fluorescent dyes and is especially helpful in identifying specific co-localized abnormalities (Figure 16F) - In patients with papillary renal cell carcinoma, it is not uncommon to find two or more anatomically distinct and histologically similar tumors at radical nephrectomy. Whether these multiple papillary lesions result from intra-renal metastasis or arise independently is unknown. Using a FISH approach, Jones et al. (2005) reported multiple papillary renal cell carcinomas arise independently. Intra-renal metastasis does not seem to playa major role in the spread of papillary renal cell carcinoma
• Methylated alleles can be amplified by methylationspecific primer pairs; unmethylated alleles can be amplified by a different set of primer pairs • The PCR products are subjected to gel electrophoresis and microsatellite banding patterns are compared
Microsatellite Instability
DNA Methylation as a Clonal Marker
• MSI is a condition of having longer or shorter microsatellite repeats than germline DNA of normal cells. This is due to failure of the mismatch repair system to correct errors in the transcription of microsatellite short sequence repeats (see Chapter 7 for more detail)
• DNA methylation is an epigenetic process involving a chemical modification of DNA without changing the DNA sequence (see Chapter 7 for more details)
• Instead of losing one heterozygous allele as seen in LOH analyses, MSI shows retention of both alleles in tumor DNA. Changes in the length of microsatellite loci are due
Fig. 16. (Opposite page) FISH is a widely used technique in clonality analysis. Panel A shows an illustration of the principle of FISH. A fluorescence dye-labeled DNA probe is hybridized to a complementary sequence on a chromosome. Panel B shows an example of isochromsome 12p (arrow), a well recognized marker for tumors of germ cell origin. Green signals show 12p and red signals show the centromere of chromosome 12. Note there are two chromosome 12 centromere signals and three 12p signals with specific patterns of signal aggregation. Panel C shows a typical bcr-abl translocation as seen in chronic myelogenous leukemia. The DNA fragments normally located on chromosome 9 (red) are transferred to chromosome 22 (green). Panel D shows a Ip36 deletion, a typical finding in about 80% of oligodendrogliomas. Most of the cells show two green (1q) and one red (1p36) signal pattern, indicating a clonal origin. Panel E demonstrates chromosomal painting with whole chromosome arms labeled with a specific color. Panel F shows FISH performed on normal urothelium using UroVysion probes specific for centromeres 3 (red), 7 (green), 17 (aqua), and 9p21 (gold) . Gains of chromosomes 3, 7, and 17 and loss of 9p21 have been observed in more than 80% of urothelial carcinomas.
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A 5'
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Fig. 17. The most important epigenetic process is mediated through DNA methylation, which silences a gene. Methylation occurs at the cytosine of a CpG dinucleotide (A). Clonal cells show an identical DNA methylation pattern . Methylation-specific PCR is a commonly used method for DNA methylation analysis (B). Genomic DNA is extracted from tumor and control tissue . Hydroquinone and sodium bisulfite convert any unmethylated cytosine to uracil (D), which allows all non-methylated cytosine bases to be identified. Methylation-specific primers are designed according to the converted (non-methylated) or non-converted (methylated) sequences. Specific primers for non-methylated or methylated sequences can only amplify the non-converted or converted sequences, respectively. The resulting gel electrophoresis shows the different methylation states of the tissue. Normal cells exhibit a non-methylated (D) DNA band, whereas the tumor tissue shows a methylated PCR product (M).
to deletion or insertion of microsatellite repeating units. Like LOH, MSI could also be used as a clonal marker in clonality analysis (Figure 18) Tumor cells derived from the same clonal origin share the same pattern of MSI and these genetic alterations can be transmitted through subsequent cell generations • The same technology can detect both MSI and LOH at a specific locus but MSI is less common than LOH in solid tumors (Figure 19)
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• Accurate classification of two or more synchronous and metachronous tumors as independent tumors or as a primary tumor with metastatic foci has important clinical implications. Kaneki et al. (2004) analyzed a series of synchronous endometrial and ovarian adenocarcinomas. Based on MSI pattern comparison, the authors concluded that most patients (82%) could be diagnosed as having either single or double clonal tumors . MSI analysis can be used to distinguish primary vs metastatic origin of tumor
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Clonality Analysis in Modern Oncology and Surgical Pathology
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Fig. 18. Schematic illustration for microsatellite alterations . Normal informative cells (yellow, A) show both alleles after PCR amplification of the microsatellite locus. Cancer cells (magenta, B and C) show alteration of the microsatellite locus either through deletion of a DNA fragment to form LOH (B) or by altering the microsatellite repeat numbers to form MSI (C). The lower box shows the gel patterns . Normal cells (A) show both alleles but tumor cells show LOH (B) and MSI (C). LOH generally represents the loss of an adjacent tumor suppressor gene. MSI represents a condition of impaired mismatch repair ability.
Viral Integration Analysis • Oncoviruses are viruses associated with malignances and include viruses with DNA and RNA genomes. Infection of oncoviruses plays an important role in the carcinogenesis of some cancers, including the associations of Epstein-Barr virus with lymphoma and nasopharyngeal carcinoma, of human papilloma virus with cervical cancer, and of hepatitis B virus with hepatocellular carcinoma
• The patterns of viral DNA incorporations are often concordant among tumor cells of the same clonal origin. Southern blot and PCR are commonly used methods for virus clonality analysis
Comparative Genomic Hybridization (CGH) • CGH gives a global overview of chromosomal gains and losses of the whole genome of a tumor
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• The characteristic gain or loss of chromosomal material in a clonal tumor will be shared by the whole clonal population and derivatives, which can be used as a clonal marker - Jiang et a1. (2005) investigated paired samples of primary cancer and pulmonary metastases obtained from patients who had undergone two consecutive surgeries. The overall CGH profiles were similar between primary carcinomas and their pulmonary metastases, indicating a common clonal origin of primary and metastasic tumors
Gene Expression Profiling!Array -Based Clonality Analysis • See section IX and Chapter 8 for more details
B
MicroRNA Signatures N
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• MicroRNA (miRNA) is a single-stranded RNA molecule encoded by genes . It is transcribed from DNA but not translated into protein (see Chapter 7 for more details) • miRNA profiles in tumors reflect the developmental lineage and differentiation state of the tumors. A tissuespecific miRNA expression signature is highly specific and reproducible and will be shared among cells with a common clonal origin
Fig. 19. MSI is a condition in which a group of cells acquires longer or shorter rnicrosatellite alleles than normal cells. Alteration in the length of a microsatellite allele is due to deletion or insertion of single nucleotides or repeating units. MSI is different from LOH in that the tumor retains both alleles. Panel A is a schematic MSI pattern showing that one allele in tumor DNA has shifted its location (arrow) relative to that which is seen in normal control DNA. Alternatively, a shift from a noninformative control (one peak in normal) to a double peaked (arrow) pattern in tumor DNA indicates the presence of MSI. Panel B is a gel photograph of MSI demonstrating that tumor (1) has an upper allele with reduced length. • CGH is a technique that permits the detection of chromosomal copy number changes in formalin-fixed and paraffin-embedded tissue . CGH gives a global overview of chromosomal gains and losses in the whole genome of a tumor CGH hybridizes green fluorochrome-labeled tumor DNA and red fluorochrome-labeled normal DNA to normal human metaphase preparations - Images of fluorescent signals are captured and the green to red signal ratio is quantified for each chromosome locus along the chromosomal axis. If the signal from a locus is skewed to red, it indicates a loss of that chromosome locus in green-labeled tumor DNA; if the signal is skewed to green, it suggests a gain of that chromosome locus in tumor DNA
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• Genome-wide profiling studies using miRNA signatures have been performed on various cancer types. Tumors have distinctive patterns of miRNA expression. miRNAexpression profiling of human tumors can aid in diagnosis, staging , prognosis, and prediction of treatment response and determination of clonal origin • Technical approaches - A set of oligoprobes corresponding to human miRNA are made into a microarray - The miRNA is extracted and reverse transcribed into eDNA. The eDNA is then PCR amplified, labeled, and hybridized to the array - The data is analyzed by an array analyzer
Protein-Based Clonality Analysis • Isoenzyme-based clonality analysis, such as comparison of different isofonns of G6PD isoenzymes (see section II, X chromosome inactivation for more details) • Proteomics (see Chapter 9 for more details) • Immunohistochemistry (IHq-based clonality analysis - Tumors from a common clonal origin express a common set of protein markers - Tissue specific antigens, such as prostate-specific antigen (PSA), thyroid transcription factor-I (TTF-l), CK7, CK20, CDX-2, OCT-4, and so on, are helpful in defining tumor origins and clonal relationships of tumors - Over-expression of oncogenes, such as ras and myc, can be readily detected in many cancers and in their metastases, and detection of these gene products has been explored as a means of clonality assessment
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TISSUE CONTAMINATION AND PATIENT IDENTITY MISMATCH TESTING Overview • Patient identity mismatches may happen before or after a specimen arrives in the laboratory. Possible sources of intra-laboratory contamination include the dissection bench, instruments, ink-marking of tissue margins, sectioning, and the water bath used in histologic sectioning • Clonality analysis is one of the most commonly used approaches for identity testing. Molecular identity testing is based on the principle that cells from the same individual share a common set of genetic characteristics (Figure 20) • Commonly used methods include microsatellite profiling (Combined DNA Index System [CODIS)), FISH for sex chromosome determination, and human leukocyte antigen system (HLA)-related genotyping
Technical Approaches • FISH (Figure 21) - FISH is usually the first step in identity testing to exclude sex-mismatched contamination. Sexmismatched tissue contaminations can be readily resolved by FISH through the detection of differing sex chromosome patterns (XX and XY) in a formalinfixed paraffin-embedded tissue section - Both host tissue (known to be from patient's sample) and suspected contaminating tissue are processed and hybridized with fluorescent-labeled DNA probes for X and Y chromosomes. The slides then are observed under fluorescence microscopy for the X and Y signals • The cells with XX signal pattern in their nuclei are from a female; the cells containing XY in their nuclei are from a male individual. A mismatched sex chromosome pattern indicates that the tissues in a sample are derived from different individuals • If the same sex chromosome pattern is observed in the known patient's tissue and the suspected contaminating tissue, further testing is needed to determine whether the tissues are derived from one or more than one individual • The marker amelogenin is also frequently used in sex determination - Amelogenin is a low-molecular-weight protein belonging to a family of extracellular matrix proteins - It is the predominant protein component in developing enamel - Amelogenin regulates the initiation and growth of hydroxyapatite crystals during the mineralization of enamel and aids in the development of cementum - The AMELX gene (from X chromosome) contains a 6-bp deletion in intron 1. PCR amplification of intron I of the AMELX gives rise to a 106-bp PCR product;
the Y chromosome gene, the AMELY, gives rise to a 112-bp PCR product - After gel electrophoresis, a male patient (XY) will show two bands (106 bp and 112 bp PCR product); a female patient (XX) will show only one band (106 bp PCR product) • Microsatellite profiling and CODIS for identity testing - Microsatellite profiling is a technique which uses polymorphisms of microsatellite loci, which are unique for each individual. Polymorphism patterns of multiple microsatellite loci are shared by every cell from the same individual, and determining this pattern is referred to as "DNA fingerprinting" or "DNA typing ." Alleles from different individuals can be unambiguously defined by the differing numbers of microsatellite repeats, which are expressed as lengths of amplified fragments (see Chapter 28 for more details) (Figure 21) - Microsatellite markers were used for human identity testing in the early 1990s. In 1997, 13 DNA polymorphic markers were identified as core markers for the US national database known as the CODIS (http://www.fbLgovlhqllab/codis/indexl.htm).This system covers microsatellite loci on chromosomes 2, 3,4,5,7,8,11,12,13,16, 18,and21 (TableS) • In the United States and United Kingdom >5 million criminal justice DNA profiles are in the database . The random match probability for each allele locus ranges from 8 x 10-4to 7.2 X 10- 19 depending on the polymorphism and number of repeats at each locus • Commercially available microsatellite panel kits and automated systems such as the ABI3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) allow the screening of multiple short tandem repeat (STR) loci in one run. With proven high specificity and sensitivity, CODIS markers have become the most commonly used loci in identity testing. Small screening panels can confirm an identity at a specified level of confidence • Brief procedures for microsatellite identity analysis (Figure 21): - The tissues are harvested separately from known host tissue or suspected contaminating tissue using tissue microdissection; genomic DNA is isolated. Patient blood DNA, if available, is also extracted as a reference - The DNA from different tissue origins is amplified using microsatellite locus-specific primers. The PCR products are separated by polyacrylamide gel electrophoresis or capillary electrophoresis - The allelic patterns from the known patient DNA sample and from the suspected contamination DNA are compared - If the tissues are derived from the same patient, both DNA samples will show identical band patterns (also
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• i
Contam ination?
I
LOH , MSI
~
. 1Il.@'2IDji1illr~@ii1 . Inconclusive results
Fig. 20. Diagnostic strategies for identifying tissue contamination or patient identity mismatches. FISH for sex chromosomes is usually the first test used to distinguish sex-mismatched samples. When the suspected contaminating tissue displays the same pattern of sex chromosomes as the patient control tissue, microsatellite markers may be employed to assess for contamination (identity mismatch). CODIS loci are usually used for microsatellite profiling. There are three possible outcomes: the suspected contaminating tissue may display a different microsatellite profile, consistent with specimencontamination; the suspected contaminant may display an identical microsatellite profile, rulingout specimencontamination; or the testmay yield inconclusive resultsdue to LOH or MSI in tumorsamples. In this latter situation, stromalcell DNA can be tested to resolvethe issue. HLA genotyping may also be employed in these cases.
identical to that of the patient blood DNA reference); simultaneous detection of different allele patterns over multiple loci strongly suggests that the tissues are from different individuals
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• HLA genotyping is useful in tests of identity mismatch or tissue contamination - The HLA system is a group of genes that reside on chromosome 6, which encode cell-surface
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A
xx
B
Patient's tissue /
® p e R Amplification .
Suspected contamination
~
XX
~
/'
t
Locus 1
Locus 2
Locus 1
Locus 2
ps
ps
p s
ps
1== ==11= I
•
~ ---+
-
@ ~
==1 II
•
P Panent'a tissue
S : Suspectedccn taminanon
o : X chromosome 0 'Y
chromosome
Histopathology-+FISH-+ONA extraction I peR -+ Microsatellite profiling
Fig. 21. Molecular identity testing is based on the principle that cells from the same individual share a common set of genetic characteri stics. FISH testing for sex chromo somes will distinguish sex-mismatched cases (A). However, there is a 50% chance that the suspected contaminating tissue is from a same gender individual. Microsatellite profiling can be used to differentiate the tissues (B). Genomic DNA is extracted from the tissues and PCR amplified followed by gel electrophoresis. Schematic allelic patterns are shown in the box on the right. P and S designate patient and suspected contaminating tissues, respectively. (I) Concordant allelic pattern s indicating the tissue is from the same individual; (II) discordant allelic patterns indicating that the tissue is from different individuals. Only informative loci are shown here for illustrative purposes. antigen-pre senting proteins and many other proteins. The chan ce of unrelated individuals having identical HLA genotypes is very low; therefore, the HLA genotype can be used as a clonal marker (see Chapter 27 for more details) - A commercial kit can be used to amplify and distinguish 21 different genotypes at a polymorphic HLA locus - Brief procedure s • DNA is extracted from a tissue section, then PCR is amplified against the HLA DQ-ulocus (6p21.3), followed by hybridization to nylon membrane strips with allele-specific HLA-DQ-u probes
• The pattern of signal dots indicates the homology to the HLA-DQ-u alleles, which reflects the genotype • Different genotypes indicate that tissue samples are from different individuals
Caveats • The ability of a single microsatellite marker to distinguish between individuals depend s on the degree of polymorphism the marker exhibits. The reported rate ranges from 59 to 91%, but absolute matching requires several polymorphic microsateIlite markers
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Table 5. The Combined DNA Index System Locus
Repeats
Location
Fragment size
TPOX
5-14
2p23-pter
102-138
O3S1358
8-20
3p21
99-147
FGA
12-51
4q28
308-464
05S818
7-16
5q21-31
133-169
CSFIPO
6-16
5q33-34
281-321
07S820
5-15
7q
205-234
08S1179
7-19
8q24.1-24.2
157-205
THO I
3-14
IIp15
146-190
vWA
10--25
12p-pter
122-182
013S317
5-16
13q22-31
157-201
016S539
5-15
16q22-24
141-173
018S51
7-27
18q21.3
262-342
021S11
24-38
21q21.1
186-244
• Since most of the analyzed materials are from formalinfixed and paraffin-embedded tissue, the rate of detectable amplification products declines with increasing amplicon length. Shorter fragments, preferably <200 bp should be used to obtain a reasonable sensitivity for the microdissected samples • The standard, or optimal, number of DNA loci that needs to be examined for tests of tissue contamination and patient identity mismatch has yet to be determined - Many studies have shown that the use of five to eight polymorphic DNA loci could well achieve a high specificity for mismatch detection - The use of nine markers achieved a power of exclusion of 99.7% in a paternity test (a power of exclusion of 99% is legally required) - The available commercial kits use three to 16 DNA markers. The Amelogenin gender determining marker is often included.
- The most frequently used markers are derived from the CODIS panel, such as FGA, THOI, TPOX , VWA, D3S1358, D8S1179, D18S5l, and D21Sll (Table 5) - The probability of random matching of unrelated individuals at 3, 4, and 8 markers is about 1.0 x 10-3, 7.8 X 10-4, and 7.4 x 10- 10, respectively. The probability of two unrelated Caucasian individuals matching at 12 loci is about 1.12 x 10- 12 based on published heterozygosity values for the known markers • The high LOH and MSI rate in tumor tissues may interfere with the microsatellite profiling tests, especially for tissues consisting predominantly of tumor cells. Alternative tissues such as stroma could be analyzed. HLA genotyping may also be used in difficult cases
IDENTIFICATION OF DONOR ORIGIN IN TRANSPLANTATION PATIENTS Overview • The incidence of cancer in solid organ transplantation recipients is reported to be 4-18%. Tumors in transplant recipients may be transmitted inadvertently through the transplanted organ • Most recipients of solid organ transplantation receive immunosuppressive therapy for prolonged periods,
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which greatly increases the risk for cancer development. Tumors may also arise de novo in the transplanted organ • Cancer incidence has been reported to be 40--50% 20 years after renal transplantation • The identification of tumor origin is of biologic, clinical, therapeutic, and possible legal importance
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A
IRecipient 1-----.
® ® xx
ITumor 1
/ <,
xx
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-----.
-
-----. 1 Donor origin 1
XY
I Donor 1-----.
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B
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R : Recipient T : Tumor 0 : Donor o : X chromosome o : Y chromoso me
xx locus 1
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R TD
R TD
R TD
R TD
peR Amplification
1=== === 1 1=== === 1 I
II
. Donor origin
. 1 _
FISH--+DNA extraction / peR --+ Microsatellite profiling
Fig. 22. Post-transplantation tumors can originate from the recipient, can be transmitted with the transplanted organ, or can arise de novo from the donor organ. FISH can be used to differentiate sex-mismatched cases by showing different sex chromosome patterns (panel A). Microsatellite profiling can distinguish donor and recipient by comparing the allelic patterns at microsatellite loci (panel B). Genomic DNA is extracted from donor (D), recipient (R), and tumor (T) and is PCR amplified. As shown in the green box, a tumor of donor origin will show an allele pattern identical to that of the donor (I). A tumor of recipient origin will show an allele pattern identical to that of the recipient (II). Only informative loci are shown here for illustrative purposes.
Technical Approaches (Figure 22) • Similar approaches to those used in patient identity mismatch testing are used (see previous discussion) • FISH - The tumor origin of sex mismatched organ allografts can be differentiated through sex chromosome patterns • Microsatellite profiling - The CaDIS is often used as a microsatellite profile panel, which can distinguish tissues of donor or recipient origin
- HLA genotyping could also be used in the analysis of tumor origin after organ transplantation (see previous discussion)
Caveats • During microsatellite profiling, donor and recipient alleles sometimes could be presented simultaneously due to the presence of recipient stroma and blood cells in the transplanted organ. Precise microdissection could help to avoid contamination with recipient cells
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• Microsatellite alleles may undergo alterations such as LOH and MSI. Multiple microsatellite loci are needed in creating a reliable microsatellite profile • Ideally, DNA samples from donor (blood or paraffinembedded tissue), recipient (blood or paraffin-embedded
tissue), and tumor are analyzed in parallel. However, in many incidences, donor DNA is not available. Accurate results could still be obtained in these circumstances if the experiments are carefully controlled with meticulous attention to the selection of markers
BONE MARROW ENGRAFTMENT TESTING Overview • Hematopoietic stem cell transplantation, also known as bone marrow transplantation, is a procedure in which hematopoietic stem cells are transferred from a donor to a recipient. Allogeneic bone marrow transplantation is primarily used for the treatment of hematopoietic malignancies and hereditary disorders (see Chapter 27 for more details) • Engraftment is the process by which transplanted donor stem cells begin to proliferate and produce blood cells within the recipient. Engraftment of bone marrow transplants usually takes between 10 and 20 days to occur • A low percentage of hematopoietic cells from the recipient may still exist after successful stem cell transplantation. This condition is called mixed chimerism (MC) and can be detected in the peripheral blood of the recipients after transplantation
i
i i i
i
i i i
Recipient allele
Donor allele
B
• Engraftment testing is used to monitor the efficiency of engraftment, to detect graft rejection, and to assess the risk for relapse and the effectiveness of therapy
Technical Approaches • FISH - This method is only suitable for recipients with a sex mismatched donor - Post-transplantation blood cells are prepared and hybridized with fluorescently labeled probes for chromosomes X and Y - The cells with donor and recipient chromosome profiles are counted and calculated for the percentage of recipient cells • Microsatellite profiling - Engraftment testing is performed using gene amplification of microsatellite markers in pre- and post-transplant samples of donor and recipient DNA - CaDIS markers are among the most frequently used markers - Pre-transplantation blood from both donor and recipient are needed to determine the informative microsatellite loci to be used in the analysis. The post-transplantation analysis establishes relative amounts of recipient and donor cells in the recipient's blood (Figure 23) - Brief procedures:
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Recipient allele
Donor allele
Fig. 23. Bone marrow engraftment analysis. The schematic illustration shows microsatellite locus amplification a short time after bone marrow transplantation (A) and several months after bone marrow transplantation (B). The main peaks in panel (A) represent donor alleles (reconstitution of the hematopoietic pool). The lower peaks on the left are from a 5% mixture of recipient DNA. There are no residual recipient cells detectable in the blood at this time. Several months after bone marrow transplantation, the test shows increased recipient alleles (red) from recipient blood cells (B), consistent with disease relapse.
Clonality Analysis in Modern Oncology and Surgical Pathology
• Genomic DNA is extracted from the donor and recipient pre-transplantation bone marrow or blood specimens, and from the recipient post-transplantation blood or bone marrow specimens • Informative microsatellite loci are selected according to the results of donor and recipient pretransplantation DNA testing • The DNAs are amplified by PCR and the DNA products are separated by capillary electrophoresis or gel electrophoresis • The donor and recipient patterns are compared and the ratio of recipient/donor cells is calculated
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• Alternatively, the relative percentage can also be inferred and calculated by comparing the recipient allele intensity to the control sample containing 5% mixed recipient DNA
Caveats • Selection of informative rnicrosatellite markers is critical for a successful engraftment test; the donor and recipient pretransplantation blood should be tested to ensure that loci are informative (two alleles are present) and non-homologous (the band sizes are different between donor and recipient) • Many laboratories use 10 or more microsatellite loci to ensure the accuracy of the test
• The relative intensity of recipient to donor is calculated by summation of two recipient allele intensities divided by summation of two recipient and two donor allele intensities
• A higher percentage or increasing level of recipient cells carries an increased risk for relapse . However, the exact cutoff value for the level of MC is yet to be established. Some studies suggest that detection of> 1% of recipient cells correlates with an increased risk of relapse
• MC = (Rl + R2)/(Rl + R2 + Dl + D2) x 100% (Rl , R2: recipient allele 1 and recipient allele 2; Dl , D2: donor allele 1 and donor allele 2)
• Measurement of chimerism in a particular sub-population can be achieved by selective enrichment of target cells using flow cytometry (see Chapter 6 for more details)
MOLECULAR DIAGNOSIS OF HYDATIDIFORM MOLE
Overview • Hydatidiform mole is a trophoblastic proliferation disease characterized by marked enlargement of villi. About one in every 1000 pregnancies is affected with much higher incidences in Asia • This condition is characterized by abnormal development of both embryonic and extra-embryonic tissues and is associated with abnormal chromosomes • Hydatidiform moles are divided into two types : complete and partial moles (Table 6) • About 20% of patients with complete moles and 5% of patients with partial moles may develop persistent gestational trophoblastic disease - Partial moles are mostly triploid (69 XXX, 69 XXY, or 69 YYX) with a genome that is almost always composed of two sets of chromosomes of paternal origin (diandric) and of a haploid maternal set. Partial moles result either from the fertilization of a single ovum by two different haploid sperms or from fertilization by a single sperm with a duplicated genome. An extra-paternal set of chromosomes results in trophoblast overgrowth with underdevelopment of the embryo. The risk for progression to choriocarcinoma is lower compared with a complete mole (Table 6, Figures 24 and 25) - A complete mole is diploid . 90% of complete moles are 46XX and 10% are 46XY. The genomes of
complete moles are purely paternal and fetal parts do not form. Complete moles result from androgenesis, in which an empty ovum is fertilized by two sperms or by one sperm that has duplicated its genome . The risk for progression to choriocarcinoma is about 2%
Technical Approaches • The basis for the molecular diagnosis of moles is the distinctive genome compositions of partial and complete moles. FISH can differentiate partial and complete moles by detection of a different numbers of sex chromosomes. Microsatellite profiles can distinguish partial and complete moles by demonstration of the allelotype and dosage differences of paternal and maternal alleles (Figure 25) • FISH detection of sex chromosomes (Figures 24 and 25) - Partial moles are composed of trisomic cells with one maternal X and two paternal sex chromosomes. FISH shows an XXY, XYY, or XXX pattern - Complete moles are composed of cells with diploid genomes that are made up entirely of paternal chromosomes. The FISH pattern could be XX or XY. FISH could not distinguish complete mole from normal pregnancy • Microsatellite profiling - CODIS loci have been used in many studies (see section V for more details)
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Table 6. Characteristics of Complete and Partial Mole Chromosome Proposed Karyotyping origin mechanism
Fetal parts
FISH
Partial mole
Maternal and paternal
69XXY (70%) Underdeveloped Trisomy Two sperm embryo fertilized one 69XXX (27%) nonnalovum 69XYY (3%)
Complete mole
Paternal
46XX (90%) Empty ovum fertilized by 46XY (10%) 2 sperms or fertilized by I sperm with duplicated genome
Do not form
Villus from normal pregnancy
Maternaland paternal
-
Embryo
46XX,46XY
Microsetellite'
Risk of choriocarcinoma
3 alleles (PPM)
Lowerrisk
Disomy
2 alleles (PP)
2%
Disomy
2 alleles (MP)
Exceedingly low
formed
aOnl y informative alleles are considered here for illustrative purpose M, maternal allele;
- The microsatellite loci from villus DNA and from a maternal control are amplified and the allele patterns are compared (Figure 25) - Partial moles show two paternal (P) alleles and one maternal (M) allele as a three allele pattern at each locus (MPP pattern). Among the three alleles demonstrated, one is of maternal origin, matching a maternal allele position (Figure 25, Table 6). Partial mole may also show an MP pattern if two paternal alleles are homozygous - Complete moles show an allelic pattern identical to the paternal allele, and may be either homozygous (P) or heterozygous (PP) (Figure 25). A maternal allele will be absent
p,
paternal allele.
- Normal villi will show one paternal and one maternal allele (MP pattern)
Caveats • FISH testing cannot distinguish a normal villus from a complete mole. Therefore, microsatellite profiling is preferred in many laboratories • For microsatellite polymorphism testing, the genomic DNA that is used for a maternal control can be obtained either from blood or from maternal tissue microdissected from paraffin sections (such as endometrium) • Because some microsatellite markers are non-informative, it is critical that multiple markers be tested to allow for accurate interpretation
CANCER OF UNKNOWN PRIMARY ORIGIN (CUP)
Overview • Cancer of unknown primary origin (CUP) is defined as a biopsy-proven metastasis for which the site of origin cannot be determined by medical history, physical examination, laboratory tests, imaging studies, and histologic evaluation • Unknown primary tumors comprise about 3-5% of all human cancers. Approximately 30,000 cases of CUP are diagnosed in the United States annually
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• CUP represents a heterogeneous collection of tumor types and clinical presentations • Identification of a primary tumor in these patients can help to predict tumor behavior and to determine appropriate therapy • Mechanisms of CUP: - The primary tumor has involuted or regressed and is no longer detectable by conventional methods when the metastasis becomes widespread
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Clonality Analysis in Modern Oncology and Surgical Pathology
Micro satellite polymorphism
FISH
Partial mo le Locus 1
Partial mole
-A-...J...AJ.PM P
I
~x
Locus 2
/
..AA..-- -
Maternal control
--.1LJJL -
Villus - - --
[
M PP
I
Non-informative Informative
Complete mole Complete mo le
+-
~
------.A...
-----AA... -
Locus 1
Locus 2
P PP Non-informative Informative
-
/ Maternal control Villus
[
• Chromosome Xof maternalorigin o ChromosomeXof paternal origin Chromosome Yof paternal origin
o Fig. 24. Mechanisms of partial and complete mole formation . Partial moles are derived from both maternal and paternal chromosomes and are mostly triploid (69 XXX, 69 XXY, and 69XYY) with two sets of paternal chromosomes (P, yellow) and a set of chromosomes of maternal origin (M, black) . Partial moles may result from two sperms fertilizing a haploid ovum or from one sperm fertilizing the ovum and duplicating its chromosomes. Complete moles are diploid and their chromosomes are purely paternal. Complete moles result from two sperms fertilizing an empty ovum or from one sperm fertilizing the empty ovum and duplicating its chromosomes. Ninety percent of complete moles are 46XX and 10% are 46XY.
- Alternatively, CUP may represent a unique tumor type in which the primary tumor has acquired a special metastatic phenotype and genotype soon after tumor initiation. These tumors have a preference for metastatic spread over local tumor growth
Technical Approaches (Table 7) Principle : cancer is a clonal process. The cells in a metastatic tumor share a similar phenotype and genotype with the tumor cells from the primary site
Clinical • Epidemiologic data (including age , sex , and race) , prior medical history, location of metastasis, careful physical examination, complete blood work -up including cancer serum marker measurements, and selected imaging studies are helpful to formulate a
Fig. 25. Molecular diagnosis of molar pregnancy using microsatellite allelotyping and FISH . DNA samples are extracted from a maternal control (blood) and from the mole (villus) . Partial moles (upper panel) show one maternal allele (Bold, M and arrow) and two paternal alleles (P). Complete moles (lower panel) show no maternal allele but may show one or two paternal alleles. Locus I represents a noninformative DNA marker as the maternal control is homozygous at this locus. Locus 2 represents an informative DNA marker as the maternal control is heterozygous at this locus. Multiple polymorphic microsatellite loci should be employed in the analyses. FISH can also be used for mole identification. As shown in the right panel, partial moles show an XXY, XXX, or XYY pattern (maternal, black; paternal, yellow) in contrast to the XX pattern in the maternal control. Complete moles are diploid with either an XX or XY complement of sex chromosomes, both of paternal origin (yellow). differential diagnosis and to rule out some common types of tumors
Pathology • Histologic evaluation using light microscopy • Hematoxylin and eosin evaluation of the metastatic lesion remains the cornerstone for the identification of a primary tumor in CUP patients • Tissue-specific protein markers can be identified using a selected IHC panel of antibodies • Numerous antibodies are now commercially available in the routine work-up for CUP (such as PSA, HMB45 , TTFl, CDX2, CK7, CK20, and OCT4)
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Table 7. Diagnostic Strategy for Discovering an Unknown Primary Tumor Clinical
• Medical history and physical examination - Age and sex - Race - Location of metatastasis - Prior medical history - Family history of cancer • Cancer serum markers such as PSA and o-fetoprotein • Imaging analysis - Chest X ray - Mammography - Computed tomography - Positron emission tomography (imaging) - Magnetic resonance imaging Pathology
• Light microscopic examination • mc using tissue specific markers such as PSA, TIFI, and OCT4 • Ultrastructural (electron microscopy) examination Molecular
• Cytogenetic andFISH analysis - Isochromosome 12p for germ cell tumor - Selected translocations - Others • Mutational screening - Oncogenes (EGFR, c-kit, or PDGFR) - Tumor suppressor gene (P53) - Metastasis suppressor gene (Kiss-Ti • Gene expression profiling/array-based analysis • CGH (Array CGH) • SAGE • Viral integration analysis (such as EBV, HPV, and HBV genome detection) • MicroRNA profiling • Proteomic profiling • Electron microscopy - Ultrastructural analysis may be useful in the diagnosis of a primary tumor in CUP patients. Characteristic ultrastructural features of tumor cells can be recognized and aid in the differential diagnosis
Molecular Cytogenetics and FISH • Characteristic chromosomal alterations may suggest a specific tumor type and organ site as these cytogenetic
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abormalities are found in high frequency in certain tumors, such as t(X;18) in synovial sarcoma and trisomy 7/17 for papillary renal cell carcinoma (refer to Table 2 in Chapter I for details) • Isochromosome 12p (i[12p]) is a marker for tumors of germ cell origin - Testicular cancer patients may have metastatic tumors of diverse histologic types that lack features of germ cell tumors. Kernek et al. (2004) found FISH for 12p amplification in routinely processed surgical specimens to be a useful adjuvant diagnostic tool in confirming the germ cell origin of metastatic tumors having the histologic appearance of somatic-type neoplasms
Mutational Screening • Overexpression of oncogenes and mutations of tumor suppressor genes are common in solid tumors. Detection of oncogenes and tumor suppressor gene mutations can be helpful in the identification of tumor primary sites and in clinical outcome prediction Gene Expression Profiling/Array-Based Analysis • DNA microarrays are used to analyze genome-wide gene expression (gene expression profiling) (see Chapter 8 for more details) • It can monitor expression levels of thousands of genes simultaneously and can discriminate different disease states through qualitative and quantitative measures • An expression profiling study begins with samples from well-defined tumors, from which a specific panel of genetic markers (molecular signature) is established. Gene expression profiles of metastasic tumors closely resemble primary tumors of the same origin. Therefore, the gene expression profile can be used to identify tumor origin. Comparison to the relative abundance of specific panels of genes known to be expressed in different tissues (tissue-specific signatures) can provide clues to the cellular origin and primary site of a metastatic tumor Comparative Genomic Hybridization (Array CGH) • See Chapters 3, 7-10, and 12 for more details
Serial Analysis of Gene Expression (SAGE) • See Chapters 3, 7-10, and 12 for more details • SAGE is a sequence-based sampling method for comprehensive analysis of the gene expression patterns. This technology does not require a pre-existing known mRNA sequence; therefore, it can be used to identify new genes and to quantify known genes • The underlying principle of SAGE technology is that a short representative sequence tag (l0-14 bp) can be used to uniquely identify a transcript, and the tag numbers directly reflect the abundance of correspondingtranscripts • The SAGE libraries are highly accurate, quantitative, and comprehensive representations of the samples from which they are derived
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• miRNAs are transcribed from RNA genes and are involved in many cellular processes including differentiation, proliferation, and the stress response
• Dennis et al. (2005) used 15 publicly available SAGE data libraries and found that a site of origin can be determined for a CUP with an 88% accuracy
Viral Integration Analysis • Identification of viral genomic sequences in a CUP is useful to establish a primary site (e.g., identification of Epstein-Barr virus DNA in a CUP is helpful for establishing a nasopharyngeal primary in the appropriate clinical setting)
miRNA Profiling • miRNAs are short 20-22 nucleotide RNA molecules that are negative regulators of gene expression (see Chapter 7 for more details)
• A distinct signature based on miRNA expression profiling can be used to identify tumor origin
Proteomic Profiling (See Chapter 9 for More Details) • Proteomic methods detect a unique panel of proteins, which can differentiate one tissue from another according to established algorithms. These methods may provide insights into specific expressions of proteins which could serve as unique tumor markers
SUMMARY CUP represents a group of biologically and clinically heterogenous tumors. Management of patients with CUP requires a truly multi-disciplinary approach and close communications between the pathologists and oncologists to ensure an effective work-up. A variety of clonality analysis methodologies can be employed to determine a site of origin
for the metastatic lesion . Gene and protein expression profiling technologies are potentially of great benefit in the identification of a primary tumor and may aid in the accurate assessment of prognosis . The identification of CUP-specific molecular signatures may offer new avenues for diagnosis and treatment.
SUGGESTED READING Abbosh PH, Wang M, Eble IN, et al. Hypennethylation of tumor suppressor gene CpG Islands in small cell carcinoma of the urinary bladder. Mod Pathol. 2007 (in press).
Bostwick DG, Shan A, Qian J, et al. Independent origin of multiple foci of prostate intraepithelial neoplasia (PIN): comparison with matched foci of prostate cancer. Cancer 1998;83:1995-2002.
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12
Fluorescence In Situ Hybridization (FISH) and Conventional Cytogenetics for Hematology and Oncology Diagnosis Vesna Najfeld,
PhD
CONTENTS I. Introduction II. Methods
12-4 l2-4
Conventional Cytogenetics 12-4 Fluorescence In Situ Hybridization (FISH) 12-4 FISH Protocol for Hematologic Malignancies 12-4 FISH Protocol of Paraffin-Embedded Tissue 12-8 FISH Protocol for Bladder Cancer 12-8 Interphase FISH 12-8 Fiber FISH 12-9 Multi-Color FISH 12-10 Comparative Genomic Hybridization (CGH) 12-11 Array CGH (aCGH) .12-12 Types of Probes 12-14 Probe Strategies 12-14 Probe Validation 12-14 Conventional Cytogenetics and Varying FISH Methodologies 12-14
III. Myeloproliferative Disorders Chron ic Myelogenous Leukemia Chron ic Phase The Ph Chromosome The BCR-ABL Fusion Gene Diagnosis Monitoring Response to Therapy
12-15 12-17 12-17 12-17 12-17 12-19 12-20
Monitoring Gleevac (Imatinib) Therapy 12-21 Blast Phase 12-21 Ph-Negative Chronic Myeloproliferative Disorders (CMPD) 12-22 Polycythemia Vera (PV) 12-22 Idiopathic Myelofibrosis 12-24 Essential Thrombocythemia 12-24 Other Ph-Negative Chronic Myeloproliferative Disorders .......... 12-24
IV. Myelodysplastic Disorders (MDS) Clonal Origin Chromosomal Abnormalities in MDS FISH Studies in MDS Current Treatment 5q-Syndrome Rearrangements of 17p Rearrangements of 3q26 Rearrangements of t(8)(pll)
V. Acute Myelogenous Leukemia Karyotype in AML AML M2 and t(8;21)(q22;q22) t( 16;21)(q24 ;q22) Rearrangements of 21q22 APL, t( 15;17)(q22 ;q21) and Variant Translocations: t( 11;17)(q23 ;q2 1), t(5;17)(q35;q21 ),t(ll ;17)(q 13;q21), and dup(17)(q21.3-q21 )
12-26 12-26 12-26 12-26 12-26 12-27 12-27 12-28 .12-28
12-29 12-29 12-29 12-31 12-31
12-32
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Molecular Genetic Pathology
Acute Myelomonocytic Leukemia with Excess Eosinophils (M4Eo);inv(16) (pI3q22) and t(16;16)(pI3;q22) t(16;21)(pll;q22) t(9;22)(q34;q 11.2) Acute Monocytic Leukemia (M5) and Translocations Involving l1q23 and Myeloid Lymphoid Lineage (MLL) Gene AML with an Increased Number of Basophils and t(6;9)(p23 ;q34) t(1;22)(p13;q13) and Acute Megakaryoblastic Leukemia (AMKL) AML in the Elderly Therapy-Related AML Monosomy 5/del(5q) Monosomy 7/del(7q) Chromosomal Gain or Loss in AML AML with Normal Karyotype
VI.
Lymphoproliferative Disorder (LPD) Difficulties in Obtaining Chromosomes from LPDs B-Lymphoid Disorders Acute Lymphoblastic Leukemia (ALL) HyperdiploidylHypodiploidy t(12;21)(pI3;q22) Other Abnormalities Involving 12p and TEUETVI t(9;22)(q34;qll.2) t(1; 19)(q23;p13 .3) Rearrangements of llq23 t(8;14)(q24;q32) in Childhood ALL Abnormalities of (9)(p21-22) Other Abnormalities Chronic Lymphocytic Leukemia (CLL) Conventional Cytogenetics in CLL FISH in CLL Deletions of 13q Trisomy 12 Deletions of 11q del (17)(pI3.1) Multiple Myeloma Plasma Cells del(13)q14.3 t(l4q;32.3) Involving IGH loci del(17)(pI3.1)/P53 Aneuploidy Hairy Cell Leukemia (HCL) B-Cell Prolymphocytic Leukemia
304
VII. Non-Hodgkin's Lymphoma 12-33 12-34 12-34
12-34 12-36 12-36 .12-37 12-37 12-37 12-38 12-38 12-39
12-39 12-39 12-39 12-39 12-39 12-41 12-41 12-42 12-42 12-43 12-43 12-43 12-43
12-43 12-43 12-44 12-44 12-45 12-45 12-45 12-46 12-46 12-46 12-47 12-49 12-49 12-49 12-49
12-49
Immunology and Genetics 12-49 Follicular Lymphoma 12-49 Burkitt's Lymphoma 12-49 Diffuse Large Cell Lymphoma .12-50 Mantle Cell Lymphoma 12-51 Marginal Zone B-Cell Lymphoma (MZBCL) and Mucosa-A ssociated Lymphoid Tissue (MALT) Lymphoma 12-52 Lymphoplasmacytoid Lymphoma (LPL) 12-52 Waldenstrom Macroglobulinemia 12-52 Hodgkin's Disease (HD) 12-52
VIII. T-Cell Leukemia and Lymphoma T-Cell Lymphoproliferative Diseases T-Cell ALL t(10;14)(q25 ;qll) T-Cell Receptor (TCR) Rearrangements t(5;14)(q35 ;q32) or t(5;14) (q34;qll) T-Cell CLL and Prolymphocytic Leukemia (PLL) T-Cell CLL/PLL Adult T-Cell LeukemialLymphoma (ATLL) Natural Killer (NK) Lymphoma! Leukemia Anaplastic Large Cell Lymphoma (ALCL)-ALK-Positive Lymphoma
IX. SolidTumors HER2 in Breast Cancer HER2 Assays The IHC Method The FISH Methods Concordance Between IHC and FISH Bladder Cancer Urovysiou'", Food and Drug Administration (FDA)-Approved Assay Protocol for Urovysion" Assay Results Obtained with
Urovysion" Chromosomal Abnormalities and Prognosi s Anticipatory Positive (AP) Results Gliomas Classification of Gliomas
12-53 12-53 12-53 12-53 12-53 12-53 12-53 12-53 12-53 12-53 12-54
12-54 12-54 12-54 12-55 12-55 12-56 12-56
12-56 12-56 12-58 12-58 12-58 12-58 12-58
FISH and Convention al Cytogenetics Genetics of Oligodendroglial Tumors Tissue Processing Neuroblastoma Ampli fication of MYCN Gene Gain of 17q Sarcoma Ewin g SarcomalPNET t(lI ;22)(q24;q I2)
12-3
12-58 12-58 12-58 12-59 12-60 12-60 12-60 12-60
Synovial Sarcoma t(X; 18)(p ll .2;q ll .2)
12-60 12-61
X. Conclusions and Future Directions
12-61
XI. Nomenclature XII. Suggested Reading
12-61 12-61
305
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Molecular Genetic Pathology
INTRODUCTION
Over the past 35 years, cytogenetic analysis of malignant hematological disorders has been one of the most rapidly growing areas in cancer. More than 45,000 cytogenetically abnormal neoplastic disorders have been reported and the evidence accumulated clearly demonstrates that karyotype information provide both biological and significant clinical value . While improved cell culture methods and the application of chromo some banding technique s had advanced our understanding of disease-specific abnormalities, molecular cytogenetics has now made it possible to identify genes involved at translocation breakpoints in specific chromosomal rearrangements. This knowledge is at the forefront of our
understanding of the molecular pathogenesis of cancer. The goal of this chapter is to illustrate specific cytogenetic events and to delineate molecular phenotype s, which are key to the diagnosis and progno sis of leukemia, lymphoma, breast cancer, bladder cancer, and sarcoma. Thus, the hypothesis put forward by Boveri at the turn of the century, namely, that an abnormal chromo some pattern is intimately associated with the malignant phenotype of the tumor cell has proved correct for many malignant disorders. Undoubtedl y, knowledge of the molecular phenotype of a disease will lead to innovative and specifically tailored treatment s. The first example of such gene-targeted therapy has already been successfully applied.
METHODS
Conventional Cytogenetics Cytogenetic analysis in malignant disease is based on studies of the tumor cells themselves. Obtaining metaphase cells of tumor specimen is sine qua non of cancer cytogenetics. In hematologic malignancie s usually a bone marrow aspirate is a required tissue while in lymphoma, it is usually a lymph node biopsy, and in solid tumors it is a tumor biopsy, either processed immediately (direct preparation) or cultured for 24-96 hours. Peripheral blood is not useful for cytogenetic analyses of the majority of patients with lymphoma and solid tumors. Amethopterin or f1uorodeoxyuridine is used to synchronize dividing cells, and ethidium bromide, results in elongated chromosomes that have an increased number of bands. Cells arrested in metaphase are obtained by exposing marrow cells or other tumor cells sequentially to mitotic inhibitors, hypotonic KCI, and fixative . The fixed cell suspension is dropped onto pre-cleaned microscope slides, which are then subjected to the most widely used banding method, i.e., trypsin-Giemsa banding (Figure IA,B) . Chromosomes obtained from leukemic bone marrow cells from lymph nodes or solid tumors may be fuzzy with indistinct bands. The criteria used to define clonal abnormalities are listed in Table 2 and described in the International System for Human Cytogenetic Nomenclature. (Ta ble I shows conventional and molecular cytogenetics methods and Table 2 shows the glossary of genetic terms.)
Fluorescence In Situ Hybridization (FISH) • FISH is a molecular method that allows detection of the number, size, and location of DNA and RNA segments within individual cells in a tissue sample. It is based on the ability of single-stranded DNA to anneal to complementary DNA
306
• In malignant disorders, the target DNA is the tumor nuclear DNA of interphase cells or the DNA of metaphase chromosomes that are fixed on a microscope slide • In FISH technology, a specific DNA segment is converted into a probe through the attachment of a fluorescent tag or a reporter molecule that later in the procedure will be conjugated with the fluore scent tag • The probe (and the target DNA) is denatured and under proper hybridization condition s recognizes and binds to the homologous sequences in the target DNA • After hybridization of the copy number and the localization of the fluore scent tags, and con sequently of the target homologous region s, are recognized under fluorescent micro scopy both in chromosme spreads and interphase nuclei. FISH technology is simple, with high sensitivity, and it phenomenal contribution to the cancer field largely relie s on its applicability to interphase cell s
FISH Protocol for Hematologic Malignancies • The basic protocol for interphase FISH is simple and in majority of situation is accomplished within 24 hours • In hematologic malignancies, target DNA (peripheral blood (PB), bone marrow (BM), ascites, spleen cells, lymph node, and bone marrow biopsy touch preparation s) is either fixed on the slide or single cell suspension of target DNA is placed on the slide. The usual fixative is methanol acetic acid (3: I vol/vol) • Following dehydration in the series of alcohol (70%, 95 %, and 100%) slides are pre-treated for more efficient hybridization using 2X sodium citrate (SSC), phosphate buffer saline (PBS)-MgHCI, and pepsin
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A small DNA fragment is used as a probe to search for homologous target sequence in chromosomal DNA
FISH application on acumulated large number of metaphase cells
A small DNA fragment is used as a probe to search for homologous target sequence on chromatin fiber
Comparative hybridization of differentially labelled total genomic DNA and normal reference DNA to normal human metaphase cell used as template
The test and normal DNA are differentially labelled and co-hybridized to a microarray
Hybridization with 24 differentially labelled chromosome-specific probes for paining of every human chromosome in a distinct color
FISH
Hypermetaphase FISH
FiberFISH
CGH
Array CGH
Multicolor karyotyping (M-FISH, SKY)
aM odified from reference Varella-Garcia 2003
Protein indigestion and/or special dye generate specific banding pattern for each schromosome
Conventional cytogenetics G-Banding
Characteristics
Detection of one or multiple rearrangements within a single metaphase cell
Disease-specialized arrays Chromosome arm-specific array Detection of DNA copy number
Detection of DNA copy number at the chromome bandlevel
Fine physical mapping DNA-protein interactions
Detection of probe on large number of chromosomes
Detection of the number copies per cell and localization of probe DNA
Identification of both numerical and structural rearrangement of the entire genome
Applications
Identification of marker chromosomey Accurate identification of all segments in complex rearrangements
Requires mitotic cells and well spread chromosomes Limited accuracy in determination of breakpoints; Intrachromosomal changes remain undetected; High cost in instrumentation High cost in probes
Not suitable for balanced translocations, inversions Automated high-throughput instrumentation Not practical for clinical use Vast amounts of data
High cost in dedicated instrument Low resolution, dependent on chromosomal condensation of normal template Balanced rearrangements and low level of inbalances cannot be detected
Cell culture not required Genome-wide screening for genetic inbalances
Genome-wide scanning;
Mainly used in research due to the complexity of the procedures involved
Not suitable for conventional cytogenetics
Tested target must be known High cost in instrumentation
Low resolution Dependent on mitotic index Mutationc cannot be detected
Limitations
High resolution mapping
Large number of metaphase cells available (50D-2000)
Cell culture not required Fast analysis and scoring Quantitative method Simple and robust Applicable to paraffin-embeded tissue
Screening for the entire genome for chromosome level abnormalities Low cost of reagents Simple and robust procedure
Advantages
Table 1. Conventional and Molecular Cytogenetics Methods Most Commonly Used in Clinical Laboratories"
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12-6
Molecular Genetic Pathology
Table 2. Glossary of Cytogenetic and Fluorescence In Situ HybridizationTerminology Aneuplody
An abnormal chromosome number, either gain or loss
Banding
A set of dark and pale segments along the length of chromosomes, resulting from a treatment with enzyme prior to staining . Each chromosome is identified by its unique sets of bands
Balanced translocation
Exchange of chromosome material that creates no extra or missing DNA
Breakpoint
A specific site on a chromosome containing a break in the DNA that is involved in chromosomal structural rearrangement such as translocation or deletion
Centromere
A constriction on the chromosome that is a site of the spindle site attachment. During the cell mitosis two copies of the DNA in each chromosome are separated by shortening of the spindle fibers attached to the opposite sides of the dividing cell. The position of the centromere determines whether the chromosome is metacentric (Xvshaped, e.g., 1,3, 19,20), submetacentric (centromere positioned more towards the short arms, e.g., 2, 4, 5, 6-12, 16-18, X) or acrocentric (inverted V-shaped, e.g., 13-15,21 ,22, Y)
Centromere enumeration probe
A highly repetitive (l (or ~) satellite DNA, located in the heterochromatin of the centromeric area of chromosomes. CEP probes target repetitive (l (or ~) sequences and produce bright compact signals. They are particularly useful for detection of numerical loss or gain of chromosomes
Clonal abnormality
Cytogenetically, two cells showing the same additional or structural abnormality or three cells with loss of the same chromosome. From the FISH aspect, any abnormality present , after probe has been validated and normal reference range established, above the normal reference range
Chromosomal rearrangements
Aberration where chromosomes are broken and rejoined
Deletion
A segment of chromosome that is missing (terminal) or a segment of chromosome missing as a result of two breakpoints and the intervening sequences are missing (interstitial)
DNA sequence
Order of nucleotides in a DNA segment, usually displayed from the 5'-triphosphate (5'end) to the 3'-hydroxyl (3'end) nucleotides
Enhancer
DNA sequences that increase the rate of transcription
Exons
Portion s of genes that encode protein
FISH
Fluorescence in situ hybridization , a method of detection of the number and location of DNA sequence (genes) in tissue section s or cell populations
Fluorochrome
A fluorescent molecule that, when conjugated to a molecule, will bind to a hapten to facilitate detection of the chromosomal probe . By definition, a fluorochrome is a molecule that will become excited by the light of one wavelength
Genotype
Genetic constitution, usually with reference to particular alleles at a locus
Gene construct
Recomb inant DNA containing a gene of interest surrounded by sequences engineered to promote a measure its expression
Gene map
Order of genes within a chromosome or entire genome
Haploid
One half of a normal complement, e.g., 23 chromosomes
Haploinsufficiency
Deletion or inactivation of one allele to produce disease due to inadequate activity of the remaining allele
Hyperdiploid
Additional chromosomes, e.g., 47 or 48 chromosomes
Hypodiploid
Loss of chromosomes, e.g., 45 or 44 chromosomes
Hybridization
A method for the rejoining (reannealing) of complementary DNA or RNA strands
Hybrid gene
Represents of fusion of two different genes as a result of a structural chromosomal rearrangement that functions as one transcriptional unit
(Continued)
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Table 2. (Continued)
•
I-FISH
Interphase FISH refers to the application of FISH to non-dividing (resting) cells
Inversion
A structural chromosomal rearrangement as a result of two breaks occurring in the same chromosome . Paracentric inversion refers to both breaks occurring on the same side of the centromere and pericentric inversion refers to breaks occurring on the opposite side of the centromere
Isochromosome
A structural chromosomal rearrangement, which consists of doubling of one of the two chromosome arams (connected by the centromere) and the loss of the other arm, the identical copies of one chromosome arm and the loss of the other arm that are connected with the centromere
Interphase
A stage of mitosis where cell is not dividing
Karyotype
Arrangement of metaphase chromosomes from a particular cell according to the size and banding so that the largest chromosome is placed first and the smallest one last (see Fig.1 for the ideogram of normal chromosomes)
Kb (kilobases)
Unit of DNAIRNA length
Locus
Unique location of a gene on a chromosome
Locus (sequence) specific probe
Probes targeted to unique sequence regions of the chromosome. They are useful for the localization of genes on normal chromosomes (gene mapping) and for detection of gene amplification, deletion, inversion, or translocation
M-FISH
Multicolor FISH karyotyping, which allows identification of 24 different human chromosomes (22 autosomes and the X and the Y chromosome) (see text)
Marker chromosome
A chromosome whose morphology cannot be identified using banding method, marker chromosomes are frequent in hematological neoplasms
Oncogene
Locus that is activated in association with tumor growth; one abnormal allele is sufficient to cause tumor formation or cancer
PCR
Polymerase chain reaction by which individual gene segments are amplified through sequential cycles of polymerization, heat denaturation, and reannealing
Pseudodiplod
A diploid number of chromosomes (46) accompanied by structural rearrangement
Recurrent abnormality
A structural or numerical abnormality observed in multiple patients with the same or similar disease. Recurrent chromosome abnormalities in hematological neoplasms have prognostic significance
Telomeric probes
Detect the repeated DNA sequences present at the end of the chromosome , which is called telomere. Telomeric DNA contains 10-15 kb ofTTAGGG repeats. Adjacent to the telomere is a region called the proximal subtelomeric region, and centromeric to it is a unique chromosome telomeric region. Chromosome-specific telomeric probes are now available. They are useful for detection of cryptic translocations involving ends of chromosome
Translocations
A structural chromosome abnormality resulting from a break in at least two chromosomes with an exchange of material. In reciprocal or balanced translocations there is no loss of chromosomal material while in unbalanced translocation there is a loss of chromosomal DNA
Tumor suppressor gene
Locus that prevents tumor growth when at least one allele is functional; loss of both alleles, first through constitutional and then through somatic mutation, is associated with tumor formation or cancer
Whole chromosome painting probes
Span the entire length of chromosome DNA sequences and, as the name implies, their target is the entire length of DNA sequences. They are useful for the identification of complex or cryptic structural rearrangements as well as for the identification of marker chromosomes
= 1000 bp of DNA
Probe, hybridization buffer, and water are mixed very well and placed on air-dry slides that contains the target DNA; the mixture is sealed with a cover slip
•
Co-denaturation of probe and target DNA takes place for 3 minutes at 73°C in Hybrite (new version, Thermabrite), an instrument that allows the most precise control of the desired temperature. If two or more probes are hybridized
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13
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11
12
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Fig. 1. (A) A normal bone marrow metaphase as seen on a microscope slide and (B) arrangements of chromosomes in an karyotype prepared from the same metaphase showing a slightly fuzzy appearance when compared with a normal karyotype obtained from PHAstimulated peripheral blood cells.
simultaneously the amount of water added to the mixture is reduced accordingly. Following denaturation the hybridization is carried over for 4 hours at 42°C on Hybrite followed by 16-20 hours hybridization at 37°C either in Hybrite or in moistured chambers in regular incubators • Post-hybridization washes may differ in the procedure and the amount of time required to perform the washes . Once cover slips are removed, slides should not be allowed to dry until completely washed. The rapid wash is highly efficient and includes incubation in O.4X SSC at 73°C for 2 minutes, followed by 2 minutes wash in 2X SSC at room temperature. Interphase nuclei are usually counterstained with Diamidinophenylindole (DAPI)
FISH Protocol for Paraffin-Embedded Tissue • Protocol for HER2 amplification in breast biopsies is provided by the Vysis insert and must be followed because this is the Food and Drug Administration (FDA) cleared kit • Tissue samples are generally formalin-fixed and paraffin embedded (on positively charged slides) for cell and tissue morphology preservation. To prepare tissue sections for clinical FISH testing, slides are deparaffinized and pre-treated to maximize probe permeability and hybridization. Currently three different deparaphization kits are available from Vysis/Abbott Molecular and are used depending on the thickness of tissue . The standard FISH protocol subsequently includes co-denaturation, for 6 minutes at 73°C in Hybrite (twice as long as single cell cytogenetic suspension) and the post-hybridization washes includes 2X SSC and 0.3 M NP-40 first at RT for 1-5 minutes and than at 73°C for 2 minutes before the staining nuclei with DAPI
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FISH Protocol for Bladder Cancer • Protocol for bladder cancer voided urine is an FDA approved kit and the package insert from Vysis/Abbott Molecular must be followed. See UroVysion protocol under bladder cancer
Interphase FISH • Interphase cytogenetics is the term used to describe detection and visualization of chromosomal abnormalities in non-dividing, interphase nucleus that allows the analysis of genome of individual cells (Figure 2). Chromatin in interphase cells is almost a tenth as condensed as in metaphase chromosomes, allowing for ordering of probes over shorter distances (50 kb-2 Mb) (Table 3) • Six aspects of interphase FISH are particularly important (Table 4) - Interphase cytogenetics allows screening of a large number of cells. This permits investigation of hematological malignancies with a low mitotic yield, such as chronic lymphocytic leukemia (CLL) or multiple myeloma (MM). It also allows investigation of paraffinembedded tissues without any mitotic figures. Most of formalin-fixed, paraffin-embedded solid tissue that has not been de-calcified may be used for I-FISH evaluation - Interphase cytogenetics permits detection of chromosomal rearrangements in hematologic malignancies in peripheral blood samples, thus obviating the need for bone marrow aspiration. For instance, in chronic myelogenous leukemia (CML), which rarely yields a large number of dividing cells in peripheral blood, conventional cytogenetics is usually uninformative. However, detection of BCR-ABL, a
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Fig. 2. Schematic representation of the cell division. Most of the clinical FISH studies are performed on non-dividing, interphase cells, while conventional cytogenetics is performed at the metaphase stage of cell division.
Table 3. Resolution of Conventional and Molecular Cytogenetics Methods
Method Conventional cytogenetics
Resolution Approximately 5 Mb
Interphase FISH
50-2 Mb
Fiber FISH
1-500 kb
molecular equivalent of the Ph chromosome, in peripheral blood using interphase FISH provides reliable, fast, and quantitative results (see Chronic Myelogenous Leukemia section) - Interphase FISH offers a quantitative assay for monitoring disease progression or detection of minimal residual disease after ablative chemotherapy or stem cell transplantation, thus allowing detection of rare and small abnormal cell population. This aspect is particularly useful for detection of micrometastasis and detection of disseminated cells in patients with early stagecancers. Studiesof disseminated cell by interphase cytogenetics
revealed a high degreeof cellular heterogeneity even in early stages of tumor progression - The use of specific probe sets allows detection of specific disease-associated abnormalities such as t(8;21), which denotes the M2 sub-type of acute myelogenous leukemia (AML) or t(lS;17) associated with acute promyelocytic leukemia ([APLj, see below) - Abnormalities can be detected accurately in archival specimens kept up to IS years at -20 to -70°C. - Lastly, simultaneous use of interphase FISH and immunophenotyping is a powerful tool for the investigation of lineageinvolvement in diseases, such as myelodysplasia and to determine which cell population carries the specific chromosome abnormality. FISH nomenclature is described in the International System for Human Cytogenetic Nomenclature
Fiber FISH • A higherresolution of chromosomal abnormalities can be achieved when fluorescently labeled probesare hybridized to extended DNAor free chromatin (chromatin strands released from their chromosomal scaffold) or free DNA fibers. DNAfibers are chromatins from which proteins such as histones are removed allowing it to unfold and extend
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Table 4. Advantages and Limitations of Cytogenetics and Interphase FISH
Method
Advantages
Limitations
Conventional cytogenetics
Provides information of the entire genome
Laborious and time consuming Low mitotic yield with poor chromosome morphology, banding and "dry tap" Requires highly skilled and trained observers Difficult interpretation of a single metaphase with non-clonal abnormality Only provides information on dividing (metaphase) cells
Interphase FISH
Dividing cells not required Cell culture not required Highly sensitive Applicable to all tissues Detection of abnormality at the single cell level May provide information when cytogenetic is uninformative
"FISHing in dark" is not a recommended clinical test
Fig. 3. Metaphase chromosomes from a near-diploid colon cancer cell line. G-banded karyotype identified three abnormal chromosomes (left) and multi-color FISH (SKY) analysis (right) enabled anotation of them as der( 10) t(10; 16), der( 16)t(8;16), and der(l 8)t(l 7;18). (Image kindly provided by Drazen Zimonjic, PhD. NCI, NIH, Bethesda.)
• The hybridized signals have the appearance of a "string of pearls" along the fiber rather than tight fluorescing spots observed in interphase cells (Figure 3). • Although at the present time fiber FISH has limited clinical applicability because it requires special techniques of target DNA preparation on a glass slide, fiber FISH is one of the most powerful tools for mapping DNA sequences because it allows the accurate fixing of gaps and overlaps between probes
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• Fiber FISH has been particulary useful for measuring the size of regions of the human genome that have been impossible to sequence, which is achieved by hybridization of the clones than flank gaps in the sequence
Multi-Color Karyotyping Multi-color karyotypin permits examination of the entire genome in a single analysis (Figure 4). In 1996, it became possible to identify 24 different human chromosomes
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Fig. 4. Multi-color spectral karyotyping analysis (SKY) of the hipotetraploid metaphase chromosome spread (A) from a breast cancer cell indicating presence of several rearranged chromosomes. Based upon their respective spectral signature chromosomes were karyotyped, (B) classified, and the precise constitution of derivative chromosomes was established (C), even though some of them were very complex and resulted from multiple recombinations. (Image kindly provided by Drazen Zimonjic, PhD. NCI, NIH, Bethesda.)
(22 autosomes and the X and the Y sex chromosomes) each with a unique color with the help of fluorochrome-specific optical filters . This method is called M-FISH. When interferometer-based spectral imaging is used, the method is called SKY-FISH or spectral karyotyping (Figure 4). • The starting point in both methodologies is the use of whole chromosome painting probes for each chromosome. Thus, each chromosome is labeled with a different combination of fluorescent dyes . The fluorochrome colors are not distinct enough for the unaided human eye to distinguish the combination with which the chromosome is labeled • In M-FISH , images are sequentially obtained using 5 different fluorochrome-specific optical filters . A computer program combines the data and displays each chromosome as if it were stained with a distinct color • The second method, termed SKY, is based on the use of an interferometer (used by astronomers to measure the light spectra of distant stars) to determine the full
spectrum of light emitted by each stained chromosome. A computer program than displays all the chromosomes simultaneously, each with its own unique color • These methods are applied with increasing frequency to resolve complex karyotypes or to detect cryptic translocations. Their clinical use is still limited because the cost of equipment and probes is beyond what can be afforded by most clinical laboratories
Comparative Genomic Hybridization (CGH) • Another powerful method used to identify locations of chromosomal gains, losses, deletions or amplifications, without prior knowledge of the chromosomal target that may be altered is CGH • Briefly, isolated DNA from leukemic bone marrow or tumor tissue is labeled with a one color fluorochrome (e.g., red) while DNA isolated from normal control tissue is labeled with a different color (e.g., green) . These differently labeled DNAs are hybridized against each
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Fig. 5. Specific DNA sequence gains and losses in a uterine fibroid tumor. The upper panel displays the CGH fluore scence ratio profiles for representati ve chromosomes. Gain s and losses observed in the upper panel can be visualized in the lower panels in the dual FITCffexas Red image as green and red intense regions respectively (see text) (Courtesy of Brynn Levy, PhD., Clinical Cytogenetics Laboratory, College of Physicians and Surgeons of Columbia University.)
other in a competitive hybridization reaction onto normal metaphase spreads. Computer-assisted image analysi s detects colors generated after hybridi zation, which indicate either equal hybridization, a relative excess, or a deficiency of the target DNA (relative to control) . The ratio of color intensity provide s a "copy number" karyotype (Figure 5) • CGH has been successfully applied to many tumors but its clinical use remain s limited because it cannot detect balanced translocations, which are the hallmark of many hematological malignancies
Array CGH (aCGH) • A newly emerging investigational method is aCGH. In aCGH metapha se chromosomes are replaced as the target by large number of mapped cloned DNA fragments and sequence-verified genom ic clone s (± 100-200 kb) arrayed on the standard glass slide as hybrid ization target. Thus, the main limitation of low resolution of CGH is substantially increa sed and radically transformed the CGH technique • In aCGH both the normal and the reference DNA are differentially labeled and co-hybridized to microarray, most often genomic DNA or eDNA, which are spotted on a glass slide (the array). The DNA copy number aberrations are subsequently measured by detecting intensity differences in the hybridization patterns of both
314
DNAs. The resolution of the analysis is restricted only by clone size and by the density of the clones on the array • The first descriptions of aCGH with large insert clone s in the late 1990s were foIlowed by the development of the whole genome array that had one clone for every megabase, which are now widely used • The flexibility of array design has also allowed the development of speciali zed arrays for applications such as telomere screening or for specific diseases such as B cell leukemia as well as for formalin-fixed, paraffinembedded tissues • The highest resolution for aCGH is now provided by oligonucleotide array that contain as many as 500,000 elements • Single nucleotide polymorphisms array are high-density oligonucleotide-based arrays that are particularly useful to identify both the loss of heterozygosity at individual nucleotides and copy number alterations. aCGH is a robust technique but technical optimizations and commercially validated platforms are needed to further improve its use as a routine diagnostic clinical application (Figure 6) • aCGH are not applicable to chromosomal abnormalities that do not involve copy number, such as balanced translocation or inversions • For these, array painting, a modification of aCGH should be applied. Array paining uses flow sorting to separate
FISH and Conventional Cyto genetics
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Fig. 6. (A) A portion of array CGH image of a human hepatocellular carcinoma (HCC) tumor. The tumor or normal liverfragmented DNA sample was labeled with Cy50r Cy3, respectively. A yellow spot indicates the intensity signal of the genomic element on that spot in the tumor equal s to that of the normal liver reference . The red or green spot indicate s intensity of the tumor, is greater or less than reference, suggesting copy number change for the genomic element on that spot. (B) Copy number change profile for a human HCC tumor. Y axis is the log2Ratio of the intensity of each gene in the order of its position on the genome in tumor sample versus that of normal liver sample. Te gained or lost regions are marked by red or green lines, respectively. Gain of Iq, and 8q and loss of 8p were observed in this tumor. (Courtesy of D. Weijia Zhang Department of Medicine, The Mount Sinai School of Medicine, New York, NY.)
the abnormal chromosomes that result from these rearrangements from the rest of the genome on the bases of their altered sizes and base ratios, after which they are hybridized to an array. In the case of balanced translocation s, differential labeling of the derivatives result in the sequences that are proximal to the breakpoint being labeled in one color, whereas sequences that are distal to the breakpoint are labeled in another color. When the two derivatives are co-hybridized to an array, the ratio of the two colors on the chromosomes that are involved in the abnormality switches from high to low (or vice versa) at the breakpoint. If a clone on array spans the breakpoint it will report an intermediate value. Array
paining is limited for research investigation but it has been used to identify inversion at the breakpoint, deletion s, or insertion s of extra material from other chromosomes • In a microchip array, labeled RNA from the sample of interest is hybridized with defined target sequences immobilized on a solid support. The advantage of this method is the ability to screen genes that are overexpres sed or underexpressed, at a particular stage of disease. The first example of the successful application of the microchip array technique was to differentiate AML from acute lymphoblastic leukemia (ALL) based solely on gene expression
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Table 5. Types of Probes for FISH Type of probe
Characteristics
Probe size
Application
Limitation
Repetitivesequence Centromeric probes (CEP)
Short sequence present in many thousand copies usually u- or p-satellite DNA subsets around centromeric region of chromosomes
500 kb-o l Mb
Numerical abnormalities; identification of chromosomes
Structural rearrangements involving other parts of chromosomes,not involving centromere, cannot be detected; centromeric probes for chromosomes 13, 21 cross-hybridizeand specific centromeric probes for 14 and 22 are not available
Pantelomeric p-Tel
Tandemlyrepeated (TTAGGG)n sequences present on all human chromosomeends
100-200 kb
To detect telomeres on all human human chromosome
Metaphase cells required; No specificityfor individual chromosome
Subtelomeric sub-T
Chromosome-specific sequences presenton specific chromosome ends
100-300 kb
Identification of chromosome-specific ends
Not suitable for other chromosomalparts
Telomeric probes
Locus-specific probes (Unique sequence) Targetspecific sequencepresent (LSI) in only one copy on chromosome
plasmid: 1-10 kb Specific chromosomal PAC,YAC, translocations, BAC: inversions deletions 80 kb-l mb
Not suitable for identification of chromosome without internal control
Wholechromosome DNA sequences of the specific painting probe chromosome to generate a (WCP) "paint" for the entire chromosome
NA
Regions under-represented are centromeric and telomeric; Not applicable to interphase FISH; Metaphasecells required
To identify chromosome or part of the chromosome in chromosomal rearrangement(s)
Types of Probes
Probe Strategies
There are three groups and five types of probes (as shown in Table 5 and Figure 7), which are usually used alone or combined to determine both numerical and structural rearrangements.
• Four strategies are used in probe design to detect chromosomal translocations in hematologic malignancies and soft-tumor tissues, as shown in Figure SA and B. They include :
• Centromeric enumeration probes (CEP), as the name implies, are used most frequently in interphase nuclei for detection of numerical chromosome anomalies • Whole chromosome painting probes (WCP) are used only on the metaphase cells and are never used in interphase cells. They are very useful in delineating complex rearrangements or the origin of marker chromosome • Subtelomeric probes and unique gene loci probes may be applied both to interphase and metaphase cells in single, dual, triple or multi-colors to determine specific chromosomal rearrangements, deletions, or amplifications
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- Conventional strategy - Extra sensitive strategy (ES) - Dual fusion strategy - Breakapart strategy • Conventional strategy: the first application of FISH technology for detection of chromosomal translocation in hematologic malignancy was in 1990 when BCR-ABL hybrid gene was identified using two-color FISH in interphase cells as well as in metaphase bone marrowderived CML cells . In the standard strategy for interphase evaluation of chromosomal translocation, a DNA probe, comprising sequences mapped proximally
FISH and Conventional Cytogenetics
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Fig. 7. Types of chromosomal probes (see text). A partial metaphase and interphase cells showing chromo somes 8 and 12 (left) after in situ hybridization with CEP probe for chromo some 12 and WCP probe for chromo some 8. A composite of two cells in metaphase and interpha se showing four copies of chromosom e 17 (right). Telomeric-specific probe for the short arms of chromo some 17 is shown in green and locus-specific probe for P53 is shown in red. Chromo somes and nuclei are counterstained with DAPI (blue).
Fig. 8. Four different probes strategies for detection of chromo somal translocation s. A normal cell after in situ hybridization with a translocation probe for detection of ETO-AMLl, showing two red and two green single signals (left, top row). Conventional fusion strategy after in situ hybridization shows one fusion (yellow) and one single color signals, red and green for normal homolog s (left middle). An ES fusion approach generate s an extra small (red) signal as well as a fusion signal (yellow) and one signal in single color, (green and red) on normal homologs (left bottom ). Dual fusion strategy generates two fusion signals (yellow) on two derivative chromo somes and one single color signal on each of two normal chromosomes (left top). Breakapart approach in a normal cell appears as two fusion signals (yellow) (left, middle). In this strategy the 3' and the 5' part of the gene are labeled in two colors. When the rearrangements occurs, the normal chromosome will show a colocalization of red and green (yellow) as a result of the proximity of the sequences on the chromosome while abnormal derivative chromo somes will each have one single red and single green signal indicating that the rearrangement occurred between the two ends of the gene separating the green and red signal on two different chromosome (left bottom row).
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to the breakpoint in one of the chromosomes involved in reciprocal translocations, is combined with differentially labeled DNA probe that includes sequences mapped distantly to the breakpoint in the other chromosome. Positive nuclei for the translocation display one dual color fusion signal, representing one of the derivative chromosomes generated by the translocation, and two single color signals, one for each of the normal alleles. This standard FISH strategy has been used in hematological malignancies and lymphoma translocations at diagnosis • ES: for detection of residual disease standard approach lacks specificity because cells with random spatial colocalization of normal signals with different colors, usually found in frequency from I to 10% of scored nuclei, are seen as false-positive. To minimize this problem, a strategy for ES method was developed in which a probe for one abnormal chromosome is designed in such way to generate an extra, smaller signal in positive nuclei. Hybridization with this strategy results in abnormal cell showing colocalization of two signals in dual color, additional two signals in one single color and one signal in another single color • A dual fusion strategy was developed not only to minimize false-positive results but also to detect additional deletions at the translocation breakpoints. Dual color dual fusion strategy includes the probe set with DNA sequences that encompass proximally and distally the translocation breakpoints on both chromosomes involved in translocation. The sequences for each chromosome are labeled with specific color, and the translocation generates fused signals in both derivative chromosomes. Positive nuclei are showing two copies of fusion signals and one copy of each of the single signals representing the normal alleles . If the third color is added for detection of particular sequences such as deletion of chromosome 9 at the site of the Ph translocation, as shown in Figure 10, right middle and bottom row, the lack of one copy of the third color is consistent with sequence deletion at the site of the translocation of one derivative chromosome • Multiple translocation partners are well known for leukemic genes such as myeloid lymphoid lineage (MLL) , RARA, or lymphoma gene ALK, and thus the fourth FISH strategy, "breakapart" probe, was developed to address this issue . In this approach, the breakapart probe includes DNA sequences mapped proximally and distally to the breakpoint within a critical gene (the 3' end and the 5' end) labeled with two different fluorochrome . In this approach, the fused fluorescent signals represent a normal gene , whereas nuclei with rearrangements within the target gene show one single color signal, one for each derivative chromosomes, regardless of which chromosome is the partner in translocation
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Probe Validation • Majority of commercially manufactured chromosomal probes for in vitro diagnostic testing are analyte-specific reagents and exempt from the FDA but must be independently validated by each laboratory • In 2006, the FDA-approved probes include: HER2/CEPl7 (Pathvysion) for detection of HER2 amplification in paraffin-embbeded breast biopsy tissue; Urovusion kit consisting of four differentially labeled probes (CEP3, CEP7, CEPI7, and PI6 at 9p21) for diagnostic and recurrence of bladder cancer in void urine; CEP8 for detection of numerical abnormalities of chromosome 8, CEPl2 for detection of gain of chromosome 12 in hematologic malignancies and dual color XY probes, which in our laboratory is used for detection of engraftment and chimerism in patients who received sex mismatched stem cell transplantation alone or in combination with disease-specific probe that was identified at diagnosis, such as BCR-ABL • According to the Standards and Guidelines for Clinical genetics Laboratories from the American College of Medical Genetics validation of probes includes both sensitivity and specificity. In the Tumor Cytogenetics laboratory at the Mount Sinai Medical Center in New York, clinical interphase testing has been performed since 1994 and the validation is done on 200 nuclei from 10 normal controls. The mean percentage of "abnormal cells" ± 3SD is used for establishing normal reference cutoff point. Probe validations process must include evaluation and scoring of nuclei by two individuals (who have been previously tested for color blindness) to minimize inter-observer differences • The standard operating procedure of the lab recommends that every clinical specimen is scored by two individuals . Stringency and quality of hybridization is evaluated by including an internal control probe . Validation of positive controls should be performed from archived material in order to determine the range of positive abnormal cells • Annual verification of validated probes is the usual practice for the clinical laboratories • When analyte-specific reagents are used for in vitro FISH studies the following disclaimer must be written in final report: This test was developed and its performance characteristics determined by (name of laboratory). It has not been cleared or approved by the
US FDA
Conventional Cytogenetics and Varying FISH Methodologies These technologies are complimentary; each has its own advantages and limitations in investigating genome rearrangements of malignant cells . While conventional cytogenetics is the comprehensive study of all chromosomes, it requires a large number of proliferating and dividing cells,
FISH and Conventional Cytogenetics
which in some diseases is difficult to obtain particularly in solid tumors . Furthermore, many small deletions or structural rearrangements are either beyond the microscopic level of detection or chromosomal banding is not distinct enough for accurate detection due to the fuzzy appearance of chromosomes obtained from leukemic or tumor tissue. Moreover, complex rearrangements involving two or more chromosomes are often difficult to decipher accurately, which leads to underestimation of chromosomal abnormalities. FISH can be used in conjunction with conventional cytogenetics both in interphase and metaphase
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cells. It is a more sensitive method and can detect rearrangements as small as I kb. The main disadvantage of interphase FISH is that it cannot be used unless a known abnormality is suspected: "FISHing in the dark" is not a recommended clinical test. When the abnormality is known , interphase FISH can pinpoint the clonal aberration at the single cell level in a very short period of time. Other investigative FISH methods provide further refinement. Undoubtedly, in the future, automation will help to more accurately classify and diagnose hematological malignancies as well as solid tumors .
MYELOPROLIFERATIVE DISORDERS Table 6 shows chromosomal probes that are currently used to detect karyotypic rearrangements and their frequencies in myeloid disorders.
Chronic Myelogenous Leukemia Chronic Phase • Chronic myeloid leukemia is a clonal hematopoietic malignancy arising from neoplastic transformation of a pluripotent bone marrow cell. Cytogenetic studies combined with clonal analysis of patients with CML have been the keystone for clonal and karyotype analysis of other human malignancies. The knowledge accumulated for the last 46 years about CML serves as molecular pathology/medicine at its best for the following reasons: • The first specific association between chromosome abnormality and malignant disease • The first description of oncogene localization at the site of chromosomal breakpoints
signature genomic rearrangement occurring in approximately 90% of patients with CML. The nature of the chromosome aberration was elucidated in 1973, when it was shown that the Ph chromosome resulted from a balanced translocation rather than a deletion as many investigators had previously assumed and it was described as balanced translocation: t(9;22)(q34;q 11.2). (Figure 9) • The Ph chromosome arises post-zygotically, as it is found only in hematopoietic tissue. The finding of the Ph chromosome in myeloid cells, erythroid cells, eosinophils, monocytes/macrophages, basophils, B-Iymphocytes, and rarely in T-cells (T-lymphocytes are long-lived cells, and thus, they may antedate the development of CML) and the absence of the Ph chromosome in cultured marrow fibroblasts, supports the concept that the Ph chromo some results from a specific rearrangement in a multi-potent hematopoietic stem cell, and that it is an acquired rather than inherited abnormality
• The first rationally designed gene-targeted therapy
• Above observations combined with glucoso-o-phosphatedehydrogenase (G6PD) studies in female patients who are heterozygous for this enzyme and have CML, provided further experimental evidence for the concept that CML is a clonal disease arising in a stem cell capable of differentiation into most of the hematopoietic cell lineages
• CML constitutes 15% of adult leukemias with approximately 4600 newly diagno sed cases per annum in the United States . The chronic phase of the disease has a median duration of 3-6 years and without treatment will progress, via accelerated phase, into the "blast crisis" phase characterized by over 30% undifferentiated blasts in the bone marrow and peripheral blood for which median survival is 18 weeks
• Studies with CML-derived B-lymphoid cell lines from G6PD heterozygous female patients with Ph-positive (Ph--)CML provided data for the hypothesi s put forward in 1982, that in at least some patients, a cytogenetically normal but genetically unstable clone proliferates. It is in this population of abnormal but not frankly malignant cells , that the distinct chromosome change (the formation of the Ph chromosome) occurs
The Philadelphia Chromosome
The BCR-ABL Fusion Gene
• The Philadelphia or the Ph chromosome, named in honor of the city of its discovery, described in 1960, is a
• The reciprocal nature of the Ph translocation was confirmed in the mid 1980s, when it was shown that its
• The first example of "hybrid" gene leading to the production of dysregulated tyrosine kinase protein • The first example of a mouse model with human aberrant gene to resemble human malignancy
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Table 6. Probes Used in Chromosomal Rearrangements Associated with Malignant Myeloid Disorders Probes
Disease
Chromosomal abnormality
Involved genes
Frequency
CML CML CML CML CMML HES,SMD
t(9;22)(q34;q11.2) t(9;22)(q34;q11.2) trisomy 8 iso(l7)(q10) t(S;12)«q33;p I3) del(4)qI2)
BCR, ABL BCR,ABL,ASS
9S-97%
PV, ET, IMF
trisomy 8, +9/+9p,de1(13)
Chronic myeloproliferati ve disorders BCR-ABL (ES or DF) BCR-ABL-ASS CEP 8 PS3 TEL-PDGFBR FIPILl
P53 PDGFBR, TEUETVI FlPILl( CHIC2)
IO-IS % 30-40% in BC
2-S % 2S%
MPD panel (S probes) CEP8, CE9/9p21
29% in PV; -SO% in IMF; -10% in ET
(q I4),del(20)(q l l q I3)
RB I, D20S 108 MDS panel (8 probes) MDS
del(Sq3l),-7/del(7q31 ),+8, del(lI )(q23), del(l2(pI3), del(l3 )(q14)del(20)(q l lq 13)
-30%
CEPI, CEP7
t-MDS
der( I;7)(q IO;p10)
70% pts have t-related hematological malignancy
SpIS .21Sq31 CEP7l7q31 MLL(BA)
t-MDS, tAML t-MDS, tAML t-MDS, tAML
del(S)(31to q33)/-S del(7)(q31-q32)/-7 t(ll ;var)(q23;var)
EGRI MLL
due to topoisomerase II chemotherapy
ETO-AMLl (DF)
AML-M2
t(8;21(q22;q22)
ETO,AMLl
PML-RARA (DF) RARA (BA)
AML-M3(APL) AML-M3(APL)
t(lS ;17)(q22q21) t(lI ;17(q23;q21» t(lI ;17)(q13;q2l) t(S;17)(q32;q21) dup( 17)(q21.3q22) inv( 16)(p13,q22) t( II ;var)(q23;var)
PML, RARA PLZF,RARA NuMA,RARA NPM,RARA STATB MYHll ,CBFB MLL
S-IO % AML, 10-30% of AML-M2; 40% childhood AML Specific for APUAPLv Specific for APLv
EGRI, CEP7l7q31 CEP8, MLL,TELlETVI I08 RB I
.nzos
Therapy -related myeloid disorders
Acute myelogenous leukemia (AML)
CBFB (BA) MLL (BA)
AML-M4(Eo) AML-MS
100% patients with M4 t(9;11)(p21;q23) in 3S% of MS and SO% ofMSa
CEP denotes chromosome enumeration probe ES denotes extra sensitive probe strategy
OF denotes double fusion probe strategy BA denotes breakapart probe strategy molecular consequence is the translocation of the ABL gene from chromosome 9, band region q34, and subsequent fusion to the breakpoint cluster region (BCR) gene on chromosome 22 , band q 11. This creates a hybrid
320
BCR-ABL gene that is transcribed into a chimeric BCRABL messenger RNA (mR NA) • Three major breakpoint locations have been identified along the BCR gene on chromosome 22 . The three resulting
FISH and Conventional Cytogenetics
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patients , there was an ABL insertion from chromosome 9 to 22q II resulting in BCR-ABL fusion product without reciprocal translocation of sequences from chromosome 22 to chromosome 9. Approximately 3-5% of patients are truly Ph-, BCR-ABL fusion-negative . They may not have CML, but rather have chronic myelomonocytic leukemia or refractory anemia with excess blasts
Diagnosis Fig. 9. The Ph chromosome is detected in over 90% of marrow cells from patients with CML. The Ph is the consequence of the balanced translocation between chromosomes 9 (left) and 22 (right). chimeric proteins are P2IO BCR-ABL, Pl90 BCR-ABL, and P230 BCR-ABL. The P210 BCR-ABL is found in the majority of patients with classic Ph+, BCR-ABL fusion-positive CML. In 50% of adult and 80% of children with ALL, in rare CML patients, and in the majority of patients with AML, there is expression ofPl90 BCR-ABL, while the expression of P230 BCR-ABL may be associated with a chronic neutrophilic leukemia variant • The concept that the BCR-ABL fusion plays a central role in the pathogenesis of CML is strongly supported by two lines of evidence - Retroviral transduction experiments in which P210 BCR·ABL was expressed in murine bone marrow cells resulted in a myeloproliferative disorder resembling CML - By demonstration that a novel agent, Gleevac (Gleevac, STI57 I , imatinib mesilate), a rationally designed tyrosine kinase inhibitor, selectively inhibit the BCR-ABL fusion protein in mice and specifically inhibit the growth of human Ph+ cells in vitro and in vivo • Imatinib provides effective and durable therapy for CML , inducing complete hematological remissions in 98% of newly diagnosed patients in the chronic phase of disease and complete cytogenetic remissions in 86% of patients • The BCR-ABL fusion has been demonstrated in both the standard as well as in variant translocation even in cases in which chromosome 9 involvement is cytogenetically not detectable or when a masked Ph chromosome is present. In the vast majority of patients the fusion of ABL and BCR takes place on chromosome 22 • In a small group of patients the BCR gene was translocated to chromosome 9 and the fusion of two genes was localized to 9q34 . The prognosis of these patients appears to be poorer but at this time the number of reports is still too small for a definitive conclusion • In the 10% of patients with CML who are Ph- by cytogenetic studies, molecular analysis revealed BCRABL fusion in about 5% and their prognosis is the same as for patients who are Ph+. In the majority of Ph%
• The Ph chromosome is detected by conventional cytogenetics in 20-30 metaphase cells, which remains the gold standard in majority of academic and commercial laboratories. In chronic phase, conventional cytogenetic analysis of bone marrow aspirate is sine qua non, since peripheral blood cells do not contain blast cells at the time of presentation. Conventional cytogenetics allows the comprehensive assessment of chromosomal aberrations. However, bone marrow aspirate is required and aspiration and the culture of proliferating cells are not always sufficient. The technique is relatively insensitive since only 20-30 metaphase cells are evaluated . Conventional cytogenetics has a routine sensitivity of about 1-5%. The advantage of conventional cytogenetics is that it will screen the entire genome and will detect additional chromosomal abnormalities that may have clinical importance • In 1990, a BCR-ABL hybrid gene was identified using two-color FISH in interphase cells as well as in metaphase bone marrow-derived cells from six patients with CML (Figure 10 left middle row). In clinical laboratory practice, the range of cells with BCR-ABL fusion at diagnosis is between 50% and 99% of marrow cells. Peripheral blood interphase FISH is particularly useful in the differential diagnosis of CML, for example, it may reliably differentiate CML from a leukemoid reaction. A high correlation between BCR-ABL fusionpositive cells in the bone marrow and the peripheral blood at the time of diagnosis has been established (r = 0.98) in our laboratory (personal observation). In the majority of patients interphase FISH of peripheral blood cells reflects the frequency of Ph-- metaphase cells in the bone marrow (Table 6) • Current state-of-the-art of I-FISH utilizes dual color BCR-ABL-ES (extra sensitive) or dual fusion , dual color BCR-ABL (Figure 10 left bottom row and right top row). The loss of an extra ABL red signal from der(9) chromosome may indicate a deletion at the site of the t(9;22) breakpoint. Indeed , using a 3 color FISH study, a substantial minority of patients with CML was discovered to have a large deletion adjacent to the Ph translocation breakpoint on the derivative 9 chromosome, on the additional partner chromosome in variant translocations or on both (Figure 10 right bottom row). The increased sensitivity of a 3-color probe demonstrated not only further heterogeneity among Ph+, BCR-ABL positive patients , but it also showed that this variable deletion may have biological consequences. For instance, patients with
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Fig. 11. Two interphase cells from a female patient who underwent stem cell transplantation. The donor was her HLAidentical brother (sex mis-matched donor). The cell on the left is the donor cell showing two large red (X) and green (Y) signals as well as two small ABL (red) and BCR (green) signals, indicated r BCR-ABL fusion negative donor cell. In contrast, the cell on the right shows only two large red signals (XX) indicating host genotype, one BCR-ABL fusion signal (yellow) as well as one single small red and green signals (normal chromosomes 9 and 22). Detection of host and donor cells in the bone marrow specimen from patients who received allogeneic stem cell transplantation is called chimerism. Fig. 10. Types of the BCR-ABL probes. Normal cell after in situ hybridization with single, ES, or dual fusion probe (top left) shows two ABL (red) and two BCR (green) signals on normal chromosomes . Single fusion BCR-ABL results in one fusion (yellow) (left, middle) with a high false-positive results. ES probe generates one extra small signal on der(9), which reduces the number of false-positive cells (left, bottom). Dual fusion BCR-ABL probe generates two fusions (yellow) on both abnormal derivative chromosomes (right, top); triple color BCR-AB-ASS (aqua) probe (right, middle) is designed to determine deletions at the site of the Ph translocation on der(9). Note the colocalization of aqua and red on nonnal chromosome 9. Deletion of der(9q) will result in one aqua signal (right, bottom cell) as well as in one fusion (yellow) signal indicating the BCR-ABL fusion with deleted 9q34 sequences. a deletion had a median survival time of 36 months while patients without detectable deletion survived >90 months. Multivariate analysis demonstrated that the prognostic importance of the deletion status was independent of age, sex, percentage of peripheral blasts, and platelet count
Monitoring Response to Therapy • The degree of tumor load reduction is an important prognostic factor for patients with CML on therapy. The standard method to monitor patients the response to therapy is conventional cytogenetic analysis of bone marrow aspirate , but as aforementioned it has limited sensitivity. Considering that patients with CML at diagnosis have a total tumor burden of 1012 leukemic
322
cells, patients without the Ph chromosome in conventional cytogenetics analysis may harbor up to 1010 leukemic cells. Nevertheless, the major advantage of conventional cytogenetics is the detection of other chromosomal abnormalities, which may indicate accelerated or blast phase of the disease or clonal proliferation of other abnormalities in Ph- cells • Hypennetaphase FISH has increased sensitivity when compared with conventional cytogenetics and may detect 31 % of BCR-ABL positive metaphase cells in patients who are Ph- as assessed by conventional cytogenetics and considered to be in complete cytogenetic remission • Interphase FISH does not depend on the cycling status of cells and novel approaches with ES and double fusion probes have cut down the number of false-positive results to about I %. However if peripheral blood cells rather than bone marrow aspirate cells are used to monitor residual disease, the high percentage of BCR-ABL fusion-negative lymphoid cells may skew the information of residual tumor load. Detection of minimal residual disease by I-FISH yields very few positives (0.1-1 %) if extra sensitive probe or dual fusion probe with or without dual color Xx/xY probes (for sex mismatched transplants) are used, and the results are concordant with RT-PCR (Figure 11). The feasibility of evaluating minimal residual disease or chimerism after stem cell in a specific cell lineage was recently validated using flowsorted cells and applying FISH to each cell lineage • In most direct comparison studies, I-FISH of peripheral blood when compared with conventional cytogenetics of
FISH and Conventional Cytogenetics
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bone marrow in patients that are treated with inteferon or Gleevac, showed a good correlations (r =091-0,97)
+
A compression study between Q-PCR, hypermetaphase FISH, interphase FISH, and conventional cytogenetics to detect residual CML cells have demonstrated a very tight correlation
+ Real-time quantitative polymerase chain reaction is by far the most sensitive method and provides the accurate measure of the total leukemia cell mass and the degree to which BCR-ABL transcripts are reduced by therapy and correlates with progression-free survival. The use of RTPCR for detection of minimal residual disease is being questioned because the presence of BCR-ABL has been detected in normal subjects. This raises the question whether cure should be defined on a functional rather than a very sensitive molecular basis
Monitoring Gleevac (Imatinib) Therapy There are three major obstacles to imatinib-based therapies for patients with Ph+, BCR-ABL fusion-positive CML.
+ Persistence of BCR-ABL fusion positive cells
+ Relapse of the disease due to the emergence of resistance to imatinib is present in approximately 20% of newly diagnosed patients. Acquired resistance to imatinib treatment has been manifestated in two ways: amplification of BCR-ABL fusion product and mutations in ABL kinase domain (Figure 12). Currently, 40 different ABL kinase domin mutations have been described
Fig. 12. Duplication oft(9;22) and the BCR-ABL amplification in a patient who developed resistance to imatininb after 3 months of therapy. Note that both chromosomes 9 are abnormal der(9) and classic Ph chromosomes as well as iso Ph chromosome are shown. Between 5 and 6 copies of the BCRABL fusion are shown on the Ph chromosomes.
+ CML patients who cannot tolerate or are resistant to imatinib may benefit from the second generation of tyrosine kinase inhibitors, such as dasatinib. Dasatinib binds to the ABL kinazs domain in a matter distinct from that of imatinib and thereby retains activity against nearly all imatinib resistant mutations. There is currently no concensus when patients with acquired mutations should be screened, which techniques should be used, and how the data should be reported • Detection of other chromosomal abnormalities in BCRABL-negative cells. As shown in Figure 13, while on treatment with imatinib, between 5% and 8% of patients will develop chromosomal abnormalities such as trisomy 8, monosomy 7 del(2q), and others in BCR-ABLnegative cells. Although it is possible that imatinib may have induced chromosomal abnormalities in BCR-ABLnegative cells, there is considerable evidence to support the other explanation, i.e., that chromosomal abnormalities were present prior to treatment and that imatinib treatment uncovered these abnormalities when significant reduction of overlying Ph+ cells occurred in response to imatinib. Presence of +8 and other chromosomal abnormalities in Ph- cells in patients treated with imatinib provide the first clinical observation that CML has a multi-step pathogenesis, and hat clonal, Ph- cells may precede development of the Ph+ clone (Figure 14)
Blast Phase
+
Blast crisis of CML may be myeloid or lymphoid
• About 15-20% appear to retain the 46, Ph+ cell line unchanged, whereas 80-85% of patients show karyotypic evolution. That is, new chromosomal abnormalities in very distinct patterns are present in addition to the new Ph chromosome • The most common changes, gain of chromosomes 8, 19, or the second Ph, and i(l7q) frequently occur in combination to produce modal chromosome numbers of 47-50 • When patients have only a single new chromosome change, this most commonly, involves the gain of the second Ph, an i(17q), or +8, or + 19 in descending order of frequency + Isochromosome l7q occurs almost exclusively in myeloid blast crisis . Other rearrangements, occurring less frequently, include monosomies of chromosomes 7, 17, and Y, trisomies of chromosomes 17 and 21 and t(3;21)(q26 ;q22) • If blast crisis is suspected, in addition to BCR-ABL probe two other probes may be used, CEP8 and P53 on 17p13.1 to determine trisomy 8 and i(l7q), which will result in deletion of one copy of P53 (Table 6)
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Ph- Chronic Myeloproliferative Disorders (CMPD) • Despite technological advances, the cytogenetic analysis of bone marrow cells from patients with chronic myeloproliferative disorders , other than CML, remains a laborious and time-consuming technology. It requires highly skilled and trained observers to interpret "fuzzy" looking chromosomes, which may have suboptimal morphology and/or banding for detection of micro deletions and translocations . In addition , between 5% and 20% of bone marrow specimens may have either a low mitotic yield or are otherwise uninformative due to "dry tap" or poor specimen collection • The unifying concept of genetic instability in CMPDs is a loss or a gain of genetic material. However, rare recurrent balanced translocations have been identified in CMPDs as shown in Table 7
Fig. 13. Trisomy 8 in a BCR -ABl__negative cell form a patient treated with imatinib (Gleevac). Top interphase cell shows lack of the BCR-ABL fusion and is showing three copies of chromosome 8 (aqua). In contrast , the lower cell is BCR-ABL fusion positive and has disomy 8 (two aqua signals). Partial karyotypes, as shown on the right, indicate trisomy 8 and t(9;22), respectively. Trisomy 8 was uncovered only in cells that were Ph- and BCR-ABL fusion negative (see text).
Polycythemia Vera (PV) • Like other chronic myeloproliferative disorders, PV is a clonal disorder arising in a stem cell capable of differentiation into most cell lineages including B-lymphoid cells. PV has a spectrum of clinical and cytogenetic manifestations overlapping with other chronic myeloproliferative disorders. Approximately 10-15% of patients with PV progress to malignant transformation with changes typical of AML
MUltisteppathogenesis of ph-positive CML
~
f8\
"Preleukemk" Ph-negative stage
~~ ~
0fA\
~ ~
V{MyelOid Erythroid
~~ -+ {;\0 \.!:J~ ~
Stem cell
-----l I -
Clonal development
Platele~ yrnphoid
~
(Tand B)
Differentiation
Ph-positive stage, - - - - i chronic phase
-+~bC~
tF
abl
Genetic Fusion of instability bcr/abl
1:9,221
I--
Acute (blast)-l phase
bcr -+ -Ph, +8, +19, i(17q) abl
Additional Karyolypic abnormolities
Fig. 14. Model for the multi-step pathogenesis of Ph+ CML in which clonal origin is the first detectable event and the Phchromosome is formed only in the descends of clonally derived CML cells. Other genetic defects are occurring later only in the Ph+, BCR-ABL fusion-positive cells .
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Table 7. Chromosomal Translocations in Ph-Negative CMPD CMPD entity
Chromosomal abnormality
Genes involved
t(9;12)(q34;p 13)
ETV6-ABL
5q33
PDGFRB
t(5;12)(q33;p13)
ETV6-PDGFRB
t(5;7)(q33;qll)
HIP1-PDGFRB
t(5;1O)(q33;q21)
CCDC6-PDGFRB
t(5;14)(q33;q32)
K1A1509-PDGFRB
t(5; 14)(q33;q32)
TRIPII-PDGFRB
t(5; 14)(q33;q22)
NIN-PDGFRB
t(5; 15)(q33;q 15)
TP53BP I-PDGFRB
t(5;17)(q33;pll)
SPECCI-PDGFRB
t(5;17)(q33;pI3)
RABEPl-PDGFRB
8pll
FGFRI
t(8;13)(p l1;q 12)
ZNF/98-FGFRl
t(7;8)(q32;pll)
TRIM24-FGFRl
t(6;8)(q27;pll)
FGFRIOP-FGFRI
ins(l2;8)(p II;p II p22)
FGFRIOP2-FGFRl
t(8; 17)(p II;q II)
MY018A-FGFRl
t(8;22)(p II ;q 11.2)
BCR-FGFRl
4ql2
PDGFRA
del(4)(q 12q12)
FlP1Ll-PDGFRA
t(4;22)(qI2;qI1.2)
BCR-FGFRi
4ql2
KiT
9p24
JAK2
t(9;22)(p24;q 11.2)
BCR-JAK2
CML-like
t(9; 12)(p24-p13)
JAK2-ETV6
CML-like
t(8;9)(p22;p24)
PCMI-JAK2
CMPD,AL
• Cytogenetic abnormalities are detected at diagnosis in about 25( and they include +9p/+9 in 33% (Figure 15), del(20)(qI2) in 33%, +8 in 20%, and del(l3)(q13) in 18%. • The frequency of chromosomal abnormalities detected by I-FISH is about 29% • The presence of rare t( 1;9) has been suggested to represent a specific PV recurrent rearrangement • The findings obtained by I-FISH also suggest that cells with +9p and monosomy for l3q 14 and 20q 12 loci may exist in PV in a dormant state. Once cells with +9p begin to divide, they appear to have a proliferative advantage compared with cells carrying del(20)(q12)
CML-like
CEL,CMML
CMML
CEL
SM
• I-FISH is an important diagnostic aid in confirming the presence of PV in patients with erythrocytosis • The set of probes used in diagnostic testing of MPDs include CEP8, CEP9/9p2l, RbI, and D20S258 for identification of the five most frequent recurrent chromosomal rearrangements associated with PhCMPD • A possible important role of JAK2 in molecular pathogenesis of MPDs is highlighted by two recent lines of observations:
- JAK2-specific acquired somatic mutation, JAK2- V6I7F, is identified in over 90% of PV, and approximately 50%
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Molecular Genetic Pathology
i(9p),i(9q)
Fig. 15. Examples of the 9p rearrangements in patients with PV showing from left to right +9, trisomy 9p, derivative (9) t(1;9), der(9;13) , tetrasomy 9p, and the isochromosome for the 9p and the 9q.
of patients with idiopathic myelofibrosis (IMF) and essential thrombocythemia (ET) - Rearranged JAK 2 was identified with three different partner chromosomes in t(8;9)(p22;p24) producing PCMI -JAK2 fusion gene in chronic and acute leukemia in t(9;22)(p24;qI1.2) producing BCR-JAK2 fusion in CML ; and in t(9;12)(p24;p13) resulting in TEL-JAK2 fusion in B-cell ALL
Idiopathic Myelofibrosis • IMF is a clonal disorder arising in a multi-potent hematopoietic progenitor cell. Bone marrow fibrosis is likely to be a secondary event in the pathogenesis. Approximately 50% of patients are chromosomally abnormal at diagnosis • del(13)(qI4) is present in about one third of patients and cytogenetic studies demonstrated 91% of reported cases to have a breakpoint at 13q12-13 to q21-22 chromosomal region (Figures 16 and 17)
326
• Other recurrent abnormalities detected at diagnosis include del(20)(q II q13) (17-22%), trisomy 8 (11-15%), + Iq (11-19%), and rearrangements of the long arms of chromosome 12 (7-11 %)
Essential Thrombocythemia • Approximately 50% of patients with ET have a clonal disease and the remaining are polyclonal at the time of diagnosis • In one of the largest cytogenetic series, 9% of 191 patients were chromosomally abnormal and chromosomal abnormalities were found both in JAK2- V617F-positive and -negative patients • The chromosomal abnormalities detected are similar as in PV and IMF
Other Ph- CMPD Table 7 shows the overview of the chromosomal aberrations involving tyrosine kinase genes in Ph- CMPDs
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FISH and Con ventional Cytogeneti cs
• Chronic eosinophilic leukemia (CEL)/Hy pereosinophilic syndrome (HES) with FlP1LJ-PDGFA CEL is close ly related to HES but it may be distinguished on the basis of clonality • The clonal origin of most CEL is under-recog nized and these cases may be classified as "idiopathic HES". Discovery of a cryptic deletion at 4q 12, associate d with FlPJL!-PDGFRA fusio n gene, identified by FISH methodology may make a correct diagnosis possible for majority of patients with chronic eosinophilic leukem ia presenting with a normal karyotype. CEL is a clonal myeloproliferative disorder in which eosinophils, mast cells, monocytes, neutroph ils, and T- and B-Iymphocytes are involved in clonal process, although the specific lineages involved may vary among patients • The chromosomal deletion in FlP1LJ-PDGFRA is only about 800 kb in size explaining why this deleti on was not previously recognized by conventional cytogenetics. Identification of the hybrid gene is of utmost importance because these patients have excellent response to imatinib therapy • Approximately 20-50% of the CEL/HES cases express FlP1LJ-PDGFRA fusion gene and highlights remarkable features of this entity: - FlP1LJ-PDGFRA is almost exclusive ly found in males (>90% males)
Fig. 16. Interphase nuclei from a patient with IMF showing a deletion of 13q 14 locus (green) and disomy for 13q34 locus, indicating a deletion of the gene and not monosomy 13.
013 I
, p11.2 <,
r: x;
./
q12.1
FlT1, FlT3
q12.3
BRCA2
RB1
ll)
~
....
q14.1 914.2 q14.3
OJ
C\J
en (")
(")
q13.3-q14.3 region commonly deleted
......
q14.3locus frequently deleted in MM and Cll
q21.1 q21.3
en (") ~
I
RB1
200 kb
I
0
0
I
I
-
130 kb
FISH
160 kb
Probes
q31
~:
q32
q34
,'"
j"
Recurrent amplification in follicular lymphoma
.•..
':!'
.x :'."'.'
-s:~">!i}". ".··L
Fig. 17. Schem atic representation of chromosome 13 showing frequently deleted region s in myelofibrosis, MM, and CLL as well as amplification of regions in follicular lymphom a.
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Molecular Genetic Pathology
- Corresponds well to elevated serum tryptase level - The presence of fusion gene is a positive predictor for response to low doses of imatinib - Development of resistance to imatinib, unlike in CML , does not seem to be a major problem • Some FIPILl-PDGFRA-negative patients also respond to imatinib therapy. The reason for this response remains unknown
• Rare other probes are currently commercially available for detection of cryptic and non-cryptic chromosomal rearrangements associated with CMPDs with FISH technology, such as PDGFRB for detection of 5q33 rearrangements involved in a number of translocations and dual color dual fusion ETVlffEL for detection of ETVlffEL-BCR fusion
MYELODYSPLASTIC DISORDERS (MDS) FISH Studies in MDS
• Like other myeloproliferative disorders, the MDS comprise a heterogeneous group of disorders with respect to peripheral blood cell counts, morphology, marrow cellularity, and prognosis. They have in common a clonal origin , dysplastic cellular morphology, abnormalities of cellular maturation, and an increased propensity to develop acute leukemia (20-40 %)
• Several FISH reports have demonstrated that FISH studies not only identifies abnormalities at the similar frequency to those detected by conventional cytogenetics, but FISH also unmasked cryptic chromosomal rearrangements in 3-15% of cases
Clonal Origin
• Figure 20 shows the set of probes used in specimens obtained from patient s with MDS
• Studie s of G6PD heterozygous females with MDS, in conjunction with cytogenetic studies as well as FISH analysis of CD34 + CD38 stem cells strongly suggests that a multi-potent stem cell capable of differentiating into both myeloid and lymphoid lineages is involved in MDS pathogenesis • MDS rarely transforms into ALL
Chromosomal Abnormalities in MDS • Acquired chromosomal abnormalities are found in 40-60% of primary and in over 95% of secondary or therapy-related cases (Figure 18) • Chromosomal rearrangements in MDS include unbalanced translocations, which often lead to the loss of genetic material. Hemizygosity for specific genes or chromosomal regions are the hallmark of MDS even though some rearrangements like +8, -5/del(5q), -7/del(7q), and del(20q) are also seen in AML • The International MDS Risk Analysis Workshop combined cytogenetic, morphologic, and clinical data from seven large risk-based studies, and defined the International Prognostic Scoring System for MDS (Figure 19) • The most common abnormalities in pediatric MDS are monosomy 7, +8, and +21 • Obtaining high-quality metaphases for conventional cytogenetic analysis in patients on treatment can be problematic in MDS, and therefore treatment response may significantly underestimate the true response rate
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• A significant proportion of both de novo and therapyrelated MDS have very complex karyotype, affecting 10 or more chromosomes, that cannot be identified using the set of "MDS-specific probes" and therefore I-FISH cannot be a substitute for conventional cytogenetics • The primary use of I-FISH in patients with MDS is to monitor residual disease after chemotherapy and/or stem cell transplantation, after diagnostic abnormality has been established • Although the use of M-FISH and SKY will allow precise characterization of complex karyotype in over 93% cases, their use in routine clinical laboratories is limited due the cost of probes, equipment, and time involved. However, in research investigations the use of these methodologies have contributed to the identification of two new recurrent translocations
Current Treatment • The treatment option for patients with MDS include: - Supportive care - Growth factors - Bone marrow transplant - VIDAZA ® (azacitidine) [Pharmion] - Revlimid" (Celgene) - Dacogem" • Treatment with Vidaza" has demonstrated the modulation of the MDS clone. Five categories of cytogenetic changes were observed during Vidaza" treatment and they include persistence of normal
FISH and Conventional Cytogenetics
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Fig. 18. Frequency, partial karyotypes, and interphase cells of chromosomal rearrangements in myelodysplasia.
5q-Syndrome • Among patients with MDS, those with an isolated deletion of the long arm of chromosome 5 (del [5q] or 5q) (Figure 21) represent a unique clinical group • They are characterized by female predominance, refractory anemia, modest neutropenia, hypolobular megakaryocytes, less than 5% blasts and a mild course. A low risk of leukemic transformation • The deletions of 5q most often observed are deletion s of regions 5q31 and 33 as the sole cytogenetic abnormality
Fig. 19. International Prognostic Scoring System for recurrent cytogenetic abnormalities identified at the diagnosis of MDS.
karyotype (37%); progression to an abnormal karyotype (18%) ; persistence of the initial abnormal karyotype (27%) ; clonal evolution (9.4%) , and normalization of the initial abnormal karyotype (9%) • Revlimid" (lenalidomide) is indicated for the treatment of patients with transfusion-dependent anemia due to low- or intermediate-I-risk MDS associated with deletion 5q cytogenetic abnormality with or without additional cytogenetic abnormalities. Durable cytogenetics remission was achieved in 76-83% patients with MDS and del(5q) treated with Revlimid'". Like Vidaza, Dacogen (decitabine) is DNA methyltransfare inhibitor and produces , at best, major cytogenetics remission in 31%
Rearrangements of 17p •
l7p deletion s are seen in 3-4% of AML and MDS patients. These patients often display several other chromo somal rearrangements
• About 30% of them are therapy related. Most of the patients who develop MDS or AML were treated with hydroxyurea, usually for a prolonged period of time . In many of the reported patients, hydroxyurea was the only cytoreductive agent. A large number of cases with therapy-related AML (tAML) or tMDS are also seen in patients with lymphoid neoplasms treated with alkylating agents. These abnormalities include monosomy 17, isochromosome 17q, and unbalanced translocations between chromosome 17 and another chromosome • The extent of 17p deletion was studied by FISH and in all cases this deletion involved the p53 gene. There appears to be a close correlation between
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Molecular Genetic Pathology
12p13, T EL
20q11 21q22. 13- q22.2
5p15 .2 5q31
5
8p1"1=q11 11q23 7p11 -q11 MLL 7q31 11 7 8
13q14 12
13
20
21
Fig. 20. Set of probes used for detection of chromosomal abnormalities from patients with myelodysplasia. The set includes 5p I5.2/5q3 1, 7q311CEP7, CEP 8, 12p 13 (TElETVlL), 13ql 4 (RBI) , D20S109, and D21S259 markers for detection of del(20)(ql lqI3) and gain of chro mosome 21, respectively.
Rearrangements of 3q26 • Eight different rearrangements involving long arms of chromosome 3, band region q26-27, were observed in blast crisis of CML , acute myeloid leukemi a, and in therapy-related MOS. The most frequent rearran gements involves two bands on chromosome 3, namely simultaneously band 3q21 and 3q26, which produces either t(3;3)(q21;q26) or inv(3)(q21q26) • The most characteristic clinical features include elevated platelet count, marked hyperplasia with dysplasia of megakaryocytes, and poor prognosis • Chromosome band q26 co ntains the locus of two genes MD SI and ecotropic viral integra tion I (EVIl ), a DNA-binding protein. In patients with AML and MDS showing 3q2 1q26 abnormalities FISH studies revealed that the breakpoint in 3q26 in t(3;3) is a large region 13-300 kb upstream of EVIl whereas the breakpoints of the inv(3) were clustered in a small region at the 3' of EVIl Fig. 2 1. A partial karyotype showing an interstitial deletion of the long arms of chromosome 5, region q14q34.
dysgranulopoiesis, for example , pseudo-pelger-Huet hypolobulation and small vacuoles in neutrophils with 17p abnormalities and p53 deletion. Median survival of these patient s is poor
330
Rearrangements of t(8)(pll) • Two distinct clinical syndromes have been associa ted with recurrent translocations involving 8p l l: stem cell myeloproliferative disorders and AML • The MPD is characterized by B-and T-cell lymphoblastic leukemia/lymphoma, myeloid hyperplasia, and peripheral blood eosinophilia
FISH and Conventional Cytogenetics
• The second group consi sts of acute myelomonocytic leukem ia, predominately associated with erythrophagocytosis. The rearrangements involved in both groups include inv(8)(p llq 13), t(8; 13)(pll;q 12), t(8;14)(pll;qll.l ), t(8;16) (p l l; p I3), t(8;19)(pll;q13), and t(8;22) (p l l ;q I3) • The MOZ gene on 8p 11 is fused to the CBP (CREB binding protein ) gene at 16pl3 in t(8;13), to the
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nuclear receptor transcriptional coactivator, TIF 2/GRIP-l/NCOA-2 (transcriptional intermediary factor 2) in inv(8), and to p300 (a homolog of CBP) in t(8;22) • Although these rearrangements can be detected cytogenetically, currently there are no FISH probe s to detect MOZ gene rearrangements
ACUTE MYELOGENOUS LEUKEMIA Karyotype in AML • Acute myeloid leukemia is a group of heterogeneous diseases with respect to clonality, chromosomal aberrations, and response to treatment. AML develops clonally and has a multi-step pathogenesis, although the pattern of stem cell differentiative expression (as detected by G6PD and other DNA or cellular markers) is heterogeneou s • In most of the patients with AML , a clonal cell population cannot be detected during remission. However, in some cases hematopoiesis remains clonal showing only one a single G6PD enzyme-type • As shown in Table 8 based on the karyotype status the two major groups of AML can be discriminated - Those with an abnormal karyotype, accounting for about 52% of patients - Those that demonstrate a normal karyotype by conventional cytogenetics compri sing 48% of AML patients • Pretreatment karyotype constitutes an independent prognostic determinant for attainment of complete remission and risk of relap se and survival • In over 6000 cytogenetically characterized patients with AML, 41 recurrent balanced and 178 unbalanced chromosomal abnormalities were described • Table 9 show the diversity and frequen cy of primary recurrent chromosomal abnormalities and Table 10 shows association of these rearran gements with the French-American-British (FAB) subtypes. On the basis of response to induction therapy, relative risk, and overall survival, three progno stic group s can be distingui shed based on the presence of specific chrom osome abnormalities irrespective of additional cytogenetic chan ges (Table 11 and Figure 22) • In contrast to primary chromosome changes , which are usually balanced reciprocal translocations, Table 12 shows common secondary rearrangements, which almost invariably lead to genomic inbalances (trisomies, monosomies, deletions, and unbalanced translocations)
• Detection of abnormal cytogenetics in morphological complete remissions has been shown recently to correlate with short overall and disease-free survival and higher relapse rate in 118 patient s evaluated through the Cancer and Leukemia B study 8461 • Although cytogenetic analysis appear s to be informative in detecting residual disease following induction therapy, as noted above, it is labor intensive but RT-PCR strategies for detect ing ETO-AMLI or CBFB-MYHII has yet to be fully validated in predicting clinical outcomes
AML M2 and t(8;21)(q22;q22) • Described for the first time in 1973, the incidence of t(8;21)(q22;q22) varies among different series and different laboratories (Figure 23A). At the time of diagno sis, t(8;21 ) is present in 10-15 % of adult patient s with de novo AML and in 6-19% of children with AML. Among the patients classified as M2 AML, t(8;21) is found in 40-50%. Among patient s with t(8;21), 30-35% also display loss of the Y chromosome in males and a loss of the X chromo some in females . Another one-third of the patient s with t(8;21) show deletion of 9q12-23 including a commonly deleted segment, which spans 7-8 Mb • Virtually all patient s with t(8;21) achieve complete remission and additional cytogenetic abnormalities, irrespective of their nature or complexity, do not have a deleterious effect on remission, relative risk, and overall survival • The t(8;21) interrupts two genes, RUNXI (AMLl , CBFA2 ) on chromo some 21, band q22 and ETO (eight, twenty-one gene; also called, MTGB-myeloid-translocated gene on no. 8, or CBFA2Tl) on chromosome 8, band q22, and join s them to form a new chimeric gene on der(8) chromo some
• RUNXI is a gene on chromosome 21, also known as CBFA2/AMLl, becau se it codes for a DNA-binding component of core-binding factor (CBF), and binds DNA through a specific sequence called the runt domain
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Table 8. Classification of AML Based on Karyotype Status" Karytype status
(%)
Abnormal
52
Type of abnormalities Balanced trans locations (25 %): t(8;2l);t(l5;17);inv(l6) Non -balanced abnormalities (27%) : -5/del(5q),del(7q), complex karyotype
Normal
NPM mutations (62%) 48
FrL 3 length mutations (23 %) MLL tandem duplications (5%) CIEBP a-mutations (8%)
"M odified from reference Hiddemann et al. 2005
Table 9. Frequencies of the Common Cytogenetics Abnormalites in Adult and Childhood AMLa Cytogenetic abnormality Norm al karyotype
Adults (n = 4257) (%)
Prognosis
Children (n = 1184) (%)
Prognosis
45 .1
Intermediate-good
23.9
Intermediate-good
Trisomy 8
9.1
Poor
9.5
-7/del(7q)
8.4
Poor/not prognostic
5.2
t(l5; I7)(q22;q2 I)
7.6
Favorable
9.9
-5/del(5q)
7.2
Not prognostic
1.2
t(8 ;21)( q22 ;q22)
5.5
Favorable
11.6
inv( 16)(p 13q22)/t(16;16)(p 13;q22)
4.7
Favorable
5.9
-y
4.1
t(llq23)
3.3
Poor
13.1
t(9;11)(p22;q23)
2.1
Poor
6.4
abn(l2p)
2.5
Favorable
NA
Trisomy 21
2.2
5.1
abn(l7p)
2.2
2
del(9q )
2.1
inv(3)(q21 q26)/t(3;3)(q21 ;q26)
2
0
del(llq)
0.9
1.3
t(9;22)(q34;q II)
0.8
t(6 ;9)(p23;q34)
0.7
I
Complex karyotype (2:3 abn)
10.7
14.3
Complex karyotype (>5 abn)
8.8
5.2
aM odified from reference Mrozek et al. 2004
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Unknown
Adverse
3.8
Intermediate
Poor
2.8
0.2
Poor
Not prognostic
Adverse
Poor
FISH and Conventional Cytogenetics
Table 10. Association of Primary Chromosomal Abnormalities in AML with FAR Subtypes FAB subtype
Chromosomal abnormality der( I ;7)(q IO;q I0)
MI -M2**
t(6;9)(p23 ;q34)
MI-M2**
t(9;22)(q34 ;q 11.2)
MI -M2
t(l6;21)(p II ;q22)
MI-M2
trisomy 4
MI-M2**
trisomy II
MI -M2 **
t(7;11)(pI5;pI5)
M2
t(8;21)(q22 ;q22)
M2
t(l5; 17)(q22;q2l)
M3 and M3 variant
t(ll ;17)(q23q21)
M3 and M3 variant
(11: 17)(q13;q13)
M3 and M3 variant
t(5;17)(q31;q21)
M3 and M3 variant
t(l ;3)(p36 ;q21)
M4
trisomy 22
M4
inv( 16)(p 13;q22)/t( 16;16)(p 13;q22)
M4Eo
Table 11. Cytogenetic Risk Categories in AML Category
Abnormality
Favorable
t(8 ;21)Q,t(l5 ;17),inv(l6) with or without other anomalies
Intermediate
Normal, +6,+8,+21,+22,-Y del(9q)
Unfavorable
-5/del(5q),-7/de1(7q),abn(3q) t(9;22), t(6;9),abn(llq), 20q or 21q, abn( 17p), complex karyotype
awithout deletion 9q or complex karyotype • The RUNX1-ETO fusion transcript may be detected by Southern blot analysis, and RT-PCR and the fusion gene may be visualized using FISH. Molecular screening with RT-PCR for t(8;21) at the time of diagnosis or relapse may reveal cryptic t(8;21), other variant transcripts, and an increased overall detection rate. However, the use of RT-PCR for detection of minimal residual disease after chemotherapy is controversial, because the hybrid transcripts were detected in long-term (up to 8 years) remissions and after stem cell transplantation
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• Two color I-FISH for detection of RUNX1-ETO fusion was first documented in 1995 and the application of this method represent an alternative tool for RT-PCR for monitoring of disease and for clonality studies
• AML-1 plays a critical role during hematopoiesis. Its disruption is associated with the development of myeloid and lymphoid leukemias. The AMLl/CBFA2 transcription factor complex is essential for the formation of definitive hematopoiesis. Chromosomal rearrangements involving AMLl, have been identified with a number of partner chromosomes, such as 1p36, 3q21,5qI3, 12p13, 12q24 14q22, 15q22, 16pll,and 17ql1.2 (see Rearrangements of 21q22). One can, therefore, view AMLl/CBFA2 as a master regulatory switch that controls development of a definitive lineage. Moreover, the AMLl transcription factor is critical for proliferation and differentiation of hematopoietic stem cells. Haploinsufficiency of AMLl has been linked to a propensity to develop AML and bi-allelic nonsense mutations in the AMLl gene and have been identified in most immature AMLs of the FAB MO subtype • The AMLlIETO oncoprotein is a dominant-negative form of AMLl, which represses the promoters of genes normally activated by the AMLl. Therefore, the AMLlIETO oncoprotein through altered transcription patterns inhibits myeloid cell differentiation, and most likely leads to development of leukemia • Similar pathways whereby transcription factors are disrupted by chromosomal translocations and fused to genes encoding other transcriptional regulators are the theme of chromosomal translocation in hematological malignancies
t(l6;21)(q24;q22) • This translocation is a rare but recurrent chromosomal abnormality associated with therapy-related AML or MDS and identified for the first time with FISH . Studies utilizing FISH and RT-PCR methods demonstrated the fusion of AMLl on 21q22 and a recently identified, MTG 16 (myeloid translocation gene on chromosome 16) on chromosome 16, which produced a AMLl-MTG J6 fusion gene on chromosome 16 • The breakpoints of both t(8;21) and t(16 ;21) occur within the same intron of the AMLl gene. AMLl-MTG J6 gene fusion results in the production of a protein that is very similar to the AMLl-ETO (AMLl-MTG8 ) protein in t(8;21)
Rearrangements of21q22 In addition to t(8;21), t(3;21), and (16;21) occurring in myeloid disorders and t(12 ;21), which is specific for pediatric ALL, there are over 15 identified partner chromosomes involving 21q22. Many, but not all, are detected in myeloid disorders whether or not they arise de novo or are therapy related.
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Molecular Genet ic Pathology
Fig. 22. Com posite of cells after in situ hybridiza tion showing chromosomal rearrangements associated with favorable prognosis (top row), interme diate prog nosis (middle row), and unfavorable prognosis (bottom row) in AML. Favorable prognosis include t(8;21), t(lS ;17), and inv(l6), intermediate prognosis include trisomy 8 and unfavorable prognosis are assoc iated with MLL rearrangements, deletion of 7q3 1 and Sq21 loci, as well as BCR-ABL fusion- positive AML.
Table 12. Secondary Aberrations Present in More Than 10% of Patients with a Given Primary Structural Abnormality inAML Primary ab erratio n
Secondary aberratio n [%] a
t(l ;3)(p36;q21)
del(5q) [1 6.7%]
t(l ;22)(p13;q13)
+19[35.7%]. +7 [28.6%], der (I )t(1;22) [28.6%]
der (l ;7)(qlO;plO)
+8 [31.8%]
inv(3)(q21 ;q26)
- 7 [46.6%, del(5q) [10.3%]
t(6;9)(p23;q3)
+8 [11.4%], +13 [11.4%]
t(8;21 )(q22;q22)
-y [39.3%]. - X[16.3%]. del(9q)
[12/7%] t(9;11 )(p21-22;q23)
+8 [17.9%]
t(9;22)(q34;qII )
+8[21/7%]. -7[16.9] der(22) t(9;22)[16.9%]. +19 [10.8%]
t(15;17)(q22;q11-1 2)
+8 [10.5%]
inv(16)(p13;q22)
+22 [16.6%]. 8[11.8]
"Percent of all patients with a given primary abnormality
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APL , t(15;17)(q22;q21) and Variant Translocations: t(11;17)(q23;q21 ), t(5;17)(q35;q21 ),t(11;17)(q13;q21 ), and dup(17)(q21.3-q21) • APL acco unts for 10% of adult myeloid leukemia and is assoc iated with structural rearrangement involving the long arms of chromosomes 15 and 17 in APL (M3) recognized for the first time in 1977 (F igu res 23B and 24). As a consequence of this translocation two derivative chromoso mes are formed: der( 15) and der( 17) • All patients with t(l S;17)(q22;q21) haveAPL (M3 and M3 variants), but the reverse is not true, about 9% of patie nts with morph ological features of APL either have a normal karyo type or have other than t(15;17) rearrangements (Ta ble 12) • In about 70-90% of patients, the t(lS; 17) is the only cytoge netic abnormality seen in leukemic metaphase cells . However, add itiona l abnormalities may accompany the t(lS ;17) among which trisomy 8 is the most freq uent abnorma lity • Cl inical, morphologic , cytogenetic. and molecular studies do not show a major difference between adult and pedi atric patients with APL • The t(lS; 17)(q22 ;q2 1) rearrangement involving the PML (promyelocytic leukemia) gene on chromoso me band I Sq22 and the retinoic acid receptor a gene (RARa) on I7q 21 is the genetic basis for approxi mately 97% of all cas es of APL
FISH and Conventional Cytogenetics
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Fig. 23. (A) A partial karyotype showing t(8;2I)(q22; q22) (left top) and an interphase cell after in situ hybridization with ETOAML probe (left, bottom row). Note two copies of co-localized signals as well as one each single signal for normal chromosomes. Both derivative chromosomes are from a patient with t(8;2I)(q22;q22)m showing ETO-AMLl rearrangement. (B) A partial metaphase from a patient with APL after in situ hybridization with WCP probes for chromosomes 15 (green) and 17 (red) (middle, top row). Note two derivative chromosomes with the mixture of red and green colors consistent with t(15 ;I7)(q22;q21). Normal chromosome 15 is "painted" in green only and normal chromosome 17 is "painted" in red only. The interphase cell from the same patient after in situ hybridization with dual color, dual fusion PML-RARA probe showing two copes of co-localized fusion consistent with PML-RARA- fusion-positive cell. (C) A partial karyotype from two metaphase cells showing a normal chromosome 16 (right, left chromosome) and inversion (16) (right chromosome, red arrow) . Interphase nuclei from the same patient with AML M4 showing CBFB rearrangement as a result of inv( 16), using "break-apart" probe strategy (right, bottom row). • About 9% of patients who are morphologically classified as APL are lacking the t( 15;17) but these still exhibit the PML-RARa fusion gene, created by insertion events or more complex rearrangements • The remaining 10% of cases include four rare variant translocations: t( 11;17)(q23;q2I), t( 11;17)(q13;q21), t(5;I7)(q35;q21), and dup(17)(q21.3-q2I) • APL is the only condition, which can be successfully treated with an agent, all trans retinoic acid (ATRA), which induces the neoplastic cells to differentiate • APL is associated with five different genetic rearrangements fusing the RARa gene with a different partner gene in each case. As shown in Figure 25 based on genomic rearrangements, FISH assay, using breakapart probe strategy with dual color RARA , can identify two different syndromes. It is important to recognize patients who may benefit from ATRA therapy because alternative therapy must be used for non-ATRA responsive patients
• Following chemotherapy detection of t(15; 17) by conventional cytogenetic study may be difficult. RT-PCR procedures may be time-consuming and they are not easily quantifiable and are limited only to detection of PML-RARa. Alternative translocations will be overlooked by these methods. Dual fusion, dual color PML-RARA probe in interphase FISH assay shows consistently clinical sensitivity of 98% and 100% specificity
Acute Myelomonocytic Leukemia with Excess Eosinophils (M4Eo); inv(16)(p13q22) and t(16;16)(p13;q22) • The bone marrow of patients with M4 Eo contains an excess of eosinophils (8-54%) or abnormal eosinophils. This FAB subtype, correlates with abnormalities of band 16q22, for example, inv(16)(pI3q22) and t( 16;16)(p13;q22)
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Molecular Genetic Pathology
• About 30( of patients have deletions of MYH11 proximal to the p-arm breakpoint, in addition to the inversion, which prevents the generation of a MYHIIICBFB fusion • Combined may-grunwald-giemsa staining with FISH, it was demonstrated clearly that the abnormal eosinophils had inv(16), and therefore , are part of the leukemic clone • Clearly, the development of both FISH and RT-PCR assays for the detection of the fusion transcripts have aided in diagnosis, monitoring of disease status, and for higher sensitivity of detection of l6q22 rearrangements • The presence of additional abnormalities such as trisomy 8 does not adversely affect clinical outcome
t(16;21)(pll;q22) • t( 16;21) is a rare chromosomal rearrangement associated with M I-M2 AML. A proportion of patients may have additional abnormalities. This translocation fuses the TLSIFUS gene on chromosome 16, band p 11 to the ERG gene on chromosome 22, and band q22. The ERG gene is a member of the ETS family of transcription factors and is a sequence-specific transcriptional activator. The presence of a fusion transcript is detected by RT-PCR, at the time of diagnosis, relapse, and during remission. These observations are consistent with the impression that patients with t(16;21) have a poor prognosis and may benefit from early detection of this chimeric gene in order to obtain more intensive therapy, such as stem cell transplantation Fig. 24. A partial karyotype from a patient with APL showing ider(17)t(15; 17)(q22;q2l ) (top row). The karyotype showed isochromosome for the long arms of chromosome 17 and deletion of the short arms. FISH studies revealed three copies of PML RARA fusion (bottom row), confirming that the first event in the pathogenesis was PML-RARA fusion, and the subsequent event was a structural rearrangement of isochromosome.
• Patients with l6q22 abnormalities have a more favorable prognosis and relatively long survival • Trisomy 22, as an accompanying abnormality was observed in a proportion of patients with inv(16) and t(16;16) • Rearrangements in inv(16) and t(16; 16) result in disruption of the myosin heavy chain (MYH11) gene at 16p13 and the core binding factor (CBFB) gene at l6q22 (Figure 23C) . Both rearrangements result in a fusion of CBFB and MYHll on l6p13. The resulting hybrid protein, CBF~-SMMHC, blocks myeloid and lymphoid differentiation during adult hematopoiesis in mouse models, suggesting a very specific role of the hybrid protein • Up to 4% of patients with CBFBIMYH11 rearrangement do not have cytogenetically detectable inv(16) or t(16 ;16)
336
t(9;22)(q34;q 11.2) • Less than 1% of patients with AML have the Ph chromosome resulting from the translocation (9;22)(q34;q 11.2) • In Ph+ AML the breakpoint is proximal to the CML-type breakpoint on chromosome 22, creating a 7.0 kb mRNA and P190 BCR-ABL protein (see Chronic Myelogenous Leukemia section) • Rare patients with AML have a late appearance of the Ph chromosome either as a sole abnormality or in a clone showing t(8;2l). It is believed that the Ph chromosome in these patients is likely to be a secondary event • The Ph+ AML patients are associated with poor outcome
Acute Monocytic Leukemia (MS) and Translocations Involving llq23 andMLL Gene • The MLL (myeloid lymphoid lineage, also called "mixedlineage leukemia" and also referred as ALLl, HRX, or HTRXl) gene, at llq23 is involved in 95% of all Ilq23 translocations including AML and ALL (Figure 26) • The MLL gene is affected in approximately 80% of all ALL cases involving infants, 3% of ALL cases involving older children, and 85% of secondary AML cases involving patients treated with topoisomerase II inhibitors
FISH and Conventional Cytogenetics
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Fig. 25. Two syndromes within APL can be distinguished as a result of RARA genomic rearrangements. Only patients with PLZFRARA fusion, a result of t( 11;17)(q23;q22), does not respond to the ATRA differentiation therapy.
Chromosome 9
Chromosome 11
Fig. 26. Rearrangements of chromosome 11 showing deletion lIq23 (left), MLL rearrangements (left center), MLL amplification (right center), and t(9;II)(p13.I;q23) in a patient with ALL. Note that in situ hybridization with the breakapart dual color dual fusion MLL probe resulted in this case in the separation of the 3' end of the gene (red) that went to chromosome 9 and the 5' end of the gene (green) remained on chromosome 11.
• Over 70 partner chromosome bands participate in MLL translocations • In addition, partial tandem duplication of the aminoterminus region of the MLL gene is associated with AML patients with or without trisomy 11 • Patients with AML and ALL and MLL rearrangements have a very poor prognosis with long-term event-free survival rates of >20% despite treatment with aggressive multi-agent chemotherapy • In contrast, patients with llq23 abnormalities not involving the MLL gene appear to have an outcome similar to those of other patients with ALL • The MLL gene is about 100 kb and encodes an I1.7-kb of cDNA and a 431-kD protein . MLL homologs have been
found in mouse, chick, pufferfish, and drosophila, which indicate that the gene has been conserved throughout evolution • Ilq23 translocations cluster in an 8.3 kb region of MLL and fuse the N-terminal portion of MLL , containing the AT-hook and methyltransferase domains, to numerous different proteins • The contribution of these various fusion partners to transformation is not known and the partner genes in the translocations do not appear to have any unifying characteristics that would clarify their role in leukemogenesis • The most common 11q23 translocation in childhood ALL as well as in adult ALL is the t(4;11)(q21;q23) creating
337
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an MLL-AF4 fusion gene followed by the t(11;19) (q23;pI3) in which an MLL-ENL fusion gene is formed • The clinical outcome of both children and adults with t(4; 11) and t(11;19) is very poor • The survival of adults with t(6;11)(q27 ;q23) and t(11;19)(q23 ;pI3.1) is short and their identification at the diagnos is of AML is considered an adverse risk for overall survival • Identical MLL rearrangements have been detected in three pairs of identical infant twins raising a possibility of in utero occurrence of MLL rearrangements. Using Guthrie card neonatal blood spots, evidence of the presence of malignant cells has been obtained for infants and young children who developed ALL and AML • There is no unifying mechanism to explain MLL translocations, deletions, amplifications, loss of heterozygosity, and microsatellite instability
AML with an Increased Number of Basophils and t(6;9) (p23;q34) • About 50% of patients with t(6;9) (p23;q34) show of basophilia in the marrow, i.e., > 1% of nucleated cells. The basophils appear to be morphologically normal. In some patients, darkly stained granules are also present in many eosinophils • The t(6;9)(p23 ;q34) is a rare cytogenetic abnormality, described for the first time in 1976, and subsequently reported in 1986 to be associated with AML and bone marrow basophilia. It is present in about 1% of patients classified as having M2, M4, or rarely Ml subtype of AML and a proportion of these patients show underlying myelodysplasia. t(6;9) has also been reported in refractory anemia with excess of blasts • Patients showing t(6;9) have a poor response to therapy and are usually young (25-30 years) at onset of the leukemia • The most common additional abnormality in these patients is trisomy 8 • Cytogenetic detection of the t(6;9) is difficult due to the exchange of chromosomal parts of almost similar length. As a result of the t(6;9), the 3' part of the CAN gene located on chromosome 9, band q34, is fused to the 5' part of the DEK gene located on chromosome 6, band p23. The resulting DEK-CAN fusion gene is on derivative chromosome 6 • The presence of the DEK-CAN fusion can be identified in blood or bone marrow cells by Southern blot analysis and PCR methods. The reciprocal CAN-DEK transcript on chromosome 9 has not been observed . DEK is a DNAbinding protein involved in transcriptional regulation and signal transduction
t(1;22)(p13;q13) and Acute Megakaryoblastic Leukemia (AMKL) • M7 FAB subtype shows diverse genetic and morphologic characteristics
338
Molecular Genetic Pathology
• The estimated frequency is 3-14% of AML and is more frequent in children than in adults. In adults , AMKL is frequently observed as secondary leukemia after chemotherapy or leukemic transformation of several CMPDs, including CML. Approximately 65% of AMKL are associated with myelofibrosis • There is no specific chromosomal abnormality associated with adult form of AMKL. Approximately 50% of AMKL patients have chromosomal abnormalities at diagnosis and diverse observed abnormalities include abnormalities 3q21-3q26, partial or total deletion of chromosomes 5 and 7, or gain of chromosomes 8 and 19 as well as t(9;22) • Adults with t(1;22)(pI3;qI3) encoding OIT-MAL fusion transcript has not been reported to date • In multivariate analysis, M7 AMKL diagnosis in adults is independent adverse progno stic factor for overall survival • In childhood AMKL three manifestations of M7 are observed: - t(1;22)(p13;q13) with M7 in children with constitutional trisomy 21 associated with GATA-l mutation . Children with constitutional trisomy 21 have 10- to 20-fold increased risk of developing leukemia. The incidence of developing M7 leukemia is up to 500 times higher in children with constitutional trisomy 21 than in children without it. However, children with constitutional +21 and AMKL have more favorable prognosis compared with patients without constitutional +21. Somatic mutation of transcription factor GATA-l in these patients leads to exclusive expression of truncated form of GATA-l - t(1;22)(p13;q 13) encoding OTT-MAL (RBMI5MKLl) fusion protein in infants without constitutional trisomy 21. t(l;22)(pI2;q13) is found in 22% of all cases of infantile M7 and in 33% of all cases of FABM7. Detection of t(1;22) is diagnostic and these patients have poor response to chemotherapy - Constitutional trisomy 21 children (or mosaic +21C) with transient myeloproliferative disorder who subsequently develop M7 leukemia. Approximately 19% of infants with transient MPD will develop M7 leukemia, at mean age of 20 months. With development of leukemia, these children acquire diverse chromosomal abnormalities, most notably, tetrasomy 21 and trisomy 8 • The t( 1;22) rearrangement was reported in a set of identical twins • Preliminary results, using gene expression profiling, provide s first insight into the molecular pathogenesis of M7 leukemia in children with and without constitutional trisomy 21. These two groups of patients were found to have distinct molecular phenotypes. Moreover, chromosome 21 genes had increased expression in patients with constitutional +21 when compared with M7 leukemia patients without constitutional +21. AMLl (RUNXl) gene localized on chromosome 21 and essential
12-37
FISH and Conventional Cytogenetics
for normal megakaryopoiesis, was expressed at lower levels in children with constitutional +21 and M7 leukemia, indicating mechanism that may contribute to a block in differentiation in AMKL
AML in the Elderly • Biology of AML changes with age • The spectrum of cytogenetic abnormalities in elderly involves higher percentage of patients with abnormalities involving -5/del(5q), -7/del(7q) and 17p and lower incidence of the translocations associated with favorable progno sis and treatment outcome • Moreover, multi-drug resistance in patients older than 75 was demonstrated in 57% while it was present in 33% of AML patients younger than 56. One of the explanation for different biology of AML in older patients may be related to the age of hematopoietic stem cell, telomere length, which tend to shorten with age and the presence of fewer normal stem cells to compete with malignant clone and to repopulate marrow following chemotherapy
Therapy-Related AML • The issue of tAML and therapy-related myelodysplasia (tMDS) following high-dose chemotherapy, radiation therapy, a combination of both, or autologous stem cell transplantation for malignant disease s has been the subject of many studies • From the cytogenetic point of view two different categories may be distinguished - Those related to medical exposure of alkylating agents who developed tAML about five years after therapy and are associated with the presence of monosomy 5/deletion (5q) or monosomy 7/deletion (7q). Many of these patients initially develop myelodysplastic features before transforming into a frank AML. These patients have a poor response to therapy - Patients with therapy-related AML without prior MDS phase often are characterized by l1q23 and 21q22 balanced rearrangements, attributed to late effects of topoisomerase II inhibitors combined with alkylating agents and radiation . The leukemia may develop within a few month s to on average three years . These patients have a more favorable prognosis
• Table 6 shows the set of probes currently used to detect most frequent therapy-related chromosomal abnormalities
Monosomy 5/del(5q) • Association of del(5q) with treatment was first reported by Rowley in 1977, who showed that patients with lymphoma who were treated with chemotherapy and/or radiation, developed myelodysplasia or acute myeloid leukemia. In therapy-related myeloid disorder s approximately 70% of patients have abnormalities of chromosomes 5 and/or 7, and approximately 22% have
Fig. 27. A partial karyotype from a patient with t-AML showing an interstitial deletion del(5)(q 15q32) (left). An interphase cell from the same patient showing two 5p15.2 signals (green), used as internal control, and one EGRJ signal (red) that is missing from the band q2l as a result of a deletion .
abnormalities of both 5 and 7 simultaneously. In contrast, in de novo AML, del(5q) occurs in 3-7%, although the frequency is increased in the elderly. At the Mount Sinai Medical Center, del(5q) was found in 29% of patients with de novo MDS and in 3.8% (22/576) of patients with de novo AML at the time of diagnosis (personal observations). However, in these patients it is associated with a favorable prognosi s (see Myelodysplastic Disorders section) • Data on 1432 patients with del(5q) showed a great heterogeneity in breakpoints. At least 42 different deletions have been described. Both interstitial and terminal breakpoints for ju st about every chromosome band has been reported. The heterogeneity of some breakpoints may be attributed to the difficulty in determining the exact breakpoints when chromosome morphology is of suboptimal quality • Although chromo somal regions 5q II , 5q 12, 5q21, 5q22, and 5q23 are frequently deleted, FISH mapping has demonstrated that 5q31 is deleted in most patients
(Figure 27) • The presence of del(5)(q31) is associated with a low tendency of karyotypic instability. Cryptic 5q31 deletion may be uncovered with FISH studies • The recurrent nature of -5/del(5q) caused many investigators to speculate that a tumor suppressor gene or genes may be located in the 5q22-23 or 5q31 band regions. The FISH method used to delineate the commonly deleted segment is currently estimated to be 300 kb in size on 5q31 .For many years a search for mutations in genes located in the chromosomal regions affected by del(5q) have been unsucces sful in part because no homozygous deletions have been detected and because 5q31 is very gene rich. Thus, rather than a typical tumor suppre ssor gene, happloinsufficiency or inactivation due to methylation may be involved
339
12-38
Molecular Genetic Pathology
Fig. 28. A partial karyotype showing a terminal deletion of chromosome 7, del(7)(q22) (left). A nuclei on the right was hybridized with CEP7 (two green signals) (internal control) and D7S522 DNA marker (red) on 7q31 band region. Only one red signal is present as a result of a terminal deletion.
leukemia, such as individuals with Fanconi's anemia, congenital neutropenia, neurofibromatosis type 1, Down syndrome, and Kostman syndrome, -7/del(7q) may be seen as the sole abnormality • One of the major advantages of I-FISH is the detection of abnormalities when conventional cytogenetic studies are uninformative. The best example is the detection of monosomy 7 in AML and primary MDS
Fig. 29. A partial karyotype from a patient with AML showing three copies of chromosome 8. Trisomy 8 in AML is associated with intermediate prognosis.
• Discordant results between cytogenetics and interphase FISH have been reported by several groups . Thus far, 58 of 222 (26%) reported patients with AML and de novo MDS had 5-67% of cells with monosomy 7, which was not detected by standard cytogenetics. An inability of myeloid cells carrying one copy of chromosome 7 to go into mitosis was demonstrated. This phenomenon is not restricted to the myeloid lineage and was detected in ALL as well • Deletions 7q are often interstitial and two regions, 7q22 and 7q33-34, have been reported as the most commonly deleted band zones
Monosomy 7/del(7q) • Loss of all of chromosome 7 (-7) or a deletion of the long arm of this chromosome [del(7q)] are observed in three conditions: - About 10% of patients with myeloid disorders including de novo AML and de novo MDS have -7/del(7q) (Figure 28). Monosomy 7 when seen in de novo MDS is remarkable because it is the only single chromosomal abnormality associated with an unfavorable prognosis (see Myelodysplastic Disorders section) - In therapy-related MDS or therapy-related AML, 52% have abnormalities of chromosome 7 - In pediatric patients with consitutional disorders associated with a predisposition to develop myeloid
340
• Similar speculations for del(5q) that a putative myeloid suppressor gene(s) may be located in the regions that are frequently deleted. Since prototypic tumor suppressor genes have not been identified in patients with either 5q or 7q deletions , an alternative explanation may be a haploinsufficiency concept, whereby the level of protein may be critical or a complex of two cooperating proteins may be affected as a result of inactivation due to methylation
Chromosomal Gain or Loss in AML • Approximately 15-20% of patients with AML show a numerical gain or loss of a single chromosome as the sole primary karyotypic abnormality. Each of the autosomes
FISH and Conventional Cytogenetics
and sex chromosomes contributes to the numerical changes • The most common trisomies in decreasing order of frequencies are gain of chromosomes 8, 22, 13,21, and II • The gain of chromosome 8, the most frequent abnormality seen in AML, was found in 5-6% of cases as an isolated abnormality and in up to 20% of all cases in association with other chromosomal changes . The incidence of +8 detected by FISH was reported to vary between 19% (31% of abnormal) and 25% (Figure 29). In a study of 447 newly diagnosed AML patients the overall incidence of trisomy 8, detected by FISH was 7%. Among the patients with an abnormal karyotype, trisomy 8 was detected in 25%. The prognosis of AML with +8 depends whether +8 occurs as an isolated abnormality or is accompanying other aberrations . In the latter situations, +8 does not appear to adversely affect the favorable outcome of patients with t(15; 17), inv(16)/t(16; 16), and t(8;2l). In contrast, patients with +8 and a complex
12-39
karyotype and/or such unfavorable aberrations such as del(5q) or -7, usually have very poor outcome. Isolated +8, has been considered to be associated with either intermediate or unfavorable prognosis
AML with Normal Karyotype • The largest group of AML patients, 48%, are presented with a normal karyotype. They are classified in the intermediate prognostic category because their survival probabilities are usually lower than those with t(8;21), inv(16)/t(16' 16), or t(15; 17) • The 5-year survival rates is between 24% and 42% • A small proportion, 4-9%, may have cryptic ETO-AML-I, PML-RARA, and CBFB rearrangements detected by FISH • Molecular analysis revealed distinct molecular aberrations in patients without detectable cytogenetic abnormalities and they include mutations of nucleophosmin (NPM) (62%) FLT3 (23%) MLL tandem duplications and CIEBP a (8%)
LYMPHOPROLIFERATIVE DISORDERS (LPD) Diffuculties in Obtaining Chromosomes FromLPD • There are two major difficulties affecting chromosome analysis in the LPDs - The small number of dividing cells and - The question relating to the origin of dividing cells This is particularly troublesome in MM and chronic lymphocytic leukemia (CLL), in which, most mitotic cells are derived from non malignant cell populations. The use of B cell mitogens and polyclonal B cell activators has increased the number of dividing cells, but these agents stimulate proliferation of malignant as well as non-malignant B cells. • Most but not all B-cell malignant disorders have translocations of the immunoglobulin (lGH) locus residing at 14q32 with multiple partner chromosomes • The rapid expansion of available probes makes FISH the method of choice for diagnostic ends because it enhances the detection rate, reveals the presence of cryptic translocations, and aids in characterization of ill-defined cytogenetic abnormalities
B-Lymphoid Disorders Acute Lymphoblastic Leukemia • A large number of specific chromosomal rearrangement and karyotype patterns in ALL are strongly associated with several clinical characteristics such as immunophenotypic features and treatment response (Table 13)
• Today cytogenetic analyses combined with FISH and/or RT-PCR investigations are now mandatory in most ALL treatment trials with genetic findings playing a pivotal role for proper risk stratification and treatment options • The frequency of abnormal karyotype in adult ALL is 64-85% and in pediatric ALL 60-69% of successful cases • The overall survival rate for adults is 22-38% whereas children have 75-80% survival rate with following risk categories: • Low risk: hyperdiploid and t(12;21)/TEL-AMLl • High risk: t(l; 19)1E2X-PBXl • Very high risk: t(9;22)/BCR-ABL and Ilq23/MLL rearrangements
Hyperdiploidy/Hypodiploidy • About 20% of children and 26% of adults with B cell ALL have a hyperdiploid number of chromosomes • Two groups can be distinguished: - Those with 51-55 chromosomes whose prognosis is poorer - Those with 56-67 chromosomes. Whose prognosis is better • Both of these groups have a more favorable prognosis than children with hypodiploidy or near-haploid ALL. In a study of 1880 children with ALL, patients with 45 chromosomes have an outcome similar to that of ALL patients with pseudodiploid or low hyperdiploid (47-50 chromosomes)
341
~
w
!'..)
11-29%
0-3% 6-30% 4-6%
10-12% 5- 7% (26% in T-ALL)
NA
NA
BCR-ABL
MLL-MLL T2
TCF3-PBXl
TEUETV6-AMLl
Pl 6 (CDKN2A,MTSl)
TEU ETVl
Not known
Not known
Not known
NA
TCRa
TRD-TLX l
Pseudodiploidy
hypodiploidy (35-44)
t(9;22)(q34;q11.2)
t(4;1l)(q21;q23)
t(l ;19)(q23; pI 3.3)
t(12;21)(p 12;q22)
Abnonnal9p
Abnonnal 12p
del(6q)
del(7p)/d el(7q)/-7
del(5q)
Trisomy 8
14qll
t(IO;14)(q24 ;qll)
CEP indica tes centrome re enumeratio n probe ES indicates extrasensitive p robe strategy OF indica tes double fusion probe strategy BA indicates breakapart probe strategy *M od ified from M rozek et al. 2004
4-9%
NA
Near haploidy(<35 )
2-3%
<2%
6-11%
3-6%
2-3%
3-7%
3 1- 50%
Rare
10-15%
NA
Low hyperdiploidy (>50)
Excellent
Excell ent
Poor
Not prognostic
Not progn ostic
Not prognostic
Favorable-unfavorable
Intermediate-
Not known
Poor
Poor
Poor
Poor
Poor
NA
Good
Good
2- 11%
NA
High hyperdiploidy (>55)
Intermediate-Good
Prognosis
15-36%
Adults freque ncy
NA
Genes in volved
Normal karyotype
Cytogenet ic abnormality
Not known
NA
Not Prognostic
Not known
2% 3-4% (17-22% in T-ALL)
Adverse
Adverse
Not Prognostic
Not Prognostic
Adverse
Good
Poor
Poor
Poor
Poor to Intermediate
Intermediate
TCRBA
TCRBA
CEP 8
CEP5IEGRI
CEP717p1217q31
C-MYB in some cases
TEUETVl BA
CEP9/9p21
TEUETV6-AMLl ES
1p36, !q25,19p, 19q
MLLBA
BCR-ABL ES, BCR-ABL DF
NA
NA
NA
Yes (CEP4, CEPIO, CEPI7)
Intermediate Poor
Yes (CEP4, CEPIO, CEPI7)
None
Available clin ical FISH probes
Good
Intermediate-G ood
Prognosis
1%
4%
6-9%
7-9%
7-11 %
20-25 %
4-5%
2%
2-6%
6%
18-26%
1-4%
10-1 1%
23-26%
3 1-40%
Children frequency
Table 13 . Frequencies of Cytogenetic Aberrations in Adult and Childhood ALL and Their Prognostic Relevance and the Availability of FISH Probes for Their Detection
o oc -c
o
::::r
;s:' ......
n
CJ ..... CJ ro ::l ro ......
c
n
$: o ro
o
~
I
!'..)
FISH and Conventional Cytogenetics
12-41
cancer. Children with the TEL-AMLl fusion gene have significantly lower rates of relapse when compared with TEL-AMLl-negative patients . TEL-AMLl-positive B-precursor ALL is characterized by a long duration of first remission, with the vast majority of relapses occurring off-therapy, and excellent cure rates • The presence of TEL-AMLl fusion appears to confer a favorable prognosis and improved survival for ALL patients treated with intensive therapy
Fig. 30. A partial karyotype showing chromosomes 12 and 21 obtained by conventional cytogenetics, destained and subjected to in situ hybridization and counterstained with DAPI (blue) . The probes used in situ hybridization were TEUETV1 (green) and AMLl. Note that TEUETV1 (green) is hybridized to 12pl3 and AMLl is hybridized to 22q22 (red). In contrast, the interphase nuclei (left) from the same patient shows TEUETVl-AMLl fusion, indicating that metaphase cells were not ALL-derived. A normal karyotype may reflect other normal hematopoietic cells present in the bone marrow from patients with ALL.
• Gain of chromosomes 4, 8, 10, 17, 18, and 21 as well as other chromosomes have been identified • Trisomy 21 is the most common numerical change in ALL with an incidence of 1-2% • Loss of chromosome 7 is frequent in adult ALL. The majority of these patients have t(9;22) as well • In contrast to children who have a favorable prognosis when a hyperdiploid karyotype is present, such a favorable constellation has not been found in adult ALL. The reason may be due to the fact that adult patients often have poor-risk chromosomal translocations, such as the Ph rearrangement, which confer a poor outcome irrespective of otherwise good-risk ploidy group. Modal chromosome numbers of 45 or less are rare, specifically the near haploid numbers of 24-36, but they do occur in <1% and confer a poor prognosis
t(12;21) (p13;q22) • In childhood ALL, the t( 12;21), as determined by conventional cytogenetics, is difficult due to the fact that the translocated portions of l2p 13 and 21q22 have virtually identical G-banding patterns making the translocations indistinguishable • In contrast, using PCR or FISH, the TEUAMLl (ETV6/CBFA2) fusion gene, the molecular consequence of t(l2;2l) is detected in 19-29R of pediatric patients with ALL (Figures 30) • The TEL-AMLl fusion, found almost exclusively in patients age 1-12 years with B-precursor ALL, represents the most frequent molecular rearrangement in childhood
• In adult ALL the TEUAMLl fusion is rare • The t(l 2;2 l)(p13;q22) fuses the helix-loop-helix domain of the TEUETV6 gene, located on 12p13 to the DNAbinding and transactivation domain of the AMLl gene, located on 21q22. The fusion gene can be visualized on 21q22 • The TEL-AMLl fusion is frequently accompanied by the loss of the other, normal, unrearranged TEUETV6 allele, and most likely, this deletion represents a subclonal evolution • The TEL-AMLl fusion gene dimerizes both with itself and with normal TEL in vitro. Fusion with TEL converts AMLl from an activator to a repressor of transcription. In addition, homozygous disruption of AMLl in mice results in a lack of definitive hematopoiesis, demonstrating a crucial role for this protein in blood cell development • In a rare group of patients with B-precursor ALL there is no fusion of TEL and AMLl. Instead bone marrow cells from these patients show 3-15 copies of the q22 band of chromosome 21, including the AMLllocus (Figure 31). These patients have apparently a normal karyotype and multiple copies of the q22 band of chromosome 21 are localized on chromosome 21. However, the number of copies is below the resolution of conventional cytogenetics. In these patients amplification of the 2lq22 band of chromosome 21 is a clonal marker of the leukemic cells
Other Abnormalities Involving 12p and TEUETV6 • Rearrangement involving 12p include deletions, duplications, and translocations and they are most often observed as a part of the complex karyotype, frequently associated with chromosome 5 and/or 7 abnormalities • Deletion of 12pl3 is much more frequent in children than in adults with ALL and it is also observed in patients with myeloid disorders • Two other genes residing on l2p were found rearranged: CCND2 is most frequently found amplified and CDKN 1B is commonly found deleted • TEL (for 'translocation, ETS, leukemia" or ETV6) gene, most frequently found in translocations, was first identified as a part of a TEL-platelet derived growth factor receptor ~ fusion (TEUPDGFRB) created by the t(5;12)(q33 ;pI3) in chronic myelomonocytic leukemia . It was detected using FISH because , as mentioned before, there are difficulties in detecting cytogenetic rearrangements at the 12p13 site. As a result of this
343
Molecular Genetic Pathology
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Fig. 31. Interphase nuclei from a child with B-cell precursor ALL showing multiple copies (amplification) of AMLl gene (red) and disomy for TEL/ETV] (green).
translocation the HLH domain of TEL is fused in frame to the PFGFRB transmembrane and tyrosine kinase domain. The fusion of TEL to a tyrosine kinase also occurs as a result of the t(9;12)(q34 ;p13), which has been observed in patients with AML, atypical CML, and ALL • The second group of TEL rearrangements involves fusion of 5' TEL to 3' part of the MN] gene located on 22q 11 in t02;22)(p13;q11). The MN1-TEL fusion contains almost all of MN] fused to the ETS domain of TEL
• TEL function in mice is essential for the establi shment of hematopoiesis of all lineages in the bone marrow. The exact role of TEL in leukemogenesis awaits elucidation because of diverse involvement of its functional domains
t(9;22)(q34;qll.2) • Approximately 1% of children and 15-30% of adults with ALL have the Ph chromosome, making it the most common structural rearrangement in adult ALL • In 50% of adult ALL and in 80% of childhood ALL, the breakpoint on 22 involved in the Ph translocation is more 5' than in CML and falls between exon el and e2 of the BCR gene , which is fused on deleted 22 with ABL gene, normally located on 9q34 on the Ph chromosome • The breakpoints within the ABL gene are scattered over a 300 kb intron, but are usually 5' of ABL exon a2 • t(9;22) , as mentioned in the section on CML , results in a chimeric mRNA and expression of a hybrid p190 BCR-ABL protein , providing a diagno stic distinction between lymphoid blast crisis of CML and de novo ALL • Prognosis in both Ph+ children and adults is very poor and identification of BCR-ABL positive leukemia, particularly in Ph- cases (see Chronic Myelogenous Leukemia section) is critical for more intensive therapies • As shown in Figure 32, I-FISH with the extra-sensitive probe may distinguish the ALL-type of BCR-ABL fusion
344
Fig. 32. A composite of two interphase nuclei after in situ hybridization with BCR-ABL-ES probe. The cell on the left is from the patient with Ph+ CML and the cell on the right is from the patient with the Ph-- ALL. The CML cell shows two copies of equal size of the BCR-ABL fusion, consistent with the blast cri sis of CML and duplication of the Ph chromosome. In contrast, the ALL cell shows one large BCRABL fusion and one, what appear s either a smaller BCR-ABL fusion or a very close localization of a smaller BCR and ABL signals. The breakpoint on the BCR in ALL is more centromeric than in CML and the ES strategy allows for distinction between the CML blast crisis and the ALL-type of the BCR-ABL fusion. producing P190 BCR-ABL chimeric protein from the CMLtype producing the P210 BCR-ABL protein • Approximately 16% of Ph+ ALL show monosomy 7 as an additional karyotypic change • Some Ph+ ALL patients may also exhibit trisomy 9 and in a fraction of these patients the BCR-ABL fusion takes place on der(9) rather than the usual 22q 11.2 site • Other additional abnormalities include gain of chromosome 8 and X, duplication of Iq, and hyperdiploidy but the outcome reported was no different except patients with monosomy 7 and deletion 9p have a particularly poor outcome
t(l;19)(q23;p13.3) • The t( 1;19) occurs in 5-6% of patients with childhood ALL but among patients with a pre-B (cytoplasmic immunoglobulin positive) immunophenotype to; 19) it is found in approximately 25%. In adults, the t(1;19) occurs in 2-3 % • More than 95% oft(1;19) creates the TCF3 (E2A)-PBX] fusion gene. The TCF3 gene (originally identified by the binding of E2A proteins to the kE2DNA sequence motif contained in the Ig kappa light-chain enhancer) on chromosome 19 is fused to PBX] (homeobox) gene on chromosome 1. The breakpoint occurs in restricted regions of E2A, in a 3.5-kb intron segment. The breakpoint region on chromosome Iq23 appears to be more dispersed and lies within an intron of at least 50 kb
12-43
FISH and Conventional Cytogenetics
• The E2A-PBXl hybrid gene is a transcriptional transactivator in vitro and induces lymphoma in transgenic mice • The t(l ;19) also occurs in approximately 1% of early B-cell progenitor cases . In contrast to patients with pre-B cell phenotype, in patients with cytoplasmic negative-early B cell phenotype, neither E2A nor PBXl is involved and their prognosis is excellent without the need for intensified therapy • In children with ALL and Ig-positive cells, early detection of t(l ;19) is crucial as such patients may benefit from more intensive therapy. However, it should be realized that results were not concordant using PCR detection of minim al residual disease between bone marrow and peripheral blood cells in children whose cells were t(l ;19)/E2A-PBXl fusion positive. Moreover, at the end of consolidation therapy, 28% of these patient s remained positive. Therefore, the use of PCR for detection of E2X-PBXl fusion is valuable for diagnosis but of questionable significance as regards detection of minimal residual disease • Approximately 3% of adult patients with ALL show t(l;19) as well as a variant t(l7;19)(q21-2;pI3). In contrast to children, 19p13 rearrangements in adults appear to fall into a standard-risk category
Fig. 33. A partial karyotype from a child with ALL showing chromosomes 11 and 19 (top row). Conventional cytogenetics could not positively identify t(1I;19)(q23 ;pI3). When chromosomes were destained from G-banding and subjected to in situ hybridization with the MLL probe using "break-apart" strategy, the MLL rearrangement was obvious since the 3' end of the gene (red) was positively identified on 19p13 (red) while the 5' of the locus remained on chromosome II (green).
Rearrangements of 11q23 • The 11q23 rearrangements were discussed in more details in the myeloid section. About 7% of adult ALL show II q23 rearrangements • The most frequently observed 11q23 and MLL translocations in adult ALL include t( 1;11)(p32 ;q23), t(4;11)(q21 ;q23), t(l0;11)(pI2-14;q23), and t(11;19)(pI3;q23) (Figure 33). The most common IIq23 abnormality is t(4;11), occurring at the rate of 3-6%, is associated both in children and adults with poor outcome
t(8;14)(q24;q32) in Childhood ALL • The t(8;14)(q24;q32) is seen in fewer than 5% of all ALL (children and adults) • Variant translocations, t(8;22)(q24;qll) and t(2;8)(pI2;q24), are seen in less than 1% of children and adults • These patients have CDlO+, CDI9+, CD20+ , surface IgM+ immonophenotype and a poor prognosis. The same translocation is found in Burkitt's lymphoma and most likely both entities represent the same disease with different manife stations. (see Burkitt's Lymphoma)
Abnormalities of (9)(p21-22) • Abnormalities of 9p, either deletions or translocation s are reported to occur with a frequency of 7-13 % with no apparent difference in childhood versus adult ALL • The most frequent deletion s include the 9p21 region, with co-deletions of two gene s, plS INK4B and p16 INK4A, as
well as the Interferon a and not all cases
Bgenes found in many, but
• Among the structural rearrangments involving the short arms of chromosome 9, t/dic(9 ;12)(pll-12;pll-13) is a rare recurrent abnormality associated with L I morphology (FAB classification), pre-B cell phenotype, and excellent prognosis
Other Abnormalities • Other chromosomal abnormalities detected in nonrandom fashion in adult ALL include deletion s, both terminal and interstitial, of the long arm of chromosome 6, and isochromosomes of7q, 9q, 17q, and 21q • Overall, patients with adult ALL showing t(9;22), t(4;11), t(8;14), -7, and +8 chromosomal aberrations have a poorer progno sis and significantly lower probability of long-term complete remission and survival than patients with a normal karyotype or patients with other chromosomal rearrangements
Chronic Lymphocytic Leukemia Conventional Cytogenetics in CLL • CLL is a clonal disease arising in a progenitor cell after the B cell pathway has diverged from the myeloid and T cell pathways • About 40-50% of patients have clonal abnormalities identified by conventional cytogenetics. This may be an underestimate because chromosomal changes may occur
345
12-44
Molecular Genetic Pathology
Table 14. Set of Probes for Detection of the Most Frequent Chromosomal Abnormalities in Cll
Probes
Chromosomal abnormality
Incidence %
Dl3S319
del(13)(qI4.3)
36-54
Good
132
Dl2Z1
trisomy 12
14-19
Intermediate
114
P53
del(l7)(p13.1)
6-16
Unfavorable
32
ATM
del(lI )(q22.3)
11-20
Unfavorable
78
C-MYB
del(6)(q23)
>5
?
?
Prognosis
Median survival (months)
Fig. 34. Prognostic risk groups in CLL based on FISH clinical test results. The top part of the diagram shows chromosomal abnormalities and their frequencies, in descendent order, from the most aggressive to the least aggressive. The bottom part of the diagram shows the frequencies of the chromosomal abnormalities identified with FISH technology at the Tumor Cytogenetics laboratory at the Mount Sinai Medical Center. The median survival of patients with these abnormalities is presented at the bottom row. in one or more cell subsets of the malignant clone . The classical cytogenetic analy sis of B-CLL has remained difficult because of the low mitotic yield of neoplastic B cells, despite the use of B cell mitogens. With the combination of immunophenotyping and cytogenetics it was demonstrated that normal metaphases obtained from BCLL specimens were often derived from non-clonal T cells
FISH in eLL • CLL is the first disease in which chromosome abnormalities detected by FISH established for the first time their prognostic significance (Table 14 and Figures 34 and 35) • Four genomic aberrations, which are independent predictors of disease progression and survival, include deletions of: - Long arm of chromosome II - Long arm of chromosome 13
346
- Short arms of chromosome 17 - Trisomy for the long arm of chromosome 12, as well as a normal karyotype
Deletions of I3q • The frequency of del(13q) by conventional cytogenetics is 10-15%. However, with the application of FISH smaller or larger deletions of band q 14 was found in up to 55% of patients over time. When multiple DNA probes , covering three different regions of band q 14 of chromosome 13 were used, the DNA markers D 13S25, located 1.6 centimorgans telomeric from RBI , and Dl3S3l9 located between RBI and D l3S25, are deleted more frequently in B-CLL than RBI • Molecular analy ses have detected deletions of 13q in cells, which had cytogenetically normal as well as abnormal 13q. Southern blot analy sis of highly purified malignant cells showed that the loss of this region may occur in >95% of malignant cells
FISH and Conventional Cytogenetics
12-45
• Follow up analysis over a 4-year period demonstrated a clonal expansion of cells with trisomy 12 as the disease progressed. These observations suggest that trisomy 12 might be relevant in the cell activation process in CLL • The survival of patients with trisomy 12 as assessed by FISH is 114 months compared with III months for patients with a normal karyotype • A long-term follow up of patients with +12 as assessed by FISH found no statistical significant difference between patients with and without chromosome 12 rearrangements after a median observation of 87 months
Fig. 35. Interphase nuclei after in situ hybridization utilizing the CLL kit from Vysis (Abbott Molecular). The kit includes probe s forCEP12, LAMPI at 13q34,andD13S319at 13qI4.3 (left), and ATM at llq22.3 and P53 (right). Note deletion of ATM locus in the right nucle i associated with unfavorable prognosis in CLL. • Homozygous deletion of D13S25 and/or D13S319 DNA segments from 13q14.3 band region is rare but consistent finding in a subset of patients with B-cell CLL • The 13q14,3 deleted segment contains a non-transcribed gene and two micro-RNA genes . Micro-RNA is made normally by cells, including B lymphocytes, and regulates the function of many genes . Two micro-RNA genes located in 13q14 region are downregulated or deleted in most cases of CLL • Trisomy 12 or 13q rearrangements are found separately in a substantial proportion of patient s with CLL. They co-exist in only 2-5% of patients suggesting that each change may have a distinct pathogenetic route • The presence of del(l3q) as the sole abnormality in CLL is associated with the most favorable prognosis, with median survival of II years
Trisomy 12 • Historically, trisomy 12 was reported as the first recurrent abnormality in CLL. It is detected by classic cytogenetics in 7-15% • FISH assessment of + 12 is reported in 15-20% of patients with CLL who frequently have atypical morphology and phenotype. FISH studies are more representative of the true incidence of trisomy 12 because the interphase FISH methodology provides information in cells independent of their cycling status. Trisomy 12 may be present as the sole abnormality or in combination with other chromosomal rearrangements. Since only a proportion of cells are trisomic, normal cells or disomic neoplastic cells are also present • Minimally duplicated segment is 12q13-q14 • The prevalence of trisomic cells varies between individual patients from 6-70%
• The observation that trisomy 12 was documented in B cells and was absent from T-Iymphocytes and CD34 + cells in the majority of patients is consistent with the original hypothesis that CLL has a clonal origin and that trisomy 12 arises in a progenitor cell already committed to the B cell pathway
Deletions of 11q • The frequency of deletions of the long arm of chromosome II in CLL as detected by conventional cytogenetics has been reported to be 58% • An interphase FISH study identified a clinical subset of B cell CLL defined by deletion of Ilq22.3-23.l in 7-10% of B cells • Among 325 patients evaluated by FISH, 33% had a 11q22-23 deletion . Their median survival was 6.5 years • FISH characterization of aberrations involved in 11q21-q23 demonstrated a minimal concensus deletion segment of 2-3 Mb, containing a number of genes including ATM at Ilq22.3. Some patients (up to 12%) have simultaneous deletion s of ATM and MLL at Ilq23 • The ATM gene is responsible for ataxia-telangiectasia and functions as a cell-cycle checkpoint regulator. Somatic disruptions of both alleles of the ATM gene by deletion or point mutation have been detected in approximately 34% of cases. This strongly suggests the pathogenic role of ATM in some patients with B CLL. A recent study revealed a discontinuous deletions at llq23.1-q23.3 indicating that genes in this region may have pathogenic significance because they constitute independent targets for amplification or deletion in cases of B-CLL • Patients with llq deletion exhibit extensive lymphadenopathy, a more rapid disease progres sion, a shorter treatment-free interval, inferior molecular remission, and reduced overall survival
del(l7)(p13.I) • Structural aberrations of chromosome 17 were observed in 4% of cytogenetically evaluable B-CLLs . This abnormality frequently affects the short arm of chromosome 17, where the TP53 tumor suppressor gene is localized in 17p13
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Molecular Genetic Pathology
Table 15. Set of Probes for Detection of Chromosomal Abnormalities in Multiple Mueloma and MGUS Frequency (%) Chromosomal abnormality
MM
MGUS
Prognosis
t(II;14)(qI3 ;q32,3)
16
15-30
Favorable
D13S319
del(l3)(qI4.3)
30-55
25-50
Intermediates/poor"
P53
del(l7)(p 13.1)
10
FGFR3-IGH
t(4;14)(pI6;q32.3)
2-15
15
Poor
IGH-MAF
t(14;16)(q32;q23)
2-5
5
Poor
Trisomy for 5,9 and 15 hyperdiploidy
40-55
40
Good
Probe CCBDlIMYEOV-IGH
5pI5.2, CEP9, CEP15
Poor
alf present alone without IG rearrangements or detected by I-FISH alone blf detected by conventional cytogenetics or metaphase FISH
• Deletions of 17p13 are detected by I-FISH in 7-20% and they are determined to be monoallelic deletions of TP53 • Deletion of TP53 is the strongest prognostic factor in B cell CLL and survival of patients with TP53 deletion is significantly shorter than that of other patients. The median survival time of patients with 17p13 deletion is only 32 months • B-CLL patients with deletion of the P53 gene are associated with refractory advanced disease and resistance to treatments and shorter survival. Therefore, alteration or deletion of TP53 provides a biological marker predictive of the clinical behavior and treatment response in B-CLL • Fewer than 5% of patients with CLL/prolymphocytic leukemia (PLL) morphology have t(11 ;14)(qI3;q32) and they usually transform into pro-lymphocytic leukemia
Multiple Myeloma Plasma Cells • MM is a malignancy of terminally differentiated B cells , which have a very low proliferation rate. This characteristic of plasma cells is the reason why cytogenetic studies of MM have been so limited until recently. The clinical course of patients with MM is best predicted by cytogenetic abnormalities obtained either by conventional cytogenetics or by FISH • Conventional cytogenetic analysis was able to detect abnormal karyotypes in only 25-30% of newly diagnosed patients, mostly in those cases with exceptionally high plasma cell proliferative rate, and recurrent chromosomal translocations were identified • When reassessed with the interphase FISH method, using a large panel of centromere-specific and
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translocation-specific probes, interphase plasma cell nuclei revealed chromosomal aneuploidy in up to 96% of the patients. Although virtually all chromosomes were found to have numerical changes, trisomy 9 was found in 52-82% while trisomy for lq was detected in 36-38%. Even during complete clinical remissions 12-71 % of cells still show a numerical gain or loss of chromosomes 3, 7, 8, 9, 11, 13, 15,21 , and X • Table 15 shows the panel of probes currently used to detect structural and numerical rearrangements in MM and monoclonal gammopathy of unknown significance (MGUS) as well as their frequencies and prognostic impact. Sufficient information is now available to result in strong recommendation for the adoption of routine molecular cytogenetic testing in myeloma patients. The strategy we employ at the Mount Sinai Medical Center in New York for patients at diagnosis is the panel of 7 probes: CCNDl-/GH, DJ3S319, P53. 5pI5.2, CEP9, and CEP15 (Figure 36). If the IGH locus is rearranged the two other translocation partners are also used in interphase FISH study to determine the exact fusion partner
del(13)(q14.3) • Interstitial deletion of either band q14 or q 14.3 (DNA marker D13S3/9) of chromosome 13 is found in 30-55% of patients with karyotypic evidence of monosomy or del(13) but also in patients with a normal karyotype (Figure 37A)
• It is associated with specific clinicopathological features , including a higher frequency of lambda-type MM , high plasma cell labeling index, female predominance, and inferior survival after standard chemotherapy. In patients with a deletion of 13q14, myeloma cell proliferation is markedly increased when compared with patients without this deletion
FISH and Conventional Cytogenetics
5p 15.2 CEP9 CEP15
11q1314q32
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13q14317p13.1
CCNDHGH
4p 163
14q32
14q32 16q23
FGFR3-IGH
IGH-MAF
Fig. 36. A diagram of chromosomes and chromosomal probes used in clinical testing for detection of numerical and structural rearrangements in patients with MM. For detection of numerical abnormalities, D5S7211D5S23 localized on 5pI5.2, CEP9, and CEPl5 probes are used . Probes D13S319 localized to 13q14.3 and P5310calized to 17pl3.l are used for detection of deletion . Other indicated probes are used for detection of three IGH rearrangements.
• Until recently, detection of the 13q14 deletion was thought to be an independent adverse prognostic feature associated with a significantly lower rate of response to conventional therapy and shorter overall survival. In a study of 1000 patients , individuals who had a deletion of 13q proved to have incurable disease whereas those without chromosome 13 abnormalities had durable complete remissions • The adverse prognostic impact of de1(l3q) detected by conventional cytogenetics vs I-FISH remains controversial. In a group of 118 points overall survival with monosomy 13/del(13q) by conventional cytogenetics and by FISH was not statistically different. In contrast , de1( 13q) alone or in combination with hypodiploidy was associated with shortest event-free survival among 231 points treated between 1989 and 1994. Moreover, the authors strongly recommended metaphase karyotyping as a part of the staging of all patients with MM at both diagnosis and relapse. This view is supported by recent comparative analysis, using conventional cytogenetics, metaphase, and I-FISH analysis in 154 evaluated patients . Interphase FISH revealed 86% of patients to have an abnormal clone, yet survival differed among patients depending on whether certain chromosome abnormalities were detected both by metaphase and interphase cells or only in interphase nuclei . Detection of chromosome
13 abnormalities in metaphase cells was associated with poor prognosis, while in contrast, chromosome 13 anomalies detected in interphase nuclei had an intermediate survival • Because presence of IGH translocations is subtle by conventional cytogenetics, metaphase FISH demonstrated that of all patients with chromosome 13 abnormalities, 57% by metaphase and 39% by interphase FISH had IGH and or/del( 17p) abnormalities. Therefore, a possibility still exist that IGH rearrangements but not de1(l3q), confer a worse prognosis and del(l3q) per se may not be associated with poor prognostic impact
t(l4q32.3) Involving 1GB Loci • In most MM patients IGH rearrangements on 14q32.3 consists of complex and heterogeneous translocations with the breakpoint involving either the switch region of IGH or the V, D, or J gene. The primary translocations are due to somatic hypermutation or errors in VDJ portion of the switch region recombination. The translocations include a promiscuous array of at least 20 non-random chromo somal partners and the characterization of these translocations has led to the identification of critical dysregulated oncogenes (e.g., BeL2, Cyclin D). In each translocation a potent enhancer is juxtaposed to a dysregulated oncogenes
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Molecular Genetic Pathology
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Fig. 37. (A) An interphase nuclei showing deletion of Dl3S319 at 13q14.3 band region (red), while other tested loci LAMPI at 13q34 (green) and RBI (aqua) at 13q14 were present in disomy (normal copy number) (B) A partial FISH karyotype of two cells from a MM cell line showing t(4;14)(pI6.3;q32.3). A normal chromosome 14 with IGH gene on 14q32.3 and der(l4) showing part of the FGFR3 gene, which is normally located on 14pI6.3. (Kindly provided by Dr. Bergsagel PL, Cornell Medical Center, New York, now Mayo Clinic, Arizona.)
• In patients with a recurrent CCNDI-IGH there is an overexpression of cyclin D I . In contrast to mantle cell lymphoma, breakpoints on II q 13 in MM are not clustered but are scattered over relatively large genomic region. Therefore , the CCNDI-IGH probe for MM involves another gene, MYEOV, and it is distinct from CCNDlIGH probe used for detection of t( II; 14) in mantle cell lymphoma. Survival and response to therapy of patients with CCNDI-IGH fusion is either borderline improvement or neutral. In contrast to other abnormalities involving IGH locus, clonal t(lI ;14) MM cells tend to be diploid • Patients with recurrent t(4;14)(pI6.3;q32.3) abnormality have dismal outcome even after the stem cell therapy • Two genes on chromosome 4p 16.3 and the IGH switch region on 14q32.3 are involved. The fibroblast growth
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factor receptor 3 gene (FGFR3) is detected on der(l4) where it is overexpre ssed along with a fusion of the MM set domain (MMST) gene, located on 4pI6.3. This is the first example of a translocation that simultaneously dysregulates two gene s with oncogenic potential , the FGFR3 gene detected on der(l4), and the MMSET gene detected on der(4 ); FGFR3 is 50-100 kb telomeric to MMSET
• Clinically there is an association between t(4;14), IgA subtype, A light chain , and immature plasma cell morphology • Translocation (14;16)(q32 .3;q23) (Figure 37B) had never been cytogenetically detected, because of the telomeric positions of both loci. However, with FISH studies the incidence of t(l4; 16) has been estimated to be
FISH and Conventional Cytogenetics
approximately 2-5%. In this translocation the MAF proto-oncogene is translocated from its normal position on 16q23 to chromosome 14, band q32.3 and is overexpressed
del(l7p 13.1)IPS3 • P53 is a powerful independent predictor of shortened survival in MM and is associated with stage III disease, clonal evolution of disease , drug resistance, and genetic instability
Aneuploidy • Analysis of numerical abnormalities revealed two broad groups of patients with MM: hyperdiploid and nonhyperdiploid. The hyperdiploid MM is associated with trisomies for chromosomes 3, 5, 7, 9, II, 15, 19, and 21 and they harbor less IOH translocations. The nonhyperdiploid MM is characterized by a very high presence of IGH translocations (>85%). Both these groups are also present in MODS, suggesting that they occur early on in the evolution of disease • The molecular basis of MM is slowly emerging but the role of these oncogenes is yet to be defined . In order to address this issue and to understand the molecular bases of aneuploidy, recent work evaluated the role of centrosome in plasma cell neoplasm. Preliminary evidence indicated that centrosome amplification is common in all stages of plasma cell neoplasm, including MODS and is probably integral to disease pathogenesis and genomic instability of MM
Hairy Cell Leukemia (HCL) • No abnormalities specific for HCL have been reported • Several abnormalities seen in other B-cell malignancies have been identified in HCL. These primarily consists of rearrangements involving the IGH locus at 14q32.3 such as t(14;18)(q32.3;q21). Deletions of 17p13, the site of the TP53 gene, have also been observed with relatively high frequency both in HCL and HCL variants. Abnormalities of chromosome II and overexpression of the CCNDI gene at II q 13, which encodes Cyclin D is found in 70% of patients
B-cell Prolymphocytic Leukemia • Similar to other B-cell LPDs, t(1I;14)(qI3 ;q32.3), trisomy 12, and structural rearrangements of I7p, most notably, i(17q)(q I0) have been reported . Perhaps, more important, is the observation that abnormalities of P53 at 17p13, have been documented in 75% of patients with B-cell prolymphocytic leukemia
Non-Hodgkin's Lymphomas (NHL) Immunology and Genetics • NHL comprise a heterogenous group of disorders characterized by localized proliferation of lymphocytes.
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Analogous to other hematopoietic disorders the pathogenesis of these malignancies is attributable to a multi-step process involving a progressive and clonal accumulation of genetic lesions. The majority of NHL are of B cell origin and involve translocations of immunoglobulin loci as shown in Table 19. IGH translocations are usually detected by cytogenetics, often in conjunction with FISH, using probes that span the IGH loci. These translocations exhibit enormous complexity with multiple and complex translocations, deletion s, and amplifications within one clone • Table 16 and Figure 38 show the frequency of recurrent chromosomal abnormalities associated with NHL and FISH probes available for their detection
Follicular Lymphoma • Approximately 80% of patients with follicular lymphoma and some patients with large cell lymphoma show t(14; I8)(q32.3;q21.3), which results in the fusion of BCL2 on 18q21 and IGH on 14q32 (Figure 38A). The breakpoints within BCL2 occur mostly within the 3' region of the gene while the breakpoints in IGH fall within the DH and J H regions. The exact underlying mechanism is not known but t(14;18) results in a dysregulated expression of BCL2 protein, one of the proteins involved in the regulation of apoptosi s. The exact formation of t(14; 18) appears to be more complex and includes V(D)J recombination mediating breaks on chromosome 14 and another undefined mechanism creating initial breaks in chromosome 18 • Although 75% of breakpoints are clustered within a remarkably narrow region of 15-20 bp at the 3' end of the BCL2 gene they are frequently missed by standard PCR. With regular and fiber FISH methods, using BCL2 breakpoint flanking probes, individual 5' and 3' breakpoints can be detected. BCL2 rearrangements are detected in 100% of patients with follicular NHL and this is well correlated with immunohistochemical staining for BCL2 protein
Burkitt's Lymphoma • Three translocations, all affecting the MYC gene at 8q24 have been recognized. In 80% of patients with Burkitt's lymphoma, a reciprocal translocation t(8;14)(q24; q32) is observed between the MYC gene and the IGH locus, while in the remainder, the reciprocal translocations t(8;22)(q24;qll) or t(2;8)(pI2;q24) are observed juxtaposing MYC to one of the light chain loci (kappa on 2p 12 and lambda on 22q II) (Figure 38B) • This was originally described in 1972 in Epstein-Barr virus (EBV)-positive tumor cells obtained from patients in Africa. Variant translocations involving MYC with a variety of other non-IG loci have since been reported • In most patients with sporadic Burkitt lymphoma the breakpoints on 8q24.1 are located 5' of the coding region of the MYC gene. In contrast, in most cases of endemic
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Molecular Genetic Pathology
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Table 16. Set of Probes for Detection of Chromosomal Rearrangements in NHl Probe
NHL
Chromosomal abnormality
Frequency
IGH-BCL2
Follicular
t(l 4;18)(q32;q21.3)
80%
MYC-IGH
Burkitt
t(8;14)(q24;q32.3)
80%
MYC, J(
Burkitt variant
t(2;8)(p12;q24)
Variants combined
MYC, A.
Burkitt variant
t(8;22)(q24;qll)
20%
BCL6
DLCL
3q27
35%
CCNDI-IGH
Mantle cell
t(lI ;14)(qI3 ;q32)
100%
API2-MALTI
MALT
t(lI ;18)(q21 ;q21.1 )
20-40 %
IGH-MALTl
MALT
t(l4;18)(q32;q21.1)
Infrequent
MALTl
MALT
t(l ;14)(p22;q32)
5%
ALK
ALCL
t(2;5)(p23;q25)
50-70%
DLCL: diffuse large eel/lymphom a; ALCL: anaplastic large eel/lymphoma; MALT: Extranodal marginal zone B-cel/ lymphoma of mucosa-associated lymphoid tissue (MALT lymphom a) Note : [CH, BCLl, MYC and MALT1 dual color break apart probes may be used f in initial screaning for those NHL's which may have these genes rearranged irrespective of their partner gene in volved in the fusion
Burkitt's lymphoma and in variant translocations, the MYC breakpoints are a considerable distance centromeric or telomeric from the MYC coding exons. As a result of the translocation, control of normal MYC is lost and the intact protein is constitutively expressed throughout the cell cycle • The MYC gene can function both as a transcriptional activator and transcriptional repre ssor. As a result of the t(8;14), there is transcription of a truncated MYC protein while in t(2;8) and t(8;22) the rearrangement of the MYC gene can occur within sequences downstream of the transcribed region . The exact molecular mechanism by which the rearranged MYC is activated has not been fully elucidated but its activation constitutes an important step in malignant transformation • An increa sed level of MYC constitutive synthesis in leukemic disorders is found not only as a result of translocation but also as a result of MYC mutation s and amplification. Approximately 65% of Burkitt 's lymphomas demon strate MYC point mutations. Overexpression of MYC has been linked to amplification of MYC genes reported in plasma cell leukemia, AML, CML , and T-cell lymphom a • A diagno sis of Burkitt's lymphoma and the rearrangements of MYC can now be made in non-dividing cells as well as paraffin-embedded lymph node biopsies using a very sensitive I-FISH assay for detection of t(8;14) with less than 2% false positive results • Recent genomic profiling study unequivocally demonstrated that only patients with MYC rearrangements should be classified as Burkitt lymphoma
352
even though some of the cases were called by pathologists as diffuse large cell lymphoma (DLCL)
Diffuse Large Cell Lymphoma • Approximately 35% of patient s with DLCL and about 5-10% of patient s with follicular lymphoma have rearrangements in the BCL6 gene normally residing at 3q27 (Figure 38D, right top row) • Translocations of 3q27 are the third most frequent translocations in NHL and at least 33 different partners have been described to partic ipate in these rearrangements. The most frequent chromosomal band partners are 2p13, 4p13 , 6p22, 7p12, 8q24, 13q14, 14q32, 18p11.2, and 22q 11. These translocations juxtapose different promoters, derived from other chromosomes with the BCL6 coding domain causing persistent expression of BCL6 • Most of the breakpoints in 3q27 occur within a to kb region. The fact that the 3q27 region is affected in different lymphomas, irrespective of the translocation partner chromosomes strongly suggests that alterations of BCL6 and not the reciprocal loci are important in the pathogenesis • The alterations in 3q27 are too small for microscopic detection, and therefore , most of these rearrangements are detected either by Southern blot analysis or by FISH
• BCL 6 function s as a transcriptional repressor of gene s containing its binding sites, and therefore , the mechani sm responsible for the malignant phenotype is transcriptional deregulation
FISH and Conventional Cytogenetics
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Fig. 38. Most frequent chromo somal rearrangements and their molecular consequences in six types of NHL. (A) t(14;18) (right, a partial karyotype) results in IGH-BCL2 fusion (left, interphase cells) in over 85% of patients with follicular lymphoma . (B) Burkitt 's lymphoma is characterized by MYC-IGH fusion in over 80% of cases. Chromosome 8 was identified by CEP8 (aqua), MYC (red), and IGH (green). Note fusion signals on der(8) and der(l4). (C) Mantle cell lymphoma is characterized by t(lI ;14) (qI 3;q32.3) (partial karyotype , right) that results in CCNDI-IGH (interphase cells left) (D) Overexpression and rearrangement of BCL6 gene localized to 3q27 is a characteri stic feature of diffuse large cell lymphoma (DLCL). (E) MALT lymphoma is characterized by t(l1 ;18) that fuses gene AP12 on chromosome II to the MALTI gene on chromosome 18. This is the only B cell lymphoma that does not include IGH locus. The interphase and metaphase cells shown here exhibit normal hybridization pattern with breakapart MALTI FISH strategy. (F) The hallmark of anaplastic large cell lymphoma (ALCL) is rearrangement of ALK gene localized on chromosome 2, band region p23. As shown in the top part of the partial karyotype the "breakapart" ALK gene is on chromosome 2, band region p23. However, in the most frequent t(2;5) translocation the 5' end of the gene (green signal) remains on chromosome 2, while the 3' end of the gene (red) is translocated to chromosome 5.
• About 3-4% of DLCL have t(l4;15)(q32;qll-13), which results in fusion of the BCL8 gene on chromo some 15 to V H segment of the IGH locus • The most common secondary abnormalities are trisomies for chromosomes 3, 5, 7, 11, 12, 18, and X, observed in > I0% of cases
Mantle Cell Lymphoma • All cases of mantle cell lymphoma (5-7% of all NHL), showing CD5 +/CD23- B-cell phenotypes, exhibit t(ll; 14)(q13;q32) abnormality (Figure 38C, left bottom row)
• The most frequent monosomies include -13, -14, and -15
• The prognosis of mantle cell lymphoma is the worst among all B-celllymphomas, and there is no therapy that can be considered as standard
• A complex karyotype may have an adverse impact on prognosis
• t(ll; 14)(q 13;q32) is also found in a wide variety of other B-cell malignancies including MM, splenic lymphoma
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with villous lymphocytes, and B-cell prolymphocytic leukemia • Most breakpoints on IIql3 are dispersed over a region of about 130 kb centromeric to the eye/in D (CCNDI) gene. At the molecular level, the BCLl locus (CCND I) on chromo some II q13 is juxtaposed to an enhancer sequence within the immunoglobulin heavy chain UGH) gene on 14q32 leading to overexpression of the eye/in D gene, which is not expre ssed in normal B- and T-cells, nor in other malignant lymphomas • The consequence of this translocation is overexpre ssion of eye/in D, a gene involved in control of the cell cycle • Each method for detection of t( II; 14) including cytogenetics, Southern blot, and PCR analyses has limitations. Cytogenetics is hampered by a low mitotic index of neopla stic B-cell s. Southern blot and PCR analyses for IIq13 rearrangements are positive in only S0-60% of patients with mantle cell lymphoma becau se the breakpoints within II q 13 are scattered along a I30-kb distance • Dual color FISH has proved to be the most sensitive assay for detection of lGH/CCNDl fusions, which was found in 100% of cases. Furthermore, the CCNDl fusion rearrangement can be detected in formalin-fixed , paraffinembedded samples making this method rapid, reliable , independent of cell cycle and applicable to all cases of mantle cell lymphoma A gain of 3q, 7p, and I2q and loss of II q 14-23 and 17p are the most frequent numeri cal changes, and unlike other lymphomas, in mantle cell lymphoma DNA amplifications of several chromosomal regions appear to be associated with a blastoid variant.
Molecular Genetic Pathology
chimeric transcript consists of S'-APl2 and 3'-MALTI located on the der(18) • Cytogenetic studies of low-grade MALT B cell lymphomas also show a recurrent (rare) t(l;I4)(p22;q32) abnormality. On the molecular level a recurrent breakpoint upstream of the promoter of BCLl 0 at Ip22 was identified in these patients
Lymphoplasmacytoid Lymphoma (LPL) • LPL is a small lymphoc ytic lymphoma with plasmacytoid differentiation (CDS- ICD I0%) characterized by t(9;14)(pI3;q32) in approximately SO% of cases. As a result of this translocation the PAX5 gene (paired homeobox S) on 9pI3 moves to the lGH locus on der(14) causing dysregulation of PAX5 • Molecular characterization of the t(9;14) revealed that the coding region of the PAX5 gene , remains intact in some patient s. In those individuals the t(9; 14) should be considered as a regulatory mutation, whereb y the PAX5 gene is brought under the control of the lGH locus. In other cases, molecular studies of the t(9;14) revealed that the breakpoint has occurred upstream of the PAX5 promoter leading to insertion of the lGH enhancer upstream of the PAX 5 gene. The PAX5 gene encode s a B-cell-specific transcription factor and PAX5 -1- mice display maturation arrest at the CD43 pro-B-cell stage with defective Ig rearrangement
Waldenstrom Macroglobulinemia • Waldenstrom macroglobulinemia is a plasma cell dyscrasia characterized by a CD I 38/CD19 phenotype with a Iympho/plasmacytic clonal expansion in the bone marrow
Marginal Zone B-cell Lymphoma (MZBCL) and Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma
• The biological nature is different from LPL because these patients do not display t(9;14)(p 13;q32)
• MZBCL and B cell lymphoma of MALT are the commonest form of extranodal NHL and may be either high or low grade . An etiological link between low-grade gastric Malt lymphoma and the lymphoid reaction associated with Helioba cter pylori infection , has been demonstrated
Hodgkin's Disease (HD)
• By interphase FISH analy sis, trisomy 3 or trisomy 18 were found in 60-80% of the analyzed cases. The pathogenic role of trisomy 3 is not known, but BCL6 residing at 3q27 is rearranged in some MZBCL, although more frequently in large B cell lymphomas • The most frequent and specific aberration occurring in MZBCL and MALT lymphomas is t(lI ;18)(q21;q21.1 ) (Figure 38D, right middle row). It is the only recurrent translocation that doe s not involve IG genes even though it present s as a B cell lymphoma. As a consequence of t( II ;18), APl2 gene on Ilq21, encoding an inhibitor of apopto sis (aka IAP2, HlAPland MIHC) and a novel gene MALTl on I8q21, characterized by several Ig-like C2-type domains , are often rearranged. The resultant
354
• There has not been much progress in delineating recurrent chromo somal abnormalities in HD. Classical HD has emerged as the term for HD characterized by the CD IS+, CD30+ phenotype • Less than I% of the cells in HD are Reed-Sternberg cells (most likely of B-cell origin). Therefore, the sparcity of dividing tumor cells for karyotype analysis has represented a major obstacle for conventional cytogenetics • With simultaneous fluore scence immunophenotyping and interpha se FISH (FICTION) all 30 HD patients studied had numerical aberrations of CD30+ • The most specific chromosomal abnormalities in HD are hyperdiploidy/tetraploidy with tremendous variations in the chromo some number indicating heterogeneity from patient to patient. Even with the use of nine different centromeric probes no specific numerical chromosomal abnormality has been identified • Deletion s of Ip, 4q, 6q, and 7q are recurrent
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FISH and Conventional Cytogenetics
T-CELL LEUKEMIA AND LYMPHOMA T-Cell Lymphoproliferative Diseases • T-cell ALL • T-cell CLLIPML as well as a group of indolent or small T-cell disorders • Large granular lymphocyte leukemia • NK leukemia/lymphoma • Anaplasic large cell lymphoma The common theme in T lymphoid malignancies is the juxtaposition of T-cell receptor genes (TCR) adjacent to a variety of transcription factors located at or near breakpoints on the partner chromosome. The chromosomal bands most frequently involved are 14q II, where TCRA and TCRD are located , 7q3S, the site of TCRB as well as 7p1S, the site of the TCRG gene. The rearrangements of TCRB and TCRG are relatively rare while 14qll rearrangements involving both TCRA and TCRD are frequent in T-Iymphoid neoplasms.
T-Cell ALL
patients with T cell ALL. It is associated with excellent outcome • The complete remission rate is 100% with a median disease-free survival of 46 months. More than 7S % of patients have a 3-year disease-free survival • The t(lO;14) , seen in about 4-7% ofT-ALL, fuses the homeobox-containing gene HOXll (TCL3) with the TCRD gene. The coding regions of HOX 11 are not disturbed by the translocation. In the variant translocation t(7; I0)(q3S;q24) HOXll is juxtaposed to the TCRB gene, which results in overexpression of normal HOXll mRNA by bringing HOXll under the influence of TCR promoter sequences
TCR Rearrangements • Table 17 shows the frequency of TCR rearrangements in T-ALL • Conventional cytogenetics may not recognize about half of the TCR~ and about one third of TCRaJo rearrangements
• In children, the overall frequency of T-cell ALL translocations is 40-S0% but there is no specific karyotype abnormality associated with T-ALL
t(5;14)(q35;q32) or t(5;14)(q34;qll)
• Three transcription factor gene s share more than 8S% homology in their basic HLH motif and they include :
• Neither of these abnormalities are recognized by conventional cytogenetics
- The TAL 1 (or SCL ) gene, located on the Ip32, is found in t(l ;14) (p32;q II) and t(l ;7)(p32;q3S). TAL 1 gene rearrangements are observed in about 3% of patients with T-ALL but are more frequent in pediatric than in adult ALL. In about 30% of these patients TALl rearrangements are not detected by conventional cytogenetics . Other rare translocations involving the TALl locus are t(1;3)(p32;p21) and t(l ;S)(p32;q3l). Another consistent rearrangement observed in 12-26% of individuals with T-cell ALL is a site-specific deletion of about 90-100 kb DNA affecting TALl and SIL genes - The TAL 2, residing at 9q32, is detected in t(7;9)(q34;q32) - LYLl gene , residing at 19p13, is identified in t(7; 19)(q24 ;p13)
• Two other genes, LMOl(RBTNl or TTGl) and LM02 (RBTN2 or TTG2) , belonging to LMO family of genes that contain two additional genes, are found at the breakpoint of rare but consistent chromosomal translocations in T cell ALL. LMO1, located at II p IS, is involved in the t(l1; 14)(p1S;qll), and LM02, located at II p 13, is involved in the t(ll; 14)(p 13;qII) and t(7;II)(q3S ;p13). As an oncogenic transcription regulator, LM02 overexpression in erythroid and T cells leads to differentiation arrest, which is a prerequisite for development of T cell malignancies
• Almost 20% of childhood T-ALL demonstrated a HOXI 1L2 (t Sq3S) gene translocations by FISH • Other variant rearrangements include inv(l4)(qllq32) as well as del(l4)(qll)
T-Cell CLL and PLL T-Cell CLUPLL • The most common chromosomal changes include inv(l4)(qllq32.1), t(l4;14)(qll;q32.1), and t(7;14)(q3S;q32.1). A TCLl (T cell leukemia I) gene, isolated from patients showing 14q32.1 rearrangements, is found to be dysregulated in these patients
Adult T-Cell LeukemiaILymphoma (ATLL) • In ATLL associated with human T-celllymphotropic virus type I (HTLV-I ) the most frequent genetic lesions include altered expression of CDKN2 (cyclin-dependent kinase inhibitor) gene on 9p21 (1S-20%) and loss of heterozygosity (LOH) at 6q1S-21
Natural Killer (NK) LymphomalLeukemia
t(lO;14)(q25;qll)
• A group of highly aggressive hemato-Iymphoid malignancies of natural killer cell lineage (CD2+ ,CD3-,CDS6+, TCR-) having a strong association with Epstein-Barr virus showed chromosomal rearrangements in over 80% by conventional karyotyping
• According to the French collaborative study, the t(10;14) is the most frequent chromosomal translocation in
• The most frequent abnormalities include del(6)(q21-23) and gain of the X chromosome. FISH, CGH, and spectral
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Table 17. Frequency of TCR Rearrangements Using Conventional Cytogenetics vs FISH FISH (%) Total
Locus
Coventional cytogenetics %
Abnormal karyotype
TCRaO
9.S
17.4
24.7
TCR~
3.1
19
26.9
TCRy
0
0
a
Q
Q
aModified from reference Cauwe/ier et a/., 2006
karyotyping confirmed the presence of del(6) in the CD3-,CD56+ tumor cells • Other less common but non-random karyotypic changes include isochromosome lq, 6p, and 17q, as well as del(llq),13q, and 17p, and trisomy 8
Anaplastic Large Cell Lymphoma (ALCL)ALK-Positive Lymphoma • ALCL is a relatively infrequent lymphoma occurring in about 2% of all adults and 13% of children with lymphoma • The molecular basis of the anaplastic large cell group of lymphomas (CD30+), defined recently as ALK-positive or "ALKoma" is the result of t(2;5)(p23;q35), which fuses part of the NPM gene on 5q35 with part of the ALK receptor tyrosine kinase gene (anaplastic lymphoma kinase-ALK) on 2p23 to produce a chimeric NPM-ALK gene. This encodes a chimeric NPM-ALK protein, which has a constitutively activated kinase
• ALK is thought to playa direct role in the malignant transformation of lymphoid cells probably by aberrant phosphorylation of intracytoplasmic substrates because accumulation of the ALK protein is only observed in cytoplasm • The t(2;5) is detected by cytogenetics in 50-70% of patients with ALCL • t(2;5) is readily identified by FISH (Figure 38 right bottom row) and the RT-PCR assay, which has been establi shed and used for detection of the chimeric gene • Overall, 43% of ALCL have NPM-ALK: 83% among pediatric and 31% among adult patients • The other cases of ALCL have variant translocations and they include rearrangements involving 2p23 region: t(1;2) (q25;p23), t(2;5)(q37;q31), t(2;19) (p23 ;p13), and inv(2)(p23q35) all associated with ALK gene expression, and those affecting 5q35 region: t(1;5)(q32;q35), t(3;5)(qI2;q35), and t(3;5) (q25 ;q34-35) • As a result of these variant rearrangements, three new fusion genes have been identified : AT/C-ALK is a fusion gene resulting from inv(2)(p23q35), which fuses ALK and 5-aminoimidazole-4-carboxamide ribonucleotide fonnyltransferase/lMP cyclohydrolase (AT/C). This molecular variant is detected by RT-PCR and is the most common ALK variant. TFG-ALK is a consequence of t(2;3)(p23 ;q21), which fuses TRK-fused gene (TFG) with ALK at the same breakpoint as found in t(2;5). In t(1;2)(q25;p23) the TPM3 gene on chromosome 1, which encodes non-muscular tropomysin, is fused to ALK, to produce the TPM3-ALK fusion gene. Molecular variants also showed chimeric protein localization in cytoplasm. Prognosis of patients with t(2;5) or variant translocations is excellent but is clearly different from ALK-negative ALCA. The true nature of ALK-negative ALCA remains obscure
SOLID TUMORS HER2 in Breast Cancer • Overexpression and/or amplification of HER2 (HER21neu or c-erbB-2Ineu), occurring in 25-30% of patients with breast cancer, is associated with poor clinical outcome, decreased responsiveness to nonanthacyline containing cytotoxic and hormonal therapie s, and shortened survival • Trastuzumab (herceptin), a recombinant monoclonal antibody that specifically targets the HER2 receptor, is a proven therapy for patients showing HER2 amplification
• HER2 status is used for select ion of patients for Trastuzumab immunotherapy
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- The HER2 oncogene encodes a protein with a molecular weght of 150,000 daltons (pI85). The gene product is a transmembrane tirosine kinase receptor belonging to a family of epidermal grow factor receptors. The ligand for HER2 has not been identified . Therefore, it has been hypothesized that the main role of HER2 may be to dimerize with the other members of HER2 family of receptors
HER2Assays • Three groups of assays are used to determine HER2 status in formalin-fixed, paraffin-embedded tissue sections and they include :
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Fig. 39. Breast biopsy tissue after hybridization with HER2 (red) and CEP17 (green) (top panel) . Cells showed a normal hybridization pattern with disomy for both loci. In contrast, three isolated cells (bottom row) from three different biopsy preparations showed disomy for CEPl 7 and numerous copies, amplification for HER2.
- Immunocytochemistry (IHC) , which measures protein overexpression - FISH method, which evaluated gene copy ampl ification - Serum ELISA test, which measure s the levels of the shed extracellular domain of the HER2 receptor protein
The IHe Method • The US FDA approved two IHC methods: - Hecepte st (Dako, Carpintera, CA) and - PATHWAY (Ventana Medical Systems Inc, Tuscon, AR) • HercepTest is currently recommended IHC method with scoring range from 0-3+. Samples scoring 3+ are regarded as unequivocally positive and Oil + as negative. Borderline 1+12+ and 2+ require FISH
confirmation as recommended by the NCCN task force. Tumors that are 2+ with a normal gene copy should be regarding as true false-positive that will not benefit from Trastuzumab therapy • Many factors may contribute to false-positive results and they include the type of fixative that preserves antigenic integrity, prolonged storage of tissue blocks, the type of antibody used in the assay, and the scoring system
The FISH Methods • FDA approved FISH tests include: - PathVysion (Vysis, AbbottIMolecular, Des Plains , IL) (Figure 39) - INFORM (Ventana Medical Systems Inc.) - HER2FISH PharmaDx (Dako)
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• PathVysion depend on scoring the exact number of HER2 hybridization signals and internal control, and centromere 17 hybridization signals per nucleus. The ratio between the two is called amplification ratio (AR). The PathVysion uses AR less than 1.8 as negative result, AR greater than 2.2 as positive result, while AR between 1.8 and 2.2 must be interrelated with caution • The INFORM HER2 assay uses the absolute number of HER2 signals and
Molecular Genetic Pathology
the value recalculated. The manufacturer recommends also that AR result of 1.9 should be reported as no amplification and for a ratio of 2.1 the results should be reported as amplification. This recommendation underscores the need for standardization and consensus for interpretation of borderline category results since accurate determination of HER2 gene status will eliminate treatment of inappropriate patents
considers ~5 .0 gene copies of HER2 as amplified
Bladder Cancer
• The HER2 FISH pharrnaDx considers ~2.0 HER2/Centromere 17 amplified
• Each year between 50,000 and 60,000 new cases of urothelial carcinoma (UC) are diagnosed in the United States. Bladder cancer is the fourth most prevalent cancer in males and the eight most prevalent Cancer in females
Concordance Between IHC and FISH • Numerous studies have compared degree of concordance between IHC and FISH as well as intra- and interlaboratory reproducibility of FISH and IHC. In the study of 2535 patients with breast cancer enrolled in North Central Cancer Treatment Group trial N9831, between 2001 and 2005, concordance of ISH and FISH findings were compared with the central testing laboratory. Concordance between the local and central laboratories to establish IHC3+ overexpression status, using HecepTest, was confirmed in 81.6%. FISH was used to detect the HER2 status in 813 patients, and conformation by central laboratory was achieved in 88.1% (85.6-90.2%, 95% CI) • FISH concordance between local and central laboratory testing was greater than IHC concordance between the local and central laboratory (P < 0.001) • Reproducibility of FISH HER testing for negative and amplified results between laboratories was also confirmed by the College of American Pathology surveys conducted between 2000 and 2004 in over 100 laboratories • When discordant cases (18.4% for IHC and 11.9% by FISH) in the North Central Cancer Treatment Group N9831 study were retested at a reference laboratory, concordance between the central and local laboratories showed a high level of agreement: 94.3% for ICH (0, I+ and 2+) and 95.2% for FISH (not amplified) • False-positive and false-negative HER2 test results can partly be attributed to the lack of concordance between IHC and FISH • IHC2+ category is most likely to be discordant when compared with FISH method because agreement rates for other IHC categories were 97% for ICH 0, 93% for IHCl+, and 89% for ICH3+ . Polysomy 17 may be one of the reasons responsible for overexpression of HER2 protein as measured by IHC method. Since Herceptinbased therapy for IHC2+ remains to be determined, an IHC2+ test result is potentionally unreliable indicator of HER2 status . The variability in definition and use of a borderline interpretation may be partly explained by inter-observer variability and different interpretation for critical cutoff values. When Pathvysion is used, the manufacturer recommends that in case of tumors showing AR of 1.8-2.2 additional 20 nuclei should be scored and
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• UC, also known as transitional cell carcinoma, arises from urothelial cells that line the bladder, ureters, renal pelvis, and proximal urethra • Two main types of UC are recognized: flat UC (20%), also termed carcinomas in situ (CIS) and more frequent, papillary UC (75-80%). Papillary tumors tend to recur but not progress, while CIS tend to recur and progress to invasive UC • UC and bladder cancer, like most epithelial tumors, derive from a step-wise progression of genetic and chromosomal events
UroVysion™, FDA-Approved Assay • The only FDA-approved test is Urovysion" FISH tests from Vysis/Abbott Molecular, which utilize centromere enumeration probes for chromosomes 3, 7, and 17 and locus specific 9p21, because these chromosomes as well as 9p21 locus were found to be the most frequently altered in UC. The multi-target, multi-colored set of probes are labeled in four different colors and are used simultaneously on voided urine specimens. The UroVysion™ kit is used for both, the diagnosis and recurrence of UC (Figure 40)
Protocol for UroVysion™ assay • The detailed protocol for UroVysion ™ is included in the Vysis/Abbott molecular package insert. It includes the following steps: - Voided urine specimen is collected in the ThinPrep urine collection kit (Cytic Corporation, Boxbourough, MA) and transported to the lab within 24 hours - Cells are pretreated in 2X SSC, protease, and PBS - Fixation of cells is in 1% formaldehyde followed by PBS wash - Co-denaturation (2 min) of target DNA (urocytes) and probes and hybridization with set of probes: CEP3 (red), CEP7 (green) , locus-specific probe 9p21 (aqua),and CEPl7 (gold) for 18-24 hours using Thermabrite
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Fig. 40. A morphologically abnormal urocyte after in situ hybridization with a set of 4 probes . Note eight copies of chromosome 3 (red), six copies of chromosome 7 (green), six copes of chromosome 17 (aqua), and no gold hybridization signals with 9p21 locus. Homozygous deletion of 9p21 is associated with recurrence but not necessarily aggressiveness or progression of disease.
- Post-hybridization washing includes O.4X SSC/O.3% NP-40 and 2X SSC/O.l % NP-40 followed by counterstaining with DAPI - Interpretation of hybridization signals : • If 4 or more of the 25 evaluated cells show gain for 2 or more chromosomes (3,7, or 17) in
the same cell the results are considered abnormal • If 12 or more of the 25 evaluated cells showed zero 9p21 signal the results are considered abnormal • If abnormal cells are not identified among 25 scored cells by two individuals the entire slide and/or sample has to be evaluated
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Results Obtained with Uro Vysion™
Classification of Gliomas
• A multi-centered, prospective, blinded trial to compare the sensitivity of multi-target FISH assay with that of voided cytology in 473 patients with gross and microscopic hematuria demonstrated that Urovysion" is significantly more sensitive than voided cytology for all grades and stages. Based on these data, UroVysion™ was FDA approved for the use in patients with hematuria
• According to the WHO histopathologic criteria, glial tumors are classified into three major types:
• The application of UroVysion TM FISH assay for UC recurrence showed, in compilation of published literature, the overall sensitivity of cytology in 48% and the overall sensitivity of FISH in 74%. More specifically, for grade 1 comparative studies showed sensitivity of cytology compared with FISH in 19% vs 58%, for grade 2, 50% vs 77%, and for grade 3, 71% vs 96% • When sensitivity of cytology was compared with FISH sensitivity, according to the stage the cumulative literature data showed for Ta: 35% vs 65%, for Tl, 66% vs 83% ,and for muscle-invasive carcinoma, 75% vs 94% • In CIS, cytology detected only 67% abnormalities vs 100% detected by FISH • Unlike conventional urine cytology and cytoscopy, which depend on subjective visible microscopic or macroscopic changes, FISH allows identification of chromosomal abnormalities associated with malignant development before phenotypic expression of those alterations
Chromosomal Abnormalities and Prognosis • Patients with homozygous 9p21 deletion are more likely to have recurrence but not necessarily aggressiveness or progression • Patients with aneuploidy of any combination for chromosomes 3, 7, and 17 as well as 9p21, suggests the possibility of both tumor recurrence and progression
- Astrocytoma - Oligodendrogliomas - Oligoastrocytomas (mixed gliomas) • Oligodendroglial tumors, comprising oligodendroglioma and mixed ologocytomas, are estimated to account for 5-18% of all gliomas and are divided into low grade (grade II and high grade [grade III])
Genetics of Oligodendroglial Tumors • The genetic hallmark of oligodendroglial tumors is the combined LOH on chromosomal arms 1p and 19q • Approximately 80% of oligodendrogliomas and 50% of oligoastrocytomas have LOH of 1p and 19q • More specifically, chromosomal regions involved are 1p36.22-p36.31 and 19q13.3 • Loss of 1p and 19q is very rare in astrocytic tumors • The -1p/-19q genotype is associated with better response to therapy and longer survival, whether therapy was given to grade II or III tumors or whether primary or recurrent
Tissue Processing • In the routine clinical molecular cytogenetics laboratory, specimen for FISH examination is paraffin-embedded brain biopsy • Tissue cells are processed the same way as other paraffinembedded tissue except, pre-treatment kit III is recommended for better hybridization due to the density of tissue • Set of 4 probes are used: dual color 1p36 and internal controllq25 and dual color 19q with 19p being an internal control. Only cells that show both signals are being scored
Anticipatory Positive (AP) Results • AP results is the term used in a setting of a FISH-positive result in the absence of concurrent detectable malignancy. In prospective investigation of such patients recurrent UC developed in 62% of 55 patients with AP results, which were 12 cases of lowgrade UC and 22 cases of high-grade Uc. In contrast, recurrent UC developed in only 5% of 155 patients of the microscopically negative, FISH negative cases
Gliomas • Gliomas are most common primary brain tumors, representing 50-70% of all adult brain tumors and constitute a heterogenous group of neoplasms with respect to morphologic appearance, biologic behavior, genetic alterations, response to therapy, and clinical outcome
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Neuroblastoma • Neuroblastoma is the most common extracranial solid tumor in infants and children. It is derived from primordial neural crest that ultimately populate s the sympathetic ganglia and adrenal medulla • The prevalence is about one case in 7000 live birth, which corresponds to approximately 700 new cases per year in the United States • Overall mortality approaches 30-60%, even with the most aggressive treatments. Although prognosis is highly variable depending on the age at diagnosis, stage of the disease , and a variety of genetic variables that might predict the clinical behavior • The current Children's Oncology Group risk stratification criteria are based on stage, age at diagnosis, MYCN gene amplification status, DNA index, and histology
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FISH and Conventional Cytogenetics
Table 18. Chromosomal Translocations and Gene Rearrangements in Soft Tissue Sarcoma and Available Probes for their Detection Tumor type
Chromosomal abnormality
Genes invo l ved
Available probes
t(11;22)(q24;q12)
EWSR1-FLll
EWSRI BA
t(21;22)(q22;q12)
EWSR1-FRG
EWSRI BA
t(7;22)(q33;q12)
EWSR1-ETVl
EWSRI BA
t(2;22)«q 13;q12)
EWSR1-FEV
EWSRI BA
t(17;22)(qI2;qI2)
EWSR1-E1AE
EWSRI BA
inv(22)
EWSR1-ZSG
EWSRI BA
Desmoplastic small round cell tumor
t(11;22)(p13;q12)
EWSRI-WTl
EWSRI BA
Extraskeletal myxoid chondrosarcoma
t(9;22)(q22;q12)
EWSR1-CHN
EWSRI BA
t(9;17)(q22;q11)
RBP56-CHN
t(9;15)(q22;q21)
CHN-TCFl2
Clear cell sarcoma
t(12;22)(q13;q12)
EWSRl-ATFl
Alveolar rhabdomyosarcoma
t(2;l3)(q35;q14)
PAX3-FKHR
t(1;13)(p36;q14)
PAX7-FKHR
t(12;16)(q13;p13)
CHOP-FUS
FUS BA
t(12;22)(q13;q12)
EWSR1-CHOP
EWSRI BA
t(X;18)(p11.2;q11.2)
SSXl-SYT
SYTBA
SSX2-SYT
SYTBA
Ewing SarcomaIPNET
Myxoid liposarcoma
Synovial sarcoma
EWSRI BA
Alveaolarsoft part sarcoma
t(X;17)(p11.2;q25)
ASPL-TFE3
Dermatofibrosarcoma protubrans (and giant cell fibroblasoma)
t(17;22)(q22;q 13)
COLlAI -PDGFB
Low grade fibromixoid sarcoma
t(17;16)(q32-34;pll)
FUS-CREB3L2
FUSBA
Angiomatoid fibrous histiocytoma
t(12;16)(q13;pll)
FUS-ATFl
FUSBA
t(12;22)(q13;q12)
EWSR1-ATFl
EWSRI BA
Congenital fibrosarcoma and mesoblastic nephroma
t(12;15)(p13;q25)
ETV6(TEL)-NTRK3
ETV6(TEL) BA
Endometrial stromal sarcoma
t(7;17)(p15;q21)
JAZF1-BAZl
Inflamatory myofibroblastic tumor
t(2p23)
ALK, multiple fusion partners
ALK
*Modified from Reference Lazar et al. 2006 PNET ind icates primitive neuroectodermal tumor BA indicates "breakapart" probe strategy
• The best characterized genetic aberrations in neuroblastoma are: - MYCN amplification - Loss of hetrozygosity at Ip36 and - Gain of 17q region
Amplification of MYCN Gene • The amplification of the MYCN gene is the most unfavorable prognostic factor in neuroblastoma. Approximately 20-30% of all patients presenting with at advanced stages show an amplification of the MYCN gene
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• Children less than 12 months at diagnosis with stage 4 or MYCN-amplified stage 3 tumors have approximately 60% mortality rate • The genes dosage is usually examined by Southern blot method, FISH, or semiquantitative PCR. Detection of MYCN by FISH is performed on tumor cells and the results are available within 2 days • The amplification of the MYCN is strongly correlated with all prognostic factors and rapid tumor progression. In contrast, the expression level of MYCN is not significantly correlated with any prognostic factor • Tumors with non-amplified MYCN gene have variable clinical behaviors that generally correlates with the patient's age at diagnosis • Children 12-18 months old with metastatic neuroblastoma have favorable outcome with high-dose therapy if their tumors were hyperdiploid and lacked MYCN amplification
• MYCN amplification is a statistically significant marker of higher risk disease within both the diploid and hyperdiploid subgroups of children
rosette structures. Ewing sarcoma does not have these structures
t(l1;22)(q24;q12) • Approximately 90% of Ewing family tumors exhibit a specific translocation t(11 ;22)(q24;q 12), which results in the expression of EWSR I-FLII chimeric protein • In a small fraction of tumors, EWSRI gene on chromosome 22 has alternative gene partners on chromosomes 2, 7, and 17 • The EWSRI gene on 22ql2 plays a recurrent major role in several other tumors such as desmoplastic small round cell tumor, clear cell tumor, and rarely in myxoid liposarcoma • The EWSRI gene spans 40 kb and has 17 exons and nearly 80% of breakpoints are found within introns 7 and 8 • Detection of the EWSRI gene rearrangements, irrespective of the fusion partner, is easily identified with interphase FISH technology in fresh specimens, frozen sections, or paraffin-embedded tissue blocks
Synovial Sarcoma
Gain of 17q • A gain of I7q region, specifically 17q21-terminal part of chromosome 17, has been shown to be correlated with the aggressiveness of neuroblastoma using either CGH or FISH • Gene dosage of Survivin gene on 17q was reported to be significantly associated with all prognostic factors
Sarcoma • Many soft tissue sarcomas have a high incidence of specific translocations as shown in Table 18.
• Synovial sarcoma is most prevalent in adolescents and young adults • In general, synovial sarcomas are highly aggressive tumors that metastasize primarily to the lungs
t(x;18)(pl1.2;qll.2) • The genetic feature of synovial sarcoma is the presence of t(X ;18)((p II ;q II). It may be detected by conventional cytogenetics of tumor tissue after a short-term culture
• The tumor is composed of undifferentiated primitive mesenchimal cells
• On the molecular level t(X ;18) result in the formation of a distinct chimeric gene SYT-SSX. The Xp II region contains closely related SSXI-SSX5 genes . The breakpoint on the X chromosome typically involves two of these genes, most frequently SSXI and SSX2 that map to Xp 11.23 and Xp 11.21, respectively. SSX1 is involved in the SYT-SSX fusion almost twice as often as the SSX2 gene
• Those Ewing sarcoma family of tumors that exhibit features of primitive neuroectodermal differentiation are classified as PNET. They have clearly recognizable
• FISH technology is used as the first line diagnostic modality for detection of SYT gene rearrangements on 18q II in synovial sarcoma
Ewing SarcomalPNET • Ewing sarcoma is a small cell cancer that occurs predominantly in the long bones of children and young adults
CONCLUSIONS AND FUTURE DIRECTIONS The analysis of cytogenetic abnormalities and FISH technology is more than a prognostic tool. Over the past 10 years it has become clear that such studies provide a better understanding of the molecular biology of leukemia and some solid tumors . Although the exponential increase of complex,
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often unrelated proteins, implicated in leukemias, lymphomas, and some solid tumors may, at first sight, appear to have little in common, the emerging scenario is that multiple genes have a common pathway. They include oncogenes, putative tumor suppressor genes, genes that act as
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transcnpnon regulating factors, and genes involved in the regulation of the cell cycle, apoptosis, and differentiation. The answer to the question of how the initial genetic damage in a hematopoietic stem cell or other stem cells causes a cascade
of other genetic events leading to development of malignancy is still unresolved. Such understanding is crucial for the design of therapeutic agents specifically targeted to arrest this process.
NOMENCLATURE p = Short arms
inv = inversion
q = Long arms
i = isochromosome
+ = When placed before the chromosome, denotes a gain of a
mar = marker chromosome
whole chromosome (e.g., +8)
con
= connected
- = When placed before the chromosome, indicates a loss of a whole chromosome (e.g., -7); in rare situations, when placed after the chromosome, as in 5q-, it indicates loss of a part of the long arms of chromosome 5
nuc ish = denotes nuclear in situ hybridization
t = translocation
nuc ish 9q34(ABLx2),22qll .2(BCRx2)(ABL con BCRxl) indicates that there are two ABL and two BCR loci, but one of each loci is juxaposed on one chromosome as a result of t(9;22) .
del = deletion der = derivative
nuc ish 21q22 (D21S65X2) indicates two copies of D21S65 DNA segment on chromosome 21
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Greaves M. Molecular genetics, natural history and the demise of childhood leukemia. EuropeanJ Cancer 1999;35:473--485. Greenberg P, Cox C, Le Beau MM, et aI. International Scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997;89:2079-2088. Grimwade D, Walker H, Olivier F, et aI. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. Blood 1998;92:2322-2333. Hiddemann W, Spiekermann K, Biske C, et al. Towards a pathogenesis oriented therapy of acute myeloid leukemia. Crit Rev Oncol Hematol. 2005;56(2) :235-245. Lazar A, Abruzzo LV, Pollock RE, Lee S, Czerniak B. Molecular diagnosis of sarcomas. Chromosomal translocations in sarcomas . Arch Pathol Lab Med. 2006; 130:1199-1207. Melnick A, Licht JD. The role of RARa and its fusion partners in acute promyelocytic leukemia. In: Ravid K, Licht 1, eds. Transcription Factors. Willey-Lyss 2001;20:327-378. Mittelman F, Johansson B, Mertens F. Mitelan Database of Chromosomes Aberrations in Cancer. http://cgap.nci.nih.gov/ ChromosomeslMitelman. Mrozek K, Hereema NA, Bloomfield CD. Cytogenetics in acute leukemia.
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Sarsody MF, Kahn PR, Ziffer MD, et al. Use of a multitarget fluoresence in situ hybridization assay to diagnose bladder cancer in patients with hematuria . J Urol. 2006;176:44-47. Schwaenen C, et al. Automated array-based genomic profiling in chronic lymphocytic leukemia : development of a clinical tool and discovery of recurrent genomic alterations . PNAS2004;101 :1039-1044. Tkachuk DC, Westbrook CA, Andreeff M, et al. Detection of bcr-abl fusion in chronic myelogenous leukemia by in situ hybridization . Science 1990;250:559-562.
Reifenberger G, Louis DN. Oligodendroglioma: toward molecular definitions in diagnostic neuro-oncology . J Neuropathol Exp Neural. 2003;62(2) :111-126.
Varella-Garcia M. Cytogenetics in solid tumors. Laboratorial tool for diagnosis , prognosis and therapy. The Oncologist 2003;8:45-58.
Rowley JD. A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinicrine fluorescence and Giemsa staining . Nature 1973;243:290-293.
Willis TG, Dyer MJS. The role of immunoglobulin translocations in the pathogenesis of B-cell malignancies. Blood 2000;96:808-822.
364
13 Instrumentation Bruce E. Petersen,
Josephine Wu, DDS, CLSp(MB), CLDir, Liang Cheng, and David Y. Zhang, MD, PhD, MPH
MD,
MD,
CONTENTS I. Nucleic Acid Extraction and Purification COBAS®Ampliprep MagNA Pure'" LC Instrument (Roche Diagno stics) MagNA Pure Compact Instrument (Roche Diagnostics) AUTOPURE LS (Qiagen/Gentra Systems; Netherlands) BioRobot M96 (Qiagen)
II. Spectrophotometers NanoDrop® ND-IOOO
13-2 13-2 13-2 13-3 13-3 13-5
13-5 13-5
peR
13-6
GeneAmp" PCR System 9600 GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) COBAS AMPLICOR® Analyzer (Roche Diagno stics)
.13-6
COBAS TaqMan 48 Analyzer (Roche Diagnostics) LightCycler
V. DNA Microarray Platforms Genet.hip" System 3000Dx Nanot.hip'" 400
VI. xMAP@ Technology
13-15 13-15
13-17 13-17 13-19
13-21
Luminex '" 100 IS System and Luminex 200 System (Luminex Corporation) ........13-21
VII. Capillary Electrophoresis Applied Biosystems 3730 and 3730 XL
III. Thermocyclers for Conventional
IV. Real-Time PCR Instruments
LightCycler 2.0 (Roche Applied Science) Genexpert'" Dx System
VIII. Gel Imaging Systems
13-23 13-23
13-24
Bio-Rad Gel DOC™ EQ, ChemiDoc™ EQ, and Chemifroc" XRS 13-24
13-7
IX. Luminometers 13-7
13-9 13-9 13-11
Digene Microplate Luminometer (DML2000)
X. Fluorescence Microscope XI. Suggested Reading
13-26 13-26
13-27 13-28
365
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The following is a brief overview of representative instruments and categories of instruments central to the practice of molecular diagnostics and related fields. Some of these instruments have been introduced in other
chapters. While a comprehensive survey of all such equipment is beyond the scope of this book, more extensive information is readily available from manufacturers' websites and brochures.
NUCLEIC ACID EXTRACTION AND PURIFICATION COBAS® Ampliprep • General information: - The COBAS ampliprep (Roche Diagnostics, Basel, Switzerland) instrument (Figure 1) uses solution-phase magnetic bead capture in the automated extraction of nucleic acids - It is appropriate for large-scale preparation of DNA and RNA samples • Principles of operation: - Processing of each sample takes place in a separate, selfcontained, single-use, disposable sample processing unit - Sample volume can range from 250-1000 ul, - All stages of nucleic acid separation are automated, including: decapping, pipeting, lysis, magnetic bead capture, washing/purification of captured nucleic acid, and resuspension/release of purified nucleic acid from beads • Procedure: - Startup procedures are performed - Reagents are loaded onto instrument - Samples are removed from storage - Consumables are loaded onto instrument - Orders are created - Samples are transferred to sample tubes (input S-tubes), which are held in a sample rack - Sample racks are loaded onto instrument Run is initiated - After completion of run, processed samples and used consumables are removed • Applications: - High-throughput extraction and purification of nucleic acids • Advantages: High throughput • Capable of continuous operation • 72 sample capacity • First 24 samples are processed in 2 hours • Each subsequent set of 24 samples can be processed in 1 hour • Can process up to 144 samples per 8 hour shift • Samples can be run overnight (20 hour on-board stability)
366
Fig. I. The COBAS Ampliprep instrument (Roche Diagnostics, used with permission). - Can automatically add polymerase chain reaction (PCR) master mix and internal control/quantitation standard (IC/QS) - Manual steps are minimized (loading and unloading only) - Machine has automated decapper, thus capped specimens may be loaded, decreasing risk of contamination - Reagents are loaded as a unit (no mixing of lots, increased reproducibility) - Continuous access for loading additional samples, reagents, and disposables - On board bar code scanner reads reagent and sample barcodes, eliminating transcription errors - Pipeting error is minimized by pipeting integrity check and clot detection - Instrument can directly load K-tubes for analysis on COBAS TaqMan® analyzers - Instrument inventories reagents and disposables prior to run • Limitations: - Only for use with plasma or serum samples - Limited to total nucleic acid extraction
MagNA Pure® LC Instrument (Roche Diagnostics) • General information: The MagNA Pure LC instrument (Figure 2) is an automated system for purification of nucleic acids following prior cell lysis.
Molecular Genetic Pathology
13-4
Fig. 3. The MagNA Pure Compact Instrument (Roche Diagnostics, used with permis sion).
(. , lI l r.'
......--... -
.
Fig. 4. The AUTOPURE LS instrument (Qiagen; Gentra System s, used with permission). • Principles of operation: - Performs cell lysis and all steps of DNA purification. (see Chapter 3 for discussion of PUREGENE chemi stry) Automated steps for blood , buffy coat, and packed cell protocol s are as follows: • Instrument reads bar codes and weighs samples (to ensure sufficient quantity) • RBCs are lysed • RBC lysate is removed • White blood cells are lysed; protein s and impurities are precipitated • Precipitate is centrifuged down ; supernatant is transferred to output tubes • DNA is precipitated using 100% isopropanol • DNA is centrifuged down; supernatant is removed • Pellet is washed with 70% ethanol • DNA hydration solution is dispensed
368
- 96 samples of whole blood , or 80 samples of 150 million cultured cells, can be processed in 8 hours - The system includes a PC workstation and software • Monitors validation steps • Perform s sample tracking • Produce s rack reports (includes sample ID, protocol description, processing run information, logged errors, and so on) • Information from rack reports can be printed , stored on CDs, and/or directly transferred to a laboratory information system - Custom protocol s can be designed for processing difficult or unusual specimen types • Procedure (for blood, buffy coat, and packed cell protocols): - Log in, select protocol and number of samples - Scan sample barcode s; transfer samples to input tubes; cap tubes - Scan barcode s on input tube caps; load input tubes into rack - Scan barcodes on output tubes; load output tubes into rack - Load rack into instrument - Initiate run After completion of processing, unload rack Rehydrate DNA by incubating at 65°C for 1-2 hours, then gently rocking at room temperature overnight • Applications: - highly purified DNA suitable for PCR, Southern blotting , archiving - Sample types : • Buccal swabs and mouthwash • Tissue homogenates • Amniotic fluid • Blood, buffy coat, and packed cells • Blood spots • Bone marrow • Cultured cells • Advantages: - Accommodate s a wide range of sample types (see above under applications) - Sample size can vary from 1-10 mL (whole blood) - High throughput High yields (typically >80%) - Extraction product is of high purity (A260/A280 ratios consistentl y 1.7-2.0) and has excellent stability (can be stored for 12+ years without degradation ) - Purified DNA is of high-molecular weight (100-200 kb) - Complete sample tracking with barcodes minimizes clerical errors and provides complete chain of custody for sample tube, input tube, output tube, and storage tube • Limitations: Restricted to total DNA extraction - Will only output to QUBESTM (50-mL polypropylene tubes), not to other containers
13-5
Instrumentation
BioRobot M96 (Qiagen) • General : - The BioRobot M96 workstation provides walk-away automation of sample preparation for applications in clinical laboratories. - The instrument performs nucleic acid isolation from blood and cell-free body fluids for 96 samples, in parallel. • Principles of Operation: Purification is performed by magnetic separation - Pipet tips function as separation chambers Up to 96 samples can be processed per run, eight samples at a time - The workstation has high-precision positioning for accurate liquid handling through eight channels and uses disposable filter-tips to eliminate carryover - Automated vacuum processing eliminates centrifugation steps, allowing walk-away automation and fast sample processing - A sample tracking system identifies and tracks bar-code-Iabeled labware for fully traceable results
- The BioRobot M96 is supplied with ready-to-run QIAamp (Qiagen) protocols as well as capability for user designed isolation of genomic DNA from blood and viral DNA and RNA from plasma and serum - Optional formats are available for laboratories with small specimen volumes: BioRobot M48, for up to 48 samples and BioRobot EZl , for up to 6 samples - The yield is about 30-60 ug DNA from I mL of blood - Built-in UV light can be used for decontamination between runs The pipetor head contains 8 high-precision syringe pumps, which operate simultaneously to allow aspiration or dispensing of small volumes of liquid (25-1000 ~L) through the disposable filter-tips. The pipetor head is also equipped with a tip guard to ensure cross-contaminationfree pipeting. The BioRobot M96 workstation is supplied with ready-torun MagAttract'" protocols that are easy to use and require minimal operator interaction, improving safety by reducing contact with potentially infectious samples.
SPECTROPHOTOMETERS NanoDrop® ND-lOOO • General information: - The NanoDrop ND-lOOO Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE) (Figure 5) is one of many spectrophotometer instruments available from a range of manufacturers. Spectrophotometers are used in the quantification of nucleic acid, protein, and cell suspension concentrations, as well as determination of DNA and protein purity. Models differ in terms of sample volume required, recoverability of the sample, and range of linearity . Nonetheless, these instruments all operate on the basis of common principles of spectrophotometry (see Chapter 3).
where A = absorbance; E = extinction coefficient (Iiter/mol-cm); b = path length, c = molarity • Nucleic acid concentration is calculated using a modified form of the Beer-Lambert equation: c = Aelb, where e =extinction coefficient (ng-cm/ul.) • Procedure: - Blanking cycle is performed, prior to running test samples, using the same solvent or buffer solution that is present in the test samples - 1-2 ~L of sample is pipeted onto a measurement pedestal, which houses a fiber optic cable
• Principles of operation: - Light source: xenon flash lamp - Detector: 2048-element linear silicon charge-coupled device (CCD) array - Wavelength range : 220-750 nm - Absorbance of each sample is measured at two different path lengths (0.2 mm, 1.0 mm) allowing for a very wide range of detection (2-3700 ng/~L dsDNA) without dilution (Figure 6) - Absorbance calculation: Absorbance = -log (Intens ity [sample]/lntensity [blank]) - Concentration calculation: • Fluorescent dye concentration is calculated using the general form of the Beer-Lambert equation: A =Ebc,
Fig. 5. The NanoDrop ND-lOOO instrument (Nanodrop Technologies Inc., used with permission).
369
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Molecular Genetic Pathology
- After measurement is completed, the sample arm is raised and surfaces that were in contact with the sample are wiped clean . The sample may be recovered with a pipet • Applications: - Measurement of nucleic acid concentration and purity - Measurement of fluorescent dye labeling density of nucleic acid microarray samples - Analysis of the purity of protein, up to 100 mg/mL (BSA) - Expanded spectrum measurement and quantitation of fluorescent dye-labeled proteins , conjugates, and metalloproteins - Bradford assay analysis of protein - Bicinchoninic acid CBCA) assay analysis of protein - Lowry assay analysis of protein - Cell density measurements - General UV and visible light-range spectrophotometry • Advantages: - Requires very small sample volume (1-2 ilL) Fig. 6. A minute (1-2 ul.) droplet of sample material is held by surface tension between a pedestal and a movable arm. Light is projected through the droplet and absorbance is measured at two different path lengths (1.0 mm and 0.2 mm) (Nanodrop Technologies Inc., used with permission).
- The sample arm is manually lowered over the sample - Measurement is initiated by command from an attached PC
- Very wide dynamic range (see principles of operation above) • Limitations - Micro-volume samples are subject to rapid evaporation; replicate measurements require reloading of fresh sample - There is risk of sample carryover if the instrument is not adequately cleaned between samples DNA samples must be homogeneous; due to microvolume sampling , heterogeneity will substantially affect reproducibility
THERMOCYCLERS FOR CONVENTIONAL PCR PCR is a process, which employs a heat stable DNA polymerase (Taq DNA polymerase) in the exponential amplification of a target DNA sequence. Three temperature dependent steps (denaturation, annealing of sequence specific primers, and elongation) are repeated in a series of cycles. Theoretically, the copy number of the target sequence doubles with each cycle . The process is described in detail in chapter 3. A thermocycler instrument produces the nece ssary temperature changes between denaturation, annealing, and elongation phase s in a series of pre-programmed steps. Reaction tubes, containing target nucleic acid and all PCR reagents, are fitted within a temperature-controlled block. In order to verify reliable operation, the temperature of each well within the block should be tested at least twice per year, using an external probe that has been calibrated again st a temperature standard.
370
The original PCR instrument was developed by Cetus Instrument Systems in 1985. Since then, various modifications have been made to increase amplification efficiency, specificity, and sensitivity. Currently many manufactures produce PCR machines, all of which are based upon the same fundamental principles. Representative instruments are described as follows .
Geneamp" peR System 9600 • General information : - The GeneAmp PCR System 9600 (Applied Biosystems; Foster City, CA), although no longer in production , remains one of the most widely used thermocycler instruments for conventional PCR applications.
Instrumentation
• Principles of operation: - The instrument contains a programmable heating and cooling block designed to heat and cool up to 96 PCR samples in a rapid and uniform manner Temperature range of block: 4.Q-99.9°C • Displayed sample temperature matches average true temperature +/-0.75 °C A heated cover is positioned over the sample block • Ensures tubes fit tightly into wells • Prevents condensation on top surface of tubes Coolant flows through 17 holes within the block • Eight are used for rapid cooling of the block (ramp cooling) • Nine are used for cold biasing the system Rapid heating is provided by a kapton heater beneath the block • Power density at the edges is greater than at the center in order to compensate for heat loss at the periphery - A key pad is used for creating, storing, editing, and running PCR programs - Indicator lights show when block is heating, hot, or cooling - The instrument can be configured for use with an optional printer • Procedure: - 0.2-mL MicroAmp reaction tubes are placed in a 96position sample tray (MicroAmp Tray) - Each tube is filled with PCR reaction mixture and all tubes are then capped - The sample tray is placed in the temperaturecontrolled block - The heated cover is secured in position - PCR program is run - After completion of PCR, sample tubes containing amplified product are removed
13-7
• Limitations: - Product detection requires post-PCR processing , which is often lengthy and increases the potential for contamination - Since PCR reactions are generally carried through to the plateau stage of amplification, accuracy of original product quantification is limited
GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) This instrument (Figure 7) offers many of the features of the GeneAmp PCR System 9600, but has the added advantages of a smaller footprint and interchangeable blocks. Networking software is available for single-source control of multiple instruments.
COBAS AMPLICOR@ Analyzer (Roche Diagnostics) • General information: - The COBAS AMPLICOR Analyzer (Figure 8) is an automated batch analyzer system combining conventional PCR with post-PCR product detection on a single instrument. - It is used widely for molecular testing in microbiology. • Principles of operation : - The instrument incorporates five components • Thermocyclers (two thermocycler units: TCA and TCB) • Automatic pipetor • Incubator • Washer
• Applications: - DNA amplification for sequencing, genotyping (allele specific PCR, restriction fragment-length polymorphism analysis, microsateIlite studies), identification of viral and bacterial pathogens, downstream cloning applications Reverse transcription PCR (RT-PCR) for expression analysis - Multiplex PCR reactions (use of multiple primer sets to simultaneously amplify multiple targets) • Advantages: - Reproducibility of cycle times - Uniformity of PCR yields - Thin-walled reaction tubes allow efficient heat transfer - Rapid heating and cooling - Oil-free operation - Low cost
Fig. 7. The GeneAmp PCR System 9700 (Applied Biosystems, used with permission).
371
Molecular Genetic Pathology
13-8
Fig. 8. The COBAS AMPLICOR Analyzer (Roche Diagnostics, used with perm ission).
• Photometer • Reads signal s at a single wavelength: 660 nm • Light source: pulsed-l ight-emitting diode (LED) • Detector: photodiode - PCR is performed using biotin-labeled primers - Amplification is followed by alkaline denaturation of the amplicon, followed by hybridization with oligonucleotide capture probes bound to magnetic microparticles, and then multiple washing steps. During washing, the microparticles are held in place by a magnet - Colorimetric detection • Bound amplicons react with avidin -conjugated horseradish peroxidase, taking advantage of the extremely high affinity between avidin and biotin molecules • Additional washing is performed • Reaction with tetramethylbenzidine sub strate produces color • Photometer performs absorbance readings at 660 nm - 48 sample capacity per run - Multiplexing capability: up to 6 different detections per sample - Incorporation of dUTP and Amplirasev (uracil-Nglycosylase) in reaction mixture prevents contamination by produ cts of previou s PCR reaction s - Results calculation • Qualitative: test result is reported as absorbance value corrected for reagent blank (Test result [A660] = A660 sample - A660 reagent blank)
372
• Test result is compared with a pre-programmed test-specific absorbance range • Result may be positive, negative, or equi vocal (gray zone ) • Quantitative: titer can be calculated on the basis of a quantitation standard • Procedure: - Nucleic acid extraction and purification steps must be performed separately, prior to loading - Samples are transferred to amplification tubes (A-tubes), along with master mix - PeR ready samples and reagents are loaded on the system - Work list data and commands are entered by operator - First test results are available 3 hours after beginning operation - Subsequent results are obtained at a rate of 50 detections per hour • Applications: - Available tests: • Human immunodeficiency virus-I • Hepatitis C virus (HCY) detection and quantitation • Hepatiti s B virus (HBY) • Cytomegalovirus (CMY) • Chlamydia trachomatis • • • •
Neis seria gonorrhoeae Mycobacterium tuberculo sis Mycobacterium avium Mycobacterium intracellulare
I nstru mentation
13-9
• Advantages : - Fully automated system - High throughput (up to 144 tests per day) - Specimens do not have to be handled manually for post-PCR processing
• Increases efficiency of procedure • Less opportunity for sample contamination • Limitations: Endpoint detection ; limited accuracy of quantitative results
REAL-TIME peR INSTRUMENTS In real-time PCR, product detection and quantitation is based on measurements made during the amplification process. This differs from conventional PCR, in which products are detected in separate steps following the completion of amplification. The general features of realtime PCR , as well as its applications and advantages, are discussed above (see Chapter 3). Real-time PCR methodologies rely on the use of fluorescent reporter molecules to produce detectable signals, the intensity of which is quantitatively related to amplicon production. Fluorescent molecules employed in real-time PCR are described above (Chapter 3) and include SYBR green, hybridization probes, and hydrolysis probes (e.g., TaqMan probes). Real-time PCR instruments generally have specific calibration protocols specified by the manufacturers.
CODAS TaqMan 48 Analyzer (Roche Diagnostics) • General information: - The COBAS TaqMan 48 Analyzer is a homogeneous real-time PCR-based system for the amplification, detection, and quantitation of DNA or RNA from clinical specimens (Figure 9) Detection is based on hydrolysis probes, which exploit the inherent 5' exonuclease activity of Taq polymerase. The mechanism of TaqMan probes is briefly summarized as follows (see Chapter 3 for detailed description) • A target-specific oligonucleotide probe conjugated with a fluorescent reporter dye and closely adjoining quencher dye (dual fluorescence-labeled probe) hybridizes with a target DNA sequence or internal standard sequence between the forward and reverse primers during the annealing phase of PCR. The probe is blocked at the 3' end to prevent extension • As a consequence of fluorescent resonance energy transfer (FRET) between the reporter and quencher dyes, the intact probe produces little fluorescent signal. However, during PCR amplification, the probe undergoes hydrolysis due to Taq polymerase 5' exonuclease activity; this causes
separation of the reporter dye from the quencher dye, and a corresponding increase in fluorescence • Fluorescence intensity progressively increases with subsequent cycles of amplification. With successive measurements of fluorescence intensity during each annealing phase, an amplification curve is produced • The original product concentration can be determined on the basis of comparing cycle threshold of the target sequence with that of an internal standard - Inclusion of Uracil-N-glycosylase (AmpErase) and dUTP as components of the PCR master mix prevents carry-over contamination from previous reactions. - TaqMan methodology may be used in the quantification of RNA; however, since RNA is not an efficient substrate for Taq DNA polymerase, amplification must be preceded by a reverse transcription step in order to generate a cDNA sequence from the target RNA strand • Principles of operation : - Thermocyclers: the COBAS TaqMan 48 Analyzer utilizes two independently controlled thermocyclers (TCA and TCB), each of which can process 24 samples
Fig. 9. The TaqMan instrument, with computer work station (Roche Diagnostics , used with permission).
373
13-10
Molecular Genetic Pathology
Excitation filter wheel with four filters
TCA
<J)
Halogen lamp
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~ ([>T
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~ ([>T
<J)
~
([>r=<
Emission filter wheel with four filters
~
cO
~D ~
cO
PhotoASIC
TCB
PhotoASIC
Fig. 10. Optical pathways of the COBAS TaqMan 48 System (Roche Diagnostics, used with permission).
in simultaneous independent runs (maximum of 48 samples, total) • During amplification, each specimen is contained within a "kinetic tube" (K-tube) • K-tubes are fitted in a holder (K-carrier), which is loaded into the thennocycler - Excitation signal (see Figure 10) • White light is produced by a tungsten halogen lamp • Light is transmitted through one of four interference filters arranged on a wheel, rotated by a stepper motor • Dedicated fiber optic pathways convey the excitation signal to each sample position, minimizing crosstalk • A shutter blocks the excitation light during nonreading time (preventing photolysis) - Fluorescence signal • A second set of fiber optics receives the fluorescence signal • These fibers are oriented at a 90° angle from the excitatory fibers to minimize interference from the excitation signal
374
• The signal passes through one of four interference filters from a second filter set, also arranged on a wheel • The signal is transmitted to one of two photo ASICs (amplification selective integrated circuits) each of which is comprised of 36 silicon photo diodes, of which 24 are utilized (one corresponding to each sample position within each thennocycler unit). Features of photo ASICs : • Read all K-tubes simultaneously • Optical range : 490-730 nm • Large dynamic signal range • Tolerate stronger signals than photomultiplier tubes (PMT) • Signals from photo ASICs are integrated and amplified - Monitoring of light source and background • Intensity of light source is monitored by a reference channel • When samples are not illuminated, data is collected for background (dark reading) • Sample readings are corrected for instrument fluctuations
Instru mentation
13-11
Fluorescence = (light reading-dark reading)/reference channel reading
• Titer < titer minimum • Sample within dynamic range of assay (titer given)
- Multiplex PCR reactions
• Titer> titer maximum • Invalid result
• Amplification and detection of multiple target sequences in the same K-tube can be performed • Detection is achieved using multiple probes, one specific to each target sequence, each labeled with different dyes • Dyes must be chosen carefully to minimize spectral overlap • Fluorescence signals can be separated using different filter combinations • Multiplex capacity allows for measurement of signal from internal standard - Results calculation • Pre-check • • • • •
Raw data acquired Baseline slope corrected Spikes removed Step correction Assigned Fluorescence Level determined
• Assigned fluorescence level =critical threshold line: level of detection at which a reaction reaches statistically significant increase in fluorescence over background • Threshold cycle (Ct value, crossing point, or "elbow"): fractional cycle number at which fluorescence reaches assigned fluorescence level • Ct value indicates beginning of exponential growth phase • Ct value is used for titer calculation • Assuming 100% amplification efficiency, a ten fold change in concentration changes Ct value by 3.3 cycles • Internal QS: • A synthetic construct of DNA or RNA designed to closely resemble the length and sequence content of the actual target, and therefore amplify with the same efficiency as the target • Has sufficient sequence differences from the target such that it hybridizes to a separate probe with a distinctive fluorescence signal • Incorporated into each sample in a precisely known amount, in quantitative assays • Carried through the sample preparation, and amplification/detection steps along with the target nucleic acid sequence • The difference between Ct values of QS and target is used in determination of target quantity • Can also correct for instrument, chemistry, and sample variances • Five possible results: • No target detected
• Procedure: - Computer, analyzer, and software are started - Sample and control orders are entered Sample and control orders are assigned to K-tube - K-tubes are loaded into K-carriers - Master mix and sample nucleic acid is manually pipeted into K-tubes - K-tubes are capped - K-carriers are loaded into one or both thermocyclers - Thermocycler lids are closed and run is initiated - Carriers are unloaded after completion of run - Results are reviewed • Applications: - Detection of viral, bacterial, and parasitic pathogens -
Determination of viral DNA copy number Monitoring drug therapy
- Quality control/assay validation - Quantitation of gene expression - Genotyping/detection of single nucleotide polymorphisms using allele-specific probes - Verification of microarray results • Advantages: - See chapter 3 for advantages of real-time PCR over conventional PCR • Limitations: - High cost of instrument and reagents in comparison with conventional PCR
LightCycier • General information: - The LightCycler (Roche Applied Science, Indianapolis, IN) (Figure 11) is a real-time PCR instrument with three fluorescence detection channels. - This system is compatible with SYBR Green I experiments, as well as single or dual color hybridization probe experiments. - The LightCycler instrument consists of a thermocycler component and a fluorimeter component. • Principles of operation (Figures 12 and 13): - Thermocycler • Samples and reagents are contained within glass capillaries, which are loaded into a carousel; the carousel fits into a thermal chamber • The carousel can accommodate up to 32 capillaries, thus the instrument can process a maximum of 32 samples per run
375
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Molecular Genetic Pathology
• Signal is focused onto individual glass capillaries as they are sequentially positioned over the fluorimeter optics by rotation of the carousel • Fluorescence detection • Dichroic mirrors divert light emitted from sample into one of 3 detection channels • Each channel is equipped with an interference filter with specific bandpass Emission maxima of relevant dyes
Channels
Bandpass (nm)
Channell
530 +/-20 nm
SYBRGreenl (521 nm) Fluorescein (525 nm)
Channel 2
640+/-20 nm
LightCycler-Red 640 (640 nm)"
Channel 3
710 +/-20 nm
LightCycler-Red 705 (705 nm)"
aUghtCycler-Red 640 and UghtCycler-Red 705 are FRET partners of fluorescein
Fig. 11. The LightCycler instrument (Roche Applied Science, used with permission).
• Reaction temperature is regulated by circulation of heated or ambient air through the thermal chamber; air temperature is determined by the voltage across a heating coil • The temperature is graphically displayed with LightCycler software • An auto correction function compensates for differences in heat capacity between air and water • A small reaction volume (10-20 ilL) and very high surface area to volume ratio of capillaries ensures rapid thermal transfer, allowing for fast temperature transition times, with about 15-20 seconds required for each cycle. Use of air as the heat transfer medium also facilitates high-speed cycling - Fluorimeter • Excitation signal • Energy source: blue LED • Signal passes through interference filter • Median wavelength of excitation signal: 470 nm o Fluorescein absorbance peak (maximum excitation) =493 nm o SYBR Green I absorbance peak (maximum excitation) = 497 nm
376
• Signal is received by photohybrid-type detectors • 32 capillaries can be measured in approximately 5 seconds • Fluorescence acquisition modes • Single: fluorescence is measured once per sample at end of selected temperature segment o SYBR Green I format: measurement at end of elongation phase o Hybridization probes format: measurement at end of annealing phase • Continuous: fluorescence of all samples is measured continuously from first sample to last o Used for melting curve analysis • Step: fluorescence of all specimens is measured between stepwise changes in temperature o Used for melting curve analysis • Dual color detection • Simultaneous detection of two target sequences in one sample • Two different acceptor dyes (LC-Red 640 and LC-Red 705) • Fluorescein serves as the FRET donor dye for both LC-Red 640 and LC-Red 705 • Dual color detection is used in the analysis of internal controls, interpretation of duplex PCR runs, and extended mutation analysis • Crosstalk between channels occurs due to overlap between emission spectra of LC-Red 640 and LC-Red 705 o Crosstalk is corrected by color compensation function
I nstru mentation
13-13
Air heating and cooling for rapid temperature ramping
---, \ ~
,, .,,., , .,,, ., , .,, , . .,, . ,,
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-+----
Carousel with capacity for 32 samples Stepper motor to position fluorimeter
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Fig . 12. Schematic diagram of thennocycler and fluorimeter components of the LightCycler instrument (Roche Diagnostics, used with permission).
,U ~--t-----
Carousel with capacity for 32 samples Stepper motor to position fluorimeter
Therman chamber Stepper motor to position samples over optics
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Filters Photohybrids Maintenance-free LED light source
Microvolume fluorimeter with rodenstock quality optics
Fig. 13. Detailed diagram of the LightCycler fluorimeter (Roche Diagnostics, used with permission).
377
13-14
Molecular Genetic Pathology
• SYBR Green I • Fluore scence is measured at the end of each elongation phase • Melting curve analysi s increases sensitivity and specificity • Sequence-specific hybridization probes (introduced in chapter 3) • Very sensitive and specific • Can detect single copy sequences in complex DNA templates • Two separate dye-labeled probes are used • The upstream probe has a fluorescence energy donor dye bound to the 3' end (fluore scein) • The downstream probe has an acceptor dye bound to the 5' end (LC-RED 640 or LC-RED 705) • While in solution, the distance between the probe s disallows energy transfer between them • When both probe s hybridize with the target in head to tail fashion, the donor and acceptor dyes are brought into close proximity permitting energy transfer from donor to acceptor dye (FRET), with emission of a specific fluorescent signal from the acceptor dye
o
The second derivative maximum method is preferred for most runs The fit points method is preferred for runs with few standard s or irregular standards
• Melting curve analysis • Useful for: o Product identification (SYBR Green 1 experiments) o
Identification of unwanted byproducts (e.g., primer dimers)
o
Mutation detection in hybridization probe experiments (a single point mutation can drastically alter melting temperature)
o
Distinguishing between wild-type, homozygous mutant s and heterozygotes
• Two separate mutation sites can be analy zed in one reaction using a dual probe assay • A polynomial, rather than linear method is recommended for all melting curve analyses • Procedure: - Program experiment protocol - Prepare master mix
• Measured fluorescence signal is proportional to the amount of product present in the reaction
- Place capillaries into adaptors that have been precooled in cooling block
• Fluorescence signal is acquired with each annealing phase
- Pipet master mix into capillaries
• Probes are displaced by Taq polymera se during elongation • The 3' end of the acceptor probe is phosphorylated to prevent extension • Determination of copy number • A standard curve is used in the determination of original copy number o The standard curve can be generated on the basis of standards, of known concentration, included with each run o Alternately, one can import an external standard curve produced in a previous run o Standard curve data points derive from the crossing points of each standard, which are plotted against the original copy number. (The "crossing point" is the first cycle at which fluorescence measurement significantly exceeds background level; this occurs in early log phase amplification ) o
o
The crossing point of a sample is related to sample concentrat ion, as determined by the standard curve The higher the original copy number, the lower the crossing point value
• The standard curve is calculated by either fit points method or second derivative maximum method
378
o
- Add sample DNNRNA to capillaries - Seal capillaries with stoppers - Centrifuge adaptors with capillaries (700g) briefly - Load capillaries into carousel - Close lid - Begin the run • Procedure modifications: - Carry over prevention with uracil DNA glyco sylase (UNG)/AmpErase • UNG and dUTP can be incorporated into reaction mix to minimize carry over of contaminants from previous reactions - Hot start • Minimizes primer dimer formation • Kits are available containing heat-activable Fastxtart" Taq DNA polymerase
• Anti-Taq polymerase antibody can also be employed in the hot-start technique - RT-PCR • With the appropriate kit, one-step RT-PCR can be performed using SYBR Green I or sequence-specific hybridization probes • Application s: - Kits for diagno stic use • Factor V Leiden (Roche Diagnostics)
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Instrumentation
Fig. 14. The GeneXpert instrument (right) , shown with computer and bar code scanner (Cepheid, used with permis sion).
• Prothrombin mutation (Roche Diagnostics) -
Kits for research use: numerous kits and analyte specific reagents are available from Roche Diagnostics for research applications in microbiology, oncology, hematology, and pharmaco-genetics, including assays for detection of single nucleotide polymorphisms (SNPs)
• Advantages: - See chapter 3 for advantages of real-time PCR over conventional PCR - Short run time (20-30 minutes) - Melting curve analysis in hybridization probe assays is exquisitely sensitive for detection of single base pair mutations • Limitations: - High cost of instrument and reagents in comparison with conventional PCR - SYBR-Green I : when there is a low number (1-100) of target sequences, signal may be barely measurable above background
• Ability to accommodate either 20 J!L capillary tubes or 100 J!L capillary tubes • Improved fan design for efficient heating and cooling of larger (100 J!L) capillary tubes with minimal increase in run time • Upgraded software
Genexpert" Dx System • General information: - The GeneXpert Dx System (Cepheid, Sunnyvale, CA) (Figure 14) integrates automated sample preparation and real-time PCR on one platform. All extraction, purification, amplification, and detection processes are performed within a single disposable cartridge (Figure 15). - This high level of integration permits largely hands-off operation with extremely rapid turnaround times . The system include s the GeneXpert instrument, personal computer, and preloaded software. • Principles of operation:
LightCycler 2.0 (Roche Applied Science) • General information: - The LightCycler 2.0 instrument is an upgraded version of the original LightCycler instrument with several added features • Six (versus three) detection channels (530 nm, 560 nm, 610 nm, 640 nm, 670 nm, 705 nm)
- Instrument • The instrument contains multiple (up to 16) "amplification modules " • Each module is independently controlled • Each module contains a dedicated fluorimeter with 4 excitation channels and 4 detection channels o Multiplexing capability
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Processing , , .......... chambers contain reagents . filters, and capture technologi es necessary to extract. purify, and amplify target nucleic acids
Optical windows enable real time optical detection
,,
,,
Reaction tube thin chamber enables very rapid therma l cycling
Valve" enables fluid transfer from chamber to chamber; may conta in nucle ic acids lysis and filtration comp onents
Fig. 15. Components of disposable cartridge for use with the GeneXpert system (Cepheid, used with permission).
• Each module performs continuous optical monitoring o Reaction is automatically stopped when target is detected
o Shortens time to results - Cartridges • Are self-contained, single-use • Can handle a range of volumes • Cartridges contain PCR reagents • Primers, probes, dNTPs , polymerase, and buffer components • Additional reagents must be introduced prior to use; e.g., washing, elution, and lysis reagents • Components of each cartridge:
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• Syringe drive • Rotary drive • Sonic hom o Produces ultrasonic energy, which lyses cells in raw specimen • Processing chambers o Contain reagents, filters, and capture mechanisms for washing, purifying, and concentrating nucleic acids • Reaction chamber (for amplification and detection) o Thin chamber facilitates efficient heat exchange for rapid thermocycling o Optical window s permit real-time 4-color detection
I nstru mentation
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- Many analyte specific reagent products are also available
• Procedure: - Preparing the cartridge • Cartridge and reagents are removed from package • Reagents and sample are introduced into designated chambers of the cartridge Performing the run • Computer, then the instrument, is turned on • System software is started • Cartridge bar code is scanned and sample identification information is entered • Command to start test is entered • Cartridge is inserted into module • Cartridge is removed at completion of test • Results are reviewed and interpreted • Applications: - Food and Drug Administration (FDA)-cleared assays • Group B Streptococcus • Methicillin-resistant Staphylococcus aureus
• Advantages: - Rapid turnaround time (test results from raw sample may be available in <30 minutes) - Minimal effort is required by operator - Cross-contamination is effectively eliminated, as each sample is contained within a separate sealed disposable cartridge during all phases of preparation and amplification/detection - Each unit of the GeneXpert instrument is independently controlled, thus different protocols can be run simultaneously - Extremely sensitive (capable of single cell detection) - Instrument has small footprint and minimal power requirement, making it relatively portable • Limitations: Cartridges are expensive and not reusable - Relatively few samples can be processed simultaneously
DNA MICROARRAY PLATFORMS DNA microarrays are of two basic types. cDNA arrays, currently used only in research, contain ssDNA probes ranging in length from 0.6 to 5 Kb, each of which may encompass an entire gene, a partial gene, or an expression sequence tag. A single slide may contain from 30,000-50,000 different probes . By contrast, Oligonucleotide arrays utilize much shorter DNA probes and have recently come into use in the molecular diagnostics laboratory. The details of oligonucleotide microarray systems are described as follows. • General features of oligonucleotide microarrays: - Each chip contains thousands of different oligonucleotide probes at specified locations • Each location contains millions of copies of a single probe • Oligonucleotide probes are 25 nucleotides in length, "25-mer" - Specificity is enhanced by combining probes to form probe sets • Each set contains 16-20 25-mers • Probes within a given probe set are complementary to different locations on a single target gene • Each set of perfectly matched (PM) probes for a given target has a corresponding set of mismatch (MM) probes. A "probe pair" is the combination of a PM probe with its corresponding MM probe • MM probes contain a single mismatched base in the middle of the sequence (usually the 13th position)
• At low levels of target concentration, MM probes show greater sensitivity to changes in target concentration than do PM probes • MM probes serve as internal controls for nonspecific binding and background noise
GeneChip® System 3000Dx • General information: - The Affymetrix GeneChip System 3000Dx ([GCS 3000Dx] Affymetrix, Santa Clara, CA), (see Figure 16), is a microarray instrument FDA cleared for use in clinical diagnostics. The system includes all necessary components for processing of affymetrix oligonucleotide chips, and consists of a scanner, autoloader, fluidics station, computer work station, and operational software. - The autoloader can hold up to 48 chips for walk-away performance. - The fluidics station performs washing and staining functions; it contains four modules and thus can process four chips simultaneously. Currently, an FDAapproved cytochrome P450 chip is available from Roche Diagnostics (AmpljChip'" CYP450 test) for use with the GCS 3000Dx System. Chips designed for other applications are in development. - The system supports DNA analysis, expression analysis, and resequencing applications . • Principles of operation : - Light source: solid-state green laser
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Molecular Genetic Pathology
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Fig. 16. The Affymetrix GeneChip System 3000Dx (Affymetrix, used with permission).
• Excitation wavelength : 532 nm Detector: meshle ss PMT Capable of genotyping up to 500,000 SNPs • Procedure (for AmpliChip CYP450 microarray): - Whole blood sample is obtained - PCR is performed - PCR products are applied to the microarray - Bound PCR products are stained with a fluorescent dye - Microarray is loaded into scanner • Chip is interrogated by laser - Software perform s data analysis • Genotypes of CYP2D6 and CYP2C19 are determined • Phenotype is predicted on the basis of the genotype • Applications: - Clinical diagno stics • Cytochrome P450 genotyping • Additional tests are in development • Diagnosis of leukemias • p53 re-sequencing assay - Research
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• Expression profiling • DNA analysis • Whole-genome scanning • Cancer genetic s research; analysis of loss of heterozygosity • Pathogen subtyping • Analysis of genes affecting pathogenicity and drug resistance • Pharmacogenetics • Analysis of molecul ar basis for variations in drug response • Advantage s (of Affymetrix oligonucleotide arrays over cDNA arrays): - Higher density of probe pairs than in cDNA arrays , thus more genes can be assayed on a single chip - Multiple independent measurements for each transcript enhance sensitivity and specificity - Commercially produced disease-specific oligochips are available • Limitations: - Only one sample can be run on one chip at one time - Costly
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Instrumentation
NanOCh ip 400
Fig. 17. NanoChip 400 instrument (Nanogen, used with permission).
NanoChip® 400 • General information: - The NanoChip 400 (NC400) system (Nanogen, San Diego, c;A), (Figures 17 and 18), is a low density DNA nucroarray platform, which utilizes fluore scence detection. - Each of the 400 test sites on a chip contains a microelectrode. The electrical charge of each test site can therefore be individually adjusted in order to facilitate DNA binding or control the stringency of hybridization reactions used in detection. There are two basic design scheme s for tests run on this type of instrumentation: forward allele-specific oligonucleotide (ASO), in which target DNA strands are electronically addressed to the test sites and then reacted with sequence-specific probes; and reverse ASO, in which the probes are electronically addressed to the test sites, and then reacted with target DNA. - The older NanoChip Molecular Biology Workstation employs the same principles of operation as the NanoChip 400, but is not fully automated and utilizes chips with 100, rather than 400 test sites. • Principles of operation: The NC400 system consists of the NanoChip instrument (which includes loader and reader), disposable NanoChip 400 array-containing cartridges, NanoChip 400 software, and an external computer
- Cartridges • 400 site array and 100 site array cartridges are available • Each site, or "pad ," consists of an independently controlled electrode with an overlying permeable gel layer • The permeable gel layer contains polyacrylamide with covalently bonded streptavidin • CMOS circuitry provides on-chip control, sensing, and data storage function s • Sealed fluidic chamber and laminar-flow fluidic channels conduct reagents to array - Optics • Excitation signals are produced by a red LED (626 nm) and a green LED (525 nm) • Emission signals: green (553 nm) and red (668 nm), are separated by green and red filters • Emission signal detection is performed by a CCD camera - Data analysis • Fluorescent signals are converted into a histogram using NanoChip data analysis software - Forward ASO • Amplified dsDNA product, with one strand biotinylated, is introduced into the cartridge • A positive charge is generated at a specific test site
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Molecular Genetic Pathology
• Dye-labeled universal reporter probes are hybridized to discriminator probes • Green reporter (Cy3) probe matches wild-type specific discriminator probes • Red reporter (Cy5) probe matches mutant specific discriminator probes • Conditions of stringency are applied such that all duplexes are denatured except for those which are perfectly matched • Thermal stringency: temperature is increased across all sites • Electronic stringency : charge can be modified or reversed at selected test sites • Chemical stringency: addition of alkaline solution • Fluorescence is detected by a cooled CCD (chargecoupled device) camera with LED filter for separation of green and red signals. • Forward ASO assays are most appropriate when large numbers of patient samples are tested for a small number of SNPs - Reverse ASO • Methodology is similar to above • However, biotinylated allele-specific oligonucleotide capture probes are electronically addressed to pads • Chip is probed with PCR products from one patient sample • Reverse ASO assays are most appropriate when a small number of patient samples are tested for a large number of SNPs • Procedure:
Fig. 18. Cartridge for use with the NanoChip 400 instrument (Nanogen, used with permission).
• DNA (negatively charged) in the overlying solution quickly migrates to the positively charged test site • Specificity of addressing can be further enhanced by producing negative charge at surrounding pads • DNA is permanently bound within the permeable gel layer overlying the test site electrode by a noncovalent streptavidin-biotin interaction • The chip is washed • The previous steps are repeated for addition of subsequent DNA products, each of which is addressed to a different test site • A denaturating wash is performed, leaving behind single-stranded DNA at the test sites • Target-specific discriminator probes are hybridized to immobilized target DNA strands
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- Warm cartridges to room temperature - Tum on instrument and PC - Prepare and load buffers • 50 mM Histidine, 0.1% Triton X-I 00, ProClin 300 • Water with 0.05% ProClin 300 • High-salt buffer • Low-salt buffer - Prime instrument using fluidics cartridge - Insert and initialize cartridge - Program assay protocol - Prepare samples and reagent packs and insert into instrument - Initiate protocol - View and analyze data using NanoChip 400 software - Rinse and shut down system • Applications: - Detection of SNPs, insertions, deletions, and other genetic variations - Pathogen detection - Gene expression profiling
Instru mentation
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• Advantages: - Electrical field accelerates molecular binding, decreasing reaction time Multiplexing capability With electronic addressing system, chips are easily customized by end users Extremely accurate genotyping results ; essentially 100% - High precision Low signal-to-noise ratio - High discrimination ratio (ability to distinguish homozygous from heterozygous) Microarray cartridge can be reused until all test sites have been utilized
- A test site can be addressed with a very minute quantity of DNA (50 ul, of a 5-50 nM DNA solution) • Limitations: - Detection system only recognizes two colors - Currently, limited number of FDA-approved kits for NC400, thus most assays performed on the NC400 must be developed by each laboratory. However, many analyte-specific reagents are available for the Molecular Biology Workstation; these include: • Hereditary hemochromatosis • Factor V Leiden • Prothrombin • Apolipoprotein E • Non-syndromic deafness (available in Europe) • Thalassemia (available in Europe)
xMAp® TECH NOLOGY xMAP technology (Luminex Corporation, Austin, TX) is a method suitable for high density, high-throughput diagnostic applications. Polystyrene microspheres, each measuring 5.6 11m in diameter, contain two different fluorophores in varying proportions in order to produce 100 different microsphere sets, each with a unique fluorescence color signature. Microspheres of a given color set are bound to a specific probe (e.g., oligonucleotide, antibody, receptor molecule, or peptide). After binding with a biotinylated analyte, followed by labeling with streptavidin-conjugated phycoerythrin, the microspheres are interrogated by lasers as they pass single-file through a flow cell, much akin to the mechanism of flow cytometry. In one format, multiplex PCR is performed, followed by primer extension using biotinylated dCTP. The primers used in the extension reaction are allele-specific, and each is labeled with a 5' tag sequence that does not hybridize to the target DNA. The extension product is then hybridized to oligonucleonucleotide probes connected to microspheres . The oligonucleotides on microspheres of a given color all contain the same "anti-tag" sequence, and therefore will only bind to DNA extension product corresponding to one allele. The bound product is then labeled with streptavidin-conjugated phycoerythrin. The presence of specific alleles or polymorphisms in the sample material is determined by correlation of the fluorescence signal intrinsic to each microsphere with the presence or absence of a corresponding phycoerythrin signal.
Lumlnex'Fl 00 IS System and Luminex 200 System (Luminex Corporation) • General information: - The Luminex 100 IS system is based on the principles of flow cytometry with various modifications to optimize performance with the xMAP microsphere format.
- These modifications include enhanced signal-processing performance, optimization of fluidics for single size microspheres, and development of software suitable for handling multiplex analyses with up to 100 analytes. Samples are contained within 96 well plates. - A full plate can be analyzed in as little as 40 minutes. - The Luminex 200 system (Figure 19) is an updated version of the 100 IS system with all of the features of the original instrument plus various improvements . • These improvements include simplified probe adjustment, easier maintenance access, and a highperformance air compressor. • Principles of operation : - Fluidics systems • A syringe-driven mechanism performs sample uptake and transfer to cuvet • Sample uptake volume = 20-200 ul, • A second fluidics pathway delivers sheath fluid to the cuvet and sample path - Optical systems • Two solid state lasers are used for excitation • Reporter laser o Excites fluorophores bound to analytes associated with the microsphere surface o Wavelength = 532 nm • Classification laser o Excites fluorophores embedded within each microsphere o Wavelength = 635 nm • While passing single-file through the cuvet, each microsphere is simultaneously interrogated by both lasers
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1,~.~.
Fig. 19: The Luminex 200 System includes the luminex 200 instrument, XY plate handling platform, SD sheath fluid delivery system, IS 2.3 Software, and PC (Luminex Corporation, used with permission).
• Fluorescence signals are received by multiple channels o Reporter channel o
Two classification channels
o
Doublet discriminator channel
• Detection is performed by photodiodes, PMT • Signals are digitized and delivered to a digital signal processor - xMAP Microspheres • See above (page 13-21) for general description • Calibrator microspheres • Used to normalize settings for each channel • Control microspheres • Used for verification of calibration and optical pathway integrity • Procedure: - Power on sheath fluid pump and instrument - Open Luminex software - Tum on lasers (using software) - Clear lines - Clean sample probe (ethanol wash) - Using software, select sample protocol and name samples - Load sample plate with samples and blanks - Start program - Results interpretation is performed by software
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• Background is subtracted based on measurements of fluorescence made between beads as they pass through the detector • Allele calls are based on mutant to wild-type ratio - After completion of run: wash lines, soak sample probe in water, and shut down instrument and sheath fluid pump • Applications: - Diagnostic • DNA based • Cystic fibrosis o Use of "liquid bead array" format for rapid detection of 40 different mutations implicated in cystic fibrosis • Cytochrome p450 o Multiplex detection of up to 100 different alleles in Cytochrome p450 family enzymes • Human leukocyte antigen o Multiplex detection of up to 100 different human leukocyte antigen alleles • • • • •
Leukemia testing Workup of thrombophilia Ashkenazi Jewish Panel Human papillomavirus (HPV) genotyping Liver fibrosis o Determination of cirrhosis risk in hepatitis C patients based on seven SNPs and gender
• Antigen-antibody interaction based
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of beads with various attachment chemistries (e.g., COOH, Avidin, DNA Zipcode). Kits are available from 21 different commercial partners in research and diagnostics
• Allergy testing • Diagnosis of autoimmune disease • Identification of infectious agents - Research
- The system is not limited to nucleic acid analysis, but is also compatible with detection formats based on antigen-antibody interactions, receptor-ligand interactions, or enzymatic reactions
• Cancer markers • Cell signaling • Gene expression profiling
- Excellent reproducibility-high volume of xMAP microspheres in a given lot facilitates assay standardization
• Cytokines • MicroRNA • Numerous other applications • Advantages: - Combines high-density multiplex analysis with high throughput - The xMAP format is very flexible . Users may purchase pre-made and validated kits or develop their own home-brew assays, selecting from a large menu
• Limitations: - Miscalls can result if an unknown or unexpected polymorphism is present at PCR primer or extension primer binding sites - Smaller multiplexing capability compared with microarray formats (limited to 50 mutant/Wild-type pairs)
CAPILLARY ELECTROPHORESIS
Applied Biosystems 3730 and 3730 XL • General Information: The general principles of capillary electrophoresis are discussed in chapter 3. Features of representative capillary electrophoresis instruments, the Applied Biosystems 3730 and 3730 XL (see Figure 20) DNA analyzers are described.
• Procedure: - Start computer workstation - Start the 3730 or 3730 XL instrument - Launch data collection software - Set up instrument • Prepare syringe • Install polymer blocks • Install capillary array
• Principles of operation: - These instruments utilize large capillary arrays for high-throughput sequencing • 3730 uses 48 capillaries; 3730 XL uses 96 capillaries • Capillary length: 36 cm or 50 em Capillaries are contained within a temperaturecontrolled oven (18-70°C) A built-in robotic loader can automatically load up to 16 microplates • Instrument can accommodate 96-well or 384-well plates - In-capillary detection is performed by dual-side illumination with argon laser - Fluorescence signal is detected by CCD - An on-board polymer delivery pump automatically repleni shes polymer after each run - Software performs data analysis (automated basecalling , genotyping) - Capillary electrophoresis instruments require both spatial and spectral calibration, using reagents provided by the manufacturer
-
• Add or change polymer • Fill reservoirs • Place reservoirs on deck Perform spatial and spectral calibration (if not previously done) Create instrument protocol, analysis protocol, results group Prepare samples and plate assemblie s Load plates in stacker
- View and archive data • Applications: De novo sequencing Re-sequencing -
Mutation detection SNP genotyping Microsatellite analysi s Amplified fragment length polymorphism analysis Loss of heterozygosity detection
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Fig. 20. Applied Biosystems 3730 XL DNA analyzer (Applied Biosystems , used with permission) . - Methylation analysis Restriction fragment length polymorphism analysis - Bacterial artificial chromosome fingerprinting - Serial analysis of gene expression • Advantages (over slab gel electrophoresis): - Smaller sample size - Highly econom ical use of reagents - Less hands-on time (no need to prepare gel)
- Shorter run time - Longer maximum read length for sequencing applications - No possibility for spill-over of sample into neighboring lanes - No difficulties with lane tracking No artifacts related to inhomogeneity of gel • Limitations: - High cost of instrumentation
GEL IMAGING SYSTEMS A variety of manufacturers offer gel documentation systems with different features and options. Two basic methods of illumination are epi-illumination (light shines down from above the platform) and transillumination (light shines up from below the platform). The illumination source may produce UV light, white light, blue light, or light of other specified wavelengths.
In addition to visualization of ethidium bromide stained gels (UV transillumination), these instruments have many other applications; for example, visualization of bacterial colonies on an agar plate (white light transillumination), and visualization of bands on TLC plates or other opaque surfaces (epi-illumination). The optimal instrument depends on the imaging requirements of the laboratory. Representative systems are described as follows .
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Bio-Rad Gel Doc™ EQ, ChemiDoc™ EQ, and ChemiDoc™ XRS • General Information : - These gel documentation systems use a CCD camera to acquire images in real-time, which allows for accurate positioning and focusing. Images can be analyzed, optimized, and printed using Bio-Rad Quantity One" software (for Windows or Macintosh). The gel or other material to be visualized is placed on a platform within a light-tight hood. Visualization is accomplished using UV or white light illumination . The ChemiDoc XRS system (Bio-Rad, Hercules, CA) is described below (see Figure 21).
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I nstru mentation
• White light transilluminator • White light conversion screen • 302 nm UV lamp • 254 nm UV lamp • 365 nm UV lamp - Peripheral component interconnect (PCI) Digitizing Card (included with system) • Converts video signal to computer image • Must be inserted into PCI slot of Macintosh or PC computer I
.
Quantity One software (included with system) is used for: • Annotation of images • Analysis of molecular weights • Other applications - Two printers are offered by Bio-Rad for use with these systems : • Analog video printer (Mitsubishi P-91W) • USB digital printer (Sony UPD895) • Procedure: - Instrument and computer workstation are powered on - Material to be visualized (e.g., a gel) is placed on the instrument platform - The platform is slid into place, closing the instrument and producing a light-tight seal - Appropriate filter is selected - Light source is activated (e.g., for UV transillumination)
Fig. 21. Molecular Imager ChemiDoc XRS System (Bio-Rad, used with permission).
Gel (or other material to be documented) is visualized in real-time - Image is acquired and optimized using Quantity One® software Image may be stored and printed
• Principles of operation: Camera • The CCD camera has a motori zed zoom lens • Zoom, focus , and iris function s are remotely controlled • Optional lense s are available for low light applications (e.g., chemiluminescence) - Illumination sources and filters • Built-in white light epi-illumination and UV transillumination • A standard filter (548-630 nm) for ethidium bromide is provided with the system • Optional filters are available for SYBR Green, green fluorescent protein , SYBR Gold, Fluorescein, CY3, Rhodamine, SYPRO Ruby, Texas Red, and HoechstlCoumarin - Optional light sources are also available:
• Applications: - Transillumination for ethidium bromide stained gels • Optional filters permit visualization of other fluorescence markers - White light epi-illumination for visualization of opaque materials - Optional white light transillumination can be used for visualization of bacterial colonies on agar media • Advantages: - Enclosed hood obviates the need for a darkroom - Enclosed hood prevents operator exposure to UV radiation - Software simplifies optimization and archiving of images - Does not require film • Limitations: - Initial cost of instrumentation greater than for traditional methods (e.g., polaroid camera)
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LUMINOMETERS Luminometers are instruments designed to detect light production in a wide range of assays, often in a 96-well microplate format. Key parameters in the function of these instruments are sensitivity, dynamic range, and capacity to minimize or reject crosstalk. • Applications include: - Reporter gene assays (e.g., detection of lucifera se activity) - Chemiluminescent assays for DNA quant ification Biomass assays - Branched DNA assays - Immunoassays using alkaline phosphatase, horseradish peroxidase, or other enzymes to produce chemiluminescent products upon reaction with substrate • Hybrid capture assays (see discussion of Digene Microplate Luminometer below)
- A PMT collects light in the visible spectrum (300-650 nm) • The current produced by the PMT is directly proportional to the number of incoming photons • If intensity is too great, an iris closes , limiting the amount of light reaching the PMT - Crosstalk (contaminating light from neighboring wells) is minimized by use of the proper microplates , use of a specialized optical pathway, and microplate mask - Signals from the PMT are amplified and converted to relative light units (RLU) - RLU values are transmitted to an external computer - Measurements and results calculation • Luminometer performs a background reading • Luminometer reads light intensity from each plate well • Software subtracts the background reading from each well reading to produce raw RLU values • Software perform s validation
Various microbiologic applications
Digene Microplate Luminometer (DML2000) • General information: - The DML2000 (Digene Corporation, Gaithersburg, MD) (Figure 22) performs measurement and analysis of light derived from chemiluminescence in Digene hybrid capture system tests such as the Digene HPV test. - The Digene HPV test (discus sed in detail in chapter 3) is a signal amplification assay, which uses HPVspecific RNA probes to hybridize with HPV DNA. The DNAIRNA hybrids are then "captured" by hybridspecific antibodies lining the wells of a microtiter plate . A second hybrid-specific antibody conjugated to alkaline phosphatase is then reacted with the immobilized DNAIRNA hybrids . Subsequently, a substrate is introduced, which upon reaction with alkaline phosphatase produces a chemiluminescent product that emits light detectable by the luminometer. Multiple alkaline phosphatase molecules are conjugated to each antibody, and multiple antibodies bind with each target DNAIRNA hybrid resulting in substantial signal amplification. - The DML 2000 system is also compatible with other hybrid capture assays from Digene including C. trachomatis, N. gonorrhoeae, and CMV. - Components of the system include the Digene Microplate Luminometer, quantitative software, computer, and peripherals. • Principles of operation: - The luminometer measures luminescence in 96-well microplates - A stepper motor moves each well sequentially over the detector
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• Results are calculated • Calibrators, negative control , and quality control samples are included with each run • A cutoff value is determined, based on the measured luminescence of the calibrator • Results are expressed as (specimen RLUlcutoff value); on the basis of this ratio, results are reported as either positive, or negative • Some laboratories also use an " indeterminate" category for results near the cutoff • Procedure: -
Tum on luminometer, computer Allow I hour for luminometer to warm up Run DML 2000 software Allow luminometer to perform self tests
-
Insert microplate Load plate layout Measurements are performed Print raw data Remove plate
- Insert additional plates (repeating above steps) - Print report - Close software, turn off computer • Applications: - For use with Digene hybrid capture tests: • HPV (High and Low risk) • CMV • C. trachomatis • N. gonorrhoeae
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Fig. 22. The DML 2000 system (luminometer is on left) shown with other components used in the Digene HPV test (Digene Corporation, used with permi ssion).
• Advantages: - Wide and very linear dynamic range • Dynamic range: 10 to 5 X 106 RLU and 20 RLU to 5 x 106RLU). R2> 0.999 - Close proximity of PMT to microwellsenhances sensitivity
• Limitations: Luminometer is sensitive to condensation due to high humidity. In environments with high humidity or significant changes in humidity, unit must be continuously left on to avoid internal condensation
FLUORESCENCE MICROSCOPE • General information: - Fluorescence is the phenomenon whereby certain chemical species can absorb light at a specific wavelength and after a brief interval (fluorescence lifetime) emit light at a longer wavelength . - Fluorescence microscopy has many research applications. In the realm of diagnostics, this technology is mainly used in fluorescence in situ hybridization (FISH) , in the diagnosis of autoimmune disorders, and some applications in microbiology. Although many manufactures produce different types of fluorescence microscope, the basic principle is the same (Figure 23). • Principles of operation: - A very high-intensity light source (usually a mercury or xenon arc lamp) induces fluorescence in the sample
- The objective lens serves a dual function (epifluorescence) • It focuses the excitatory light beam on the sample • It collects light emitted from the sample - A "filter cube" composed of an excitation filter, an emission filter, and an intervening dichroic mirror separate s the excitatory signal from the emis sion signal • The excitation filter selects a wavelength appropriate for excitation of a target fluorochrome • The emission filter is selective for the emission signal wavelength, and thus blocks any contaminating light from reaching the oculars or camera tube • The dichroic mirror reflects the excitatory signal, such that it enters the objective lens, while transmitting (rather than reflecting) the emission signal
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t Filter cube
-----IL....------l Excitation signal source
Objective lens
Slide
Fig. 23. Schematic diagram illustrating the essential optical components of a fluore scence microscope.
• Each filter cube is optimized for a given fluorochrome, in terms of its selectivity for excitatory and emission signal wavelengths • To allow for detection of different fluorochromes, multiple filter cubes are mounted on a turret. A specific filter cube is selected by rotating the turret The emission signal is focused by a projection eyepiece to produce a fluorescence image The image is photographed and can be processed by image analysis software • Procedure:
- See chapter 12 • Applications: FISH Infectious diseases • Direct fluorescent antibody for detection of specific microorganisms
• Acridine orange fluorescent stain for non-selective detection of microorganisms • Auramine-rhodamine stain for detection of acid fast bacilli Indirect fluorescent antibody • Detection of microorganisms • Diagnosis of autoimmune disorders Numerous research applications in cell biology • Advantages: See chapter 12 for discussion of advantages of FISH in detection of chromosomal abnormalities Fluorescent stains are generally more sensitive than conventional stains for the detection of microorganisms • Limitations: - More expensive than conventional microscopy
SUGGESTED READING C&L Instrumentslast accessed June 19, 2007, Inc: Tutorial-fluorescent microscope optics: http://www.fluorescence.comltutoriallfm-optic.htm. Feng L, Nerenberg M, Electronic microarrayfor DNA analysis. Gene Therap Mol Bioi. 1999;4:183-191.
Heller C. Principles of DNA separation with capillary electrophoresis. Electrophoresis 200I;22(4):629-64 3. Heller MJ, An active microelectronics device for multiplexDNA analysis. Eng Med Bioi IEEE . 1996;15(2):100-104.
Holland PM, Abramson RD, Watson R, et al. Detectionof specific polymerase chain reaction product by utilizing the 5' ~3 ' exonuclease
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activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci . 1991 ;88:7276-7280. Keen-Kim D, Grody WW, Richards CS, Microelectronic array system for moleculardiagnostic genotyping: Nanogen NanoChip400 and molecular biology workstation. Expert Rev Mol Diagn. 2006;6(3) :287-294. Kohane IS, Kho AT, Butte AJ. Microarrays for an Integrative Genomics. Cambridge, MA:MIT Press; 20m , pp. 69-88. National Center for Biotechnology Information (NCBI)last accessed June 19, 2007, Microarrays: chipping away at the mysteries ofscience and medicine. http://www.ncbi.nlm.nih.gov/Aboutlprimer/microarrays.html.
14 Genetic Inheritance and Population Genetics Tatiana Foroud,
PhD and
Daniel l. Koller,
PhD
CONTENTS
I. Polymorphisms ..................................14-2 II. Hardy Weinberg Law ..........................14-2 III. Autosomal Recessive Inheritance ......14·3 IV. Autosomal Dominant Inheritance ......14·6 V. X-Linked Dominant Disorders ............14-7
VI. X·Linked Recessive Disorders ............14-8 VII. Gene Mapping and Recombination ........................ 14-8
VIII. Disorder with Complex Genetic Inheritance....................................14-11
IX. Suggesting Reading ..........................14-11
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Molecular Genetic Pathology
POLYMORPHISMS • Variation in DNA sequence produces genetic polymorphisms (Greek: many forms) . A polymorphism is any DNA sequence in which the less common allele occurs at a frequency of 1% or greater. The first polymorphisms that were identified were those defining the serologic blood groups. Subsequently, many more polymorphisms have been identified • Single nucleotide polymorphisms (SNPs) are a single base pair change in the DNA sequence that can be found in either the coding or non-coding region of the DNA. Many of the originally identified polymorphisms were SNPs;
however, most were in the coding region and resulted in a phenotypic effect. Many of the SNPs used today do not result in any noticeable phenotypic effect. SNPs are estimated to occur, on average, every 300 bp, suggesting that there are over 10 million SNPs in the human genome • Another type of polymorphism is a simple sequence repeat. This type of polymorphism is often termed a microsatellite and consists of alleles defined by a variable number of two, three , or four tandem nucleotide sequences. These polymorphisms are commonly used to map disease genes
HARDY WEINBERG LAW • Population genetics is defined as the study of genes in populations as distinguished from the study of the behavior of genes in families. However, this is a rather narrow definition since this area of genetics encompasses, for example, inbreeding and prediction of recurrence of a disorder in families • The fundamental law in population genetics is the Hardy Weinberg Law. It is named after the English mathematician G.H. Hardy and the German physician W. Weinberg who independently published their versions of the law in 1908 • The Hardy Weinberg Law has important implications in the field of population genetics and for risk assessment for members of families with genetic disorders. The importance of the Hardy Weinberg Law lies in its ability to take information about the frequency of alleles in the population and then make predictions about the frequency of genotypes in the population • For example, this law allows us to estimate the frequency of gene carriers in a population. Then, this risk or frequency can be used to estimate the probability that another family member will be affected with a particular disease. The Hardy Weinberg Law defines the frequencies of genotypes in a randomly mating population based on the frequencies of the alleles at a locus. By random mating, we mean that matings occur without regard to the genotypes of the individuals
• The following illustrates the Hardy Weinberg Law with random mating for a locus with two alleles: A and a. The frequency of the two alleles are p and q, respectively, and the sum of the two allele frequencies is 1. There are three possible genotypes at this gene. An individual can have two copies of allele A (i.e., homozygous) and thus have genotype AA. The individual could have one copy of the A allele and one copy of the a allele (i.e., heterozygous), and thus have genotype Aa. The individual could have two copies of allele a (i.e., homozygous) and have genotype aa • If the assumptions of the Hardy Weinberg Law are met (Sidebar A), then the frequency of these three genotypes (AA, Aa and aa) in the population can be estimated using the allele frequency estimates of A and a obtained from the same population. The relationship between the estimated allele frequencies and the genotypic frequencies are the basis of the Hardy Weinberg Law and are shown below in Figure 1. Therefore, the Hardy Weinberg Law allows us to estimate the frequency of the three genotypes at this locus in the population to be: Frequency of AA =p2, Frequency of Aa = 2pq, and Frequency of aa = q2 • The Hardy Weinberg Law also holds for loci with more than two alleles . The frequency of a given homozygote, AjA j, is pj2 and for a heterozygote, AjAj , is 2PPj
Sidebar A: Assumptions of the Hardy Weinberg Law • Random mating - Individuals select their mate at random and do not select individuals with particular disease or clinical traits • Consta nt mutation rate - The frequency at which a normal allele mutates to an abnormal allele is the same as the rate at which an abnormal allele sustains another mutation • No selection
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Individuals of all geno types reproduce at similar rates (i.e., there is no selection aga inst the reproduction of a particular genotype) • No fluctuation of gene frequencies due to migration or random causes - The composition of the population genotypes does not alter due to the entrance or departure of individuals with certain genotypes, or due to chance, in small populations (genetic drift)
Genetic Inheritance and Population Genetics
14-3
Male Gametes A Allele
a Allele
Frequency of A
Frequency of A
allele, p
allele, q
Female
A allele
Frequency of A allele, p
AA (p x p)
Aa (p x q)
Gametes
a allele
Frequency of a allele, q
Aa (p x q)
aa (q x q)
Fig. I. Transmission of gametes .
Sidebar B: Using the Hardy Weinberg Law to Estimate Genotypic Frequencies Consider the P-globin locus to have two alleles. The normal allele is termed pA and an abnormal allele, ps. Individual s who have two copie s of the pA allele are considered normal. Individuals who have one normal allele and one abnormal allele, with genotype pA ps, have sickle trait. Those individuals who have two abnormal copies of the beta globin gene, genotype ps ps, have sickle cell disease. Using the counting method, the frequency of the pAallele is estimated to be ([2{884} + I {112})/[2 x 1000]) = 0.94 while the frequency of the ps allele is 0.06. Therefore, in a sample of 1000 individuals we would expect the three genotype s to occur in the following frequencie s
Phenotype Genotype Norm al
I3 A I3 A
Sickle trait
I3 A 13 5
Sickl e cell anemi a
135 13 5
Frequency of genotype
Number of individuals
p2= (0.94)2 = 0.884
884
2pq = 2(.94) (.06) = 0.112
(p = (0.06)2
112 4
= 0.004
AUTOSOMAL RECESSIVE INHERITANCE • There are many disorders with developmental disabilities that are inherited with an autosomal recessive pattern of inheritance. Individuals are only affected with the condition if they have inherited two abnormal alleles at the disease gene . Typically, the abnormal allele for an autosomal recessive disorder is denoted by an a and the normal allele is indicated by A. A characteristic pattern of autosomal recessive inheritance is horizontal transmission. This means that affected individuals are only observed in a single generation, and typically are siblings • When studying autosomal recessive disorders, the only individuals whose genotypes are known with certainty are typically those who are affected with the disease, and therefore must have genotype aa. Individuals who are unaffected may be either homozygous for the normal allele or might be heterozygous. Those individuals who have one normal and one abnormal allele at the disease gene are often termed "carriers"
• In populations meeting the assumptions of the Hardy Weinberg Law, we can estimate the frequency of a heterozygous individual to be 2pq. If the frequency of individuals affected with a particular autosomal recessive disease is I in 2500. Then, using the Hardy-Weinberg Law, we would assume the frequency of affected individuals =q2 = 1/2500. From this formula, we would estimate the frequency of the disease allele (q) to be the square root of I in 2500, which is I in 50. We can estimate that the frequency of the normal allele must be 49/50 . Using the frequency of the two alleles, we can now estimate the frequency of a disease allele carrier for this particular disease to be: Frequency of disease allele carrier = 2pq = 2(0.02) (0.98) = 0.0392 . However, since the disease is relatively rare, the frequency of the normal allele (p) is very close to I and the frequency of the disease allele (q) is very small. Then, the frequency of a carrier = 2pq is approximately 2( I)q = 2q. For this particular disease , estimating the frequency of
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heterozygotes as 2q results in an estimated carrier frequency of I in 25, which is 0.04. Thus, the frequency of a gene carrier in a population
meeting the assumptions of the Hardy Weinberg Law is simply twice the frequency of the disease allele (Table 1)
Sidebar C: Phenylketonuria (PKU) PKU, which has a frequency of 1/1 0,000. Then, the frequency of (/ = 1/10,000 and the frequency of the disease allele is: q =-V 1/1 0,000 = 1/100 =0.01. From this, we can estimate the
frequency of heterozygous disease allele carriers to be twice the disease allele frequency (2q) , which is 1150.
• With the diagnosis of an individual with an autosomal recessive disorder, many unaffected family members will be interested in determining whether or not they have also inherited a copy of the mutant disease allele and what the risk of disease is for their children
• The remaining individual in the family, the unaffected son, could have two possible genotypes. He could have inherited two normal alleles and have genotype AA or he could have inherited one normal and one abnormal allele and have genotype Aa. Both genotypes would result in an individual without symptoms of the disease ; therefore, we cannot determine an individual's genotype through a careful physical examination or clinical evaluation
• In Figure 2, a common clinical scenario is shown. In the left portion of the figure, a family is shown consisting of two parents and their two children . Their daughter has an autosomal recessive disorder. The affected daughter is fully shaded to denote that she is affected with the disease and must have two abnormal alleles at the disease gene. Her unaffected parents are half shaded to indicate that they must be carriers • The right panel of Figure 2 illustrates the potential transmission of alleles from the parents to their children . Since each heterozygous parent will, on average, transmit the abnormal allele 50% of the time, we would expect that 25% of their children will inherit two normal alleles (genotype AA) and be unaffected. We would also expect, on average, that 50% of their children will inherit one normal and one abnormal allele (genotype Aa) and also be unaffected . On average, 25% of their children will inherit from each parent an abnormal allele, resulting in genotype aa and an affected child. Thus, in this hypothetical scenario , the genotype of three individuals is known with certainty. The parents both have genotype Aa and the affected child has genotype aa
• We can estimate the likelihood that the son has genotype AA or Aa. Assuming that he cannot have genotype aa, we must recompute the risk that he is heterozygous at the disease locus, by only considering the two genotype s that he can be, which are AA and Aa. Using the right portion of Figure 2, we see that the each of the boxed genotypes is equally likely. Therefore, if we eliminate the possibility that he is aa, the likelihood that he is AA is one of the three remaining boxes and is 1/3. The possibility that he is Aa is two of the remaining three equally likely boxes, which is 2/3. This can be shown mathematically using the following formula: Probability the son has genotype AA, since he is unaffected =P(AA)/(P[AA] + P[Aa)) =(1/4)/(1/4 + 1/2) = (1/4)/(3/4) = 1/3. Probability the son has genotype Aa, since he is unaffected = P(Aa)l(P[AA] + P[Aa]) = (1/2)1(1/4 + 1/2) = (1/2)/(3/4) =2/3
Table 1. Frequencies of Genotypes, Genes, and Carriers for an Autosomal Recessive Disorder Disease frequency (q2)
Gene frequency (q)
Carrier frequency (2q)
1140
1120
80
1110,000
1/100
1150
200
1140,000
11200
1/100
400
1/250,000
11500
11250
1000
111600
Carrier frequency/disease frequency
An important point to note is that carriers are much more common than individuals with the disorder; the rarer the disorder, greater is the relative frequency of carriers (last column).
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Paternal alleles 1
I.
~ 1
II.
2
2
A Maternal alleles
a
A~ a~
?
Fig. 2. (A) Pedigree segregating an autosomal recessive disorder. (B) Transmission of alleles in autosomal recessive inheritance.
Fig. 3. Risk assessment for a family segregating an autosomal recessive disorder.
• Familie s having a member with an autosomal recessive disorder often wish to know the risk of disease to other family members. Figure 3 illustrates a common scenario. In this family, a couple has come for more information regarding their risk to have a child with a disorder similar to that of the wife's sister
second generation is heterozygous at the disease gene. Using the Hardy Weinberg Law, we would use the estimate of the frequency of the disease allele in the population (q), and from this information , estimate the probability that he is a carrier for 2q (Step 1)
• To address this question , we need to address three questions. - What is the probability the father in the second generation is a carrier? - What is the probability the mother in the second generation is a carrier? - If they are both carriers, what is the probability their child will have the autosomal recessive disease ? • If we apply the steps listed above sequentially, we first have to estimate the probability that the father in the
• His wife has an affected sister. We know that she does not have symptoms of the disease; therefore, we know that her genotype is not aa. Therefore, her genotype must be AA or Aa. We have shown that the probability that she would have genotype AA is 1/3 and the probability that she would have genotype Aa is 2/3 (Step 2) • Even if both parents are carriers, it is still only 25% of their children who will have the disease (Step 3) • Therefore, the probability that this couple would have an affected child is the product of each of these probabilities. The answer would therefore be: (2q) (2/3) (1/4) = q/3
Sidebar D: Counseling A Family With PKU The family shown below comes to learn more about their risk to have a child with PKU. The wife has in a previous union had a child with PKU. She now has a new partner and wants to know the probability that another child will have PKU Since the woman has already had a child with PKU, we know that she must be heterozygous at the PKU locus. Her new partner does not have any family history of PKU. Therefore, the probability that he is a carrier of a PKU mutation is the same as from the general population (i.e., 2q). To estimate the frequency of a gene carrier, we use the information that the frequency of PKU is 1/10,000, so the frequency of heterozygotes in the population is 1/50. Therefore, the probability that the woman would have a child with PKU with her new partner is: (1/50) (I) (1/4) = 1/200
• If a disease is rare , then the probability that two individuals who are heterozygous for the disease gene will marry becomes exceedingly rare. The probability that two individuals might both be carriers for the mutant allele increases if the two individuals have both
?
inherited the allele from a common ancestor (i.e., consanguinity). Consanguinity is more common in certain communities or countries. As a result, the descendents of a founder, who was heterozygous for recessive mutant allele, may have a higher frequency of
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1-1
11-1
11I-1
1-2
11-2
111-2
11-4
11-3
111-3
111-4
111-5
111-6
Fig. 4. Pedigree with consanguinity segregating an autosomal recessive disorder.
a rare disease. In general, there is a higher frequency of consanguinity among parents of children with rare autosomal recessive disorders. When consanguinity occurs, it is not appropriate to utilize the Hardy Weinberg Law to estimate the probability that an individual in the pedigree is the carrier for an autosomal recessive disorder • Consider the pedigree shown in Figure 4. The parents of individual IlI-2 are both carriers of the autosomal recessive disorder. Individual II-2 inherited his disease allele from one of his parents . Therefore, if we arbitrarily assign the disease gene to his father, 1-1, then his father is a heterozygote. The probability that his daughter (II-3) inherited the disease alIele is 1/2. If II-3 inherited the
disease allele, the probability that she transmitted the disease allele to her daughter (IlI-4) is 1/2. So, the risk that IlI-4 is a carrier is (112) (1!2) = 1/4. The probability that IlI-3 is a gene carrier is 2/3, since both his parents are carriers and he is not affected. Then, using the three steps to calculate risk, the probability that this couple would have a child with the same disorder as IlI-2 is (1/4) (2/3) (1/4) = 1/24 • Consanguinity results in a higher frequency of recessive disorders . One cause of consanguinity is incest between a parent and child. The result of incest among such firstdegree relatives is a 40% risk for a handicapped child. The most common handicaps associated with incest are mental retardation and seizures
AUTOSOMAL DOMINANT INHERITANCE • Unlike autosomal recessive inheritance where the genotype of the affected person is unambiguous, in the case of autosomal dominant inheritance it is the affected individual who may have an ambiguous genotype . They could have either the AA or Aa genotype (Figure 5) • Since individuals who are homozygous for the disease allele are very rare, the vast majority of affected individuals have the genotype Aa. Therefore, we can estimate the frequency of the disease alIele by assuming that all affected individuals are Aa. Thus, the frequency of affected individuals is simply the frequency of heterozygotes , as estimated using the Hardy Weinberg Law. While the exact estimate of heterozygotes would be 2pq, we note that the frequency of the normal allele (p) is
398
Genotype
AA
Aa
aa
p2
2pq
q2
t
t
t
Affected
Affected
Unaffected
Fig. 5. Hardy Weinberg Law for an autosomal dominant disorder. Note that p is the disease allele.
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close to I, as we did for the autosomal recessive situation, so the frequency of the (heterozygous) affected individuals can be approximated as 2p • For example, the disorder dentinogenesis imperfecta is an autosomal dominant disorder. The frequency of affected individuals is 1/8000. Therefore, 2p = 1/8000 and we estimate the frequency of the disease allele (p) to be 1/16,000. In other words, the frequency of an autosomal dominant disorder is twice the gene frequency • In some instances, an individual may have an autosomal dominant disorder and there may not be any family history of the disorder. In this instance, it is likely that the individual who is affected has had a mutation in one of the alleles of the disease gene. This mutation occurred spontaneously in either the egg or sperm, which produced this individual. As a result, neither of the parents has the mutation in the other cells of their body, and hence they do not have the disease phenotype
• New mutations are more likely in disorders that are very severe, especially those that are lethal in childhood, or prevent reproduction . Diastrophic dysplasia and osteogenesis imperfecta, type II, both of which are perinatal lethal conditions leading to severe bone and cartilage abnormalities, are virtually all the result of new mutations. New mutations are more likely in disorders that limit the ability to reproduce (i.e., reduce fitness of the individual) . Specifically, if the disease remains at a constant frequency in the population, then those disease alleles that are lost as a result of affected individuals decreased fitness must be replenished through new mutations • As an example, individuals with achondroplasia have only 20% as many children as individuals of normal height. It is estimated that >80% of cases of achondroplasia are the result of new mutations. It appears that the frequency of mutation increases with paternal age
X-LINKED DOMINANT DISORDERS • When a disorder has X-linked dominant inheritance, both male and females may be affected with the disorder. Importantly, affected males would have a mutation in their only X chromosome while an affected female would typically have a mutation in at least one of their two X chromosomes • Using the principles of the Hardy Weinberg Law, we can estimate the frequency of each genotypic group. Since males have only one X chromosome and females have two X chromosomes, we have to consider the frequency of the genotypes in the two sexes separately, as shown in Figure 6 • In the left panel of Figure 6, the frequency of affected males is simply the frequency of the mutant allele (p).
The frequency of unaffected males is simply the frequency of the normal allele in the population (q). On the other hand , the frequency of unaffected females is the square of the frequency of the normal allele (q2), as was true for an autosomal dominant disorder. Similar to the autosomal dominant situation, affected females can have one of two possible genotypes (AA or Aa), although the vast majority will be heterozygous. Since most of the affected females will be heterozygotes, the frequency of affected females can be estimated as the frequency of heterozygotes, which is 2pq, which is approximately 2p. Using this approximation, for a rare, X-linked dominant disorder, the ratio of affected female to affected male will be about 2: 1 (i.e ., 2p:p)
Females
Males A
a
p
q
M
Aa
aa
2pq
q2
t
t
t
Affected
Affected
Affected
Fig. 6. Hardy Weinberg Law for an X-linked dominant disorder.
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Males
Females
A
a
AA
Aa
aa
p
q
p2
2pq
q2
t
t
Affected
Affected
Fig. 7. Hardy Weinberg Law for an X-linked recessive disorder.
X-LINKED RECESSIVE DISORDERS • When a disorder has X-linked rece ssive inheritance, the vast majority of the affected individuals will be males (Figure 7). Similar to X-linked dominant inheritance, since males have only one X chromosome, the frequency of an affected male is simply the frequency of the disease allele in the population (q) (left panel, Figure 7). Using the principles of the Hardy Weinberg Law, we can estimate the frequency of each genotypic group in the females . Females are only affected if both of their alleles are mutant; therefore, the frequency of affected females is q2. Similar to autosomal recessive inheritance, females who are homozygous for the
normal allele or heterozygous for the normal allele will be unaffected • When considering a rare, recessive X-linked disorder, the frequency of homozygous females is the square of the frequency in males (i.e., q2 vs q), and therefore homozygous affected females are extremely rare. Another important principle is that the frequency of heterozygous carrier females is approximately 2q, or twice the frequency of affected males. Lastly, it should be noted that 1/3 of the mutant alleles are in males while 2/3 of the mutant alleles are in females . Therefore, there are twice as many carrier females as affected males
GENE MAPPING AND RECOMBINATION • During the past decade there has been substantial progress made in elucidating the genes that contribute to many genetic disorders. This has been accomplished through the use of genetic linkage analysis , an experimental method in which many families having affected members are studied so as to identify a chromosomal region inherited by all affected family members in a family and which is not inherited by unaffected family members • The basic principle underlying genetic linkage analysis is the study of the segregation of homologous chromosomes in meiosis. A gene, typically a disease gene, is considered linked to a chromosome if the gene of interest cosegregates with a gene or marker known to be on that chromosome. During meiosis I, homologous chromosomes pair together and cross over occurs (Figure 8). On average, about 30-40 cross overs (about 1-2/chromosome) occur during a meiotic division. Genes that are close together on a chromosome will tend to be inherited together, violating Mendel's second law of independent segregation. Genes that are far apart on a chromosome will be more likely to have a cross over occur between them. Genes on different chromosomes will segregate independently of each other
400
• The observed result of crossing over is recombination, often denoted by the symbol theta (9). The recombination fraction (9) can take values between 0 and 0.5. When the
Fig. 8. Crossing over of chromosomes during meiosis .
Genetic Inheritance and Population Genetics
Normal
Normal
A
A
disease gene is located and sequentially refine its location by analyzing more markers in that region. The Lod Score Method will also determine how far a disease gene is from the marker being tested by estimating e, the percentage of recombination between the disease gene and the marker. Thus, if a disease gene is estimated to be 1 cM from a marker, then there is typically recombination between the disease gene and the marker on 1% of the chromosomes during meiosis
Fig. 9. Phase for a double homozygote.
Disease
Normal
A
A
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• For all autosomes , that is chromosomes 1-22, an individual has a pair of chromosomes, with one member of the pair maternally inherited and the other paternally inherited . When studying the segregation of marker and disease alleles, we are interested in determining which allele, at each of the two loci, is on the same chromosomal strand. The orientation of the four alleles (two at the disease locus and two at the marker locus) on the pair of chromosomes is termed phase (see Figure 9 for an illustration)
Fig. 10. Phase for a single heterozygote.
recombination fraction is 0.0, this means the two loci are so close together on the chromosome that recombination never occurs between them. When the recombination fraction is 0.50, this indicates the two loci are segregating independently and are either far apart on the same chromosome or are on different chromosomes • The estimate of recombination between two loci can be used to approximate the physical distance between the two loci. A recombination fraction of 1% corresponds to roughly 1 cM or 1 million bp of DNA • The identification of common DNA sequence variation has rapidly altered the study of genetics. The identification of these sequence variants results in a polymorphism (Greek: many forms) of the DNA sequence . These sequence variations are commonly termed genetic markers and have become an indispensable tool in the study of genetics • A disease gene can be mapped to a chromosome by studying the cosegregation of the disease gene with a marker of interest in families having individuals affected with the disease. This approach is termed the Lod Score Method . Using the Lod Score Method , it is possible to determine a small region on a chromosome where a
• Phase is an important concept in the Lod Score Method and refers to the alleles at each locus, which are on the same chromosome. It is essential that phase be known, or at least estimated, in order to determine whether or not recombination has occurred during meiosis. If a marker and disease gene are physically near each other on the same chromosome, recombination is less likely to occur between them. Through the examination of many meiosis, it is possible to perform a statistical test, which will determine whether a disease gene and the marker being tested are close together on the same chromosome. This provides the first piece of information needed to determine the location of a disease gene within the human genome • For individuals who are homozygous at both loci, it is easy to set phase, unambiguously. In fact, the two chromosomes are identical at these loci
• In some instances, the individual is heterozygous at only one of the two loci. For example, the individual could be heterozygous at the disease locus, which would correspond to an affected individual for an autosomal dominant disorder or a carrier for an autosomal recessive disorder. At the marker locus, the individual would then be homozygous (see Figure 10). In a second example, the individual could be homozygous at the disease locus and heterozygous at the marker locus. For individuals who are heterozygous
Disease
Normal
Disease
Normal
A
B
B
A
Fig. 11. Possible phase for a double heterozygote.
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AlA
BIB
II
AlA
AlB
7
III
AlA
AlB
AlA
AlB
AlB
AlA
AlB
Fig. 12. Example of an autosomal dominant disorder with marker data.
at only one of the two loci, phase can still be set unambiguously. Importantly, the two chromosomes will not be identical • If the individual is heterozygous at the disease locus and also heterozygous at the marker locus, there are two possible ways to arrange the disease and marker genotypes (Figure 11). These two possibilities are mutually exclusive • The pedigree (Figure 12) is segregating an autosomal dominant disorder. The first thing to consider in a pedigree of this type is what each person's genotype is at the disease locus. The affected individualsare all heterozygous at the disease locus (i.e., Did), since this is an autosomal dominant disorder and the unaffected individuals are all
homozygous (did). Once we know this, we can begin to determine the genetic phase of each individual • It is typically easiest to start with those individuals whose phase is unambiguous. For an autosomal dominant disease, first assign the phase to the individuals who are unaffected, or who are affected and are homozygous at the marker. This allows the phase of I:I, 1:2, II:2, III:2, III:4, and III:? to be determined • Then, since you know that II:I inherited his disease allele from his father, I:I, we can determine the phase for II: I. Similarly, since we know that II:2 must have given each of her children a normal allele at the disease locus and an A allele at the marker, the phase of all the affected children can now be determined
II
AlA
7 III
AlA
AlB
AlA
AlB
AlB
AlA
AlB
PhaseA
NR
NR
NR
NR
R
NR
NR
Phase B
R
R
R
R
NR
R
R
Fig. 13. Example of an autosomal dominant disorder with marker data.
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• Reviewing the pedigree , we see that individuals III: I, 2, 3, 4, 6, and 7 all are non-recombinant while individual III:5 has had a recombination between the disease and the marker on the chromosome he inherited from his affected father • When there is data on only two generation s of individuals, the phase of the marker and disease locus cannot be determined unambiguou sly. For example , consider the pedigree shown in example 2, which is the same as the previous pedigree, but without generation I (Figure 13) • When we try to determine the phase of the unaffected mother, 1:2, her phase is still known unambiguously,
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since she is not a double heterozygote. However, the phase of individual I:1 is now unclear. He can be either of the 2 phases shown in Figure 11 • Now when we consider the offspring in generation II, it is ambiguous whether they are recombinant or nonrecombinant. This can only be determined if we set the phase of the father, I: I. So, what we must do is determine for each child their status (recombinant or nonrecomb inant) twice, once with the father 's phase set as shown on the left in Figure 11 and then again when the father 's phase is set as shown on the right panel of Figure 11
DISORDER WITH COMPLEX GENETIC INHERITANCE
• There are a number of common disorders, such as autism, epilepsy, cardiovascular disea se, and dementia, which clearly have a genetic contribution, but which are not inherited in a simple Mendelian fashion . These disorders are often con sidered to have complex genetic inheritance. It is hypothesized that multiple genes contribute to disease susceptibility and that the effect of a gene or genotype may vary depending on the
environment in which the individual is placed. The identification of the susceptibility gene s or allele that contribute to these disorders will lead to new challenges for the field of genetics and geneti c counseling. Individuals who inherit a susceptibility allele (s) may be at an increased risk to develop the disorder, but it will be difficult to quantify the exact risk to that individual or their offspring
SUGGESTING READING Nussbaum RL, McInnes RR, Willard UF. Thompson and Thompson Genetics in Medicine. Zth Edition. Philadelphi a: Saunders, 2007.
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15 Genetic Counseling Kimberly A. Quaid,
PhD
and Lisa J. Cushman,
PhD
CONTENTS
I. Definition of Genetic Counseling
15-2
II. Role and Training of Genetic Counselors
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III. History of Genetic Counseling
15-2
IV. Models of Genetic Counseling
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Eugenic Mod el MedicallPreventive Model Decision-Making Model Psychotherapeutic Model
V. Common Genetic CounselingTerms VI.
Common Genetic Counseling Problems
VII. The Process of Clinical Genetics and Genetic Counseling Prior to Clinic Visit Clinic Visit Follow-Up Care
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15-3 15-4 15-4 15-4 15-4 15-4
VIII. Determining Genetic Risks
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Recurrence Risk Based on Known Genot ype 15-4 Autosomal Recessive Disorders 15-4 Autosomal Domin ant Disorders 15-4 X-Linked Recessive Disorders 15-4 X-Linked Domin ant Disorders 15-5 Mitochondrial Disorders 15-5 Recurrence Risks Using Empiric Data 15-6 Structural Chromo some Rearrangements 15-6 Autosomal Domin ant Conditions with Germline Mosaicism 15-6 Risk Assessment in Cases With Consanguinity 15-6 Baye sian Analy sis in Risk Estimation 15-6 Auto somal Dominant Disorders 15-6 Auto somal Recessive Disorders 15-7 X-Linked Recessive Disorders 15-7
IX. Use of Molecular Genetics in Risk Assessment X. Suggested Reading
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D EFINI TIO N OF GENETIC COUNSELING
• A communication process, which deals with the human problems associated with the occurrence, or the risk of occurrence, of a genetic disorder in a family. Involves an attempt by one or more appropriately trained persons to help the individual or family to: - Comprehend the medical facts, including the diagno sis, probable course of the disorder, and available management - Appreciate the way heredity contribute s to the disorder and the risk of recurrence in specified relatives
- Understand the alternatives for dealing with the risk of recurrence - Choose the course of action, which seems to them appropriate in view of their risk, their family goals, and their ethical and rel igiou s standards and to act in accordance with that deci sion - To make the best possible adjustment to the disorder in an affected family member and/or to the risk of recurrence of that disorder (ASHG, 1975)
ROLE AND TRAIN ING OF GENETIC COUNSELORS
• Major role in the investigation and management of genetic disorders
• Board certification by the American Board of Genetic Counseling
• Graduates of 2-year Masters level training program s in medical genetics and coun seling
H ISTORY OF GENETIC COU NSELING • In 1865, Gregor Mendel finds that individual traits are determined by discrete factor s, later called gene, that are inherited from parent s - In 1883, Franci s Galton suggests that eugenic s (Greek meaning "well born") become s the study of social policies that may improve or impair racial qualitie s of future generations, either physically or mentally - In 1906, Bateson suggests the term "genetics" for the biologic and medical study of heredity
• By the mid-1940s, heredit y clinics were being established • By the 1950s, medicine began to focus on prevention and clinics established to advise people about inherited traits
- In 1969, Sarah Lawrence College established first program in genetic counseling
• In 1971, first 10 "genetic associates" graduate from Sarah Lawrence
- In 1975,term "geneticcounseling"coined by SheldonReed
MODELS OF GENETIC COUNSELING
Eugenic Model • Goal of eugenicists to improve the human species by better breeding • In 1904, Genetic s Record s Office opened at Cold Spring Harbor • In 1907, the state of Indiana passed the first sterilization law • When approved by a board of experts, the law mandated the sterilization of imbeciles, idiots, criminal s, and others in state institutions
• Sterili zation of the unfit became a signature policy in the Nazi' s quest for racial purity and superiority
MedicallPreventive Model • Nazi excesses lead to a retreat from eugenics practice s • Structure of DNA discovered in 1953 • Few diagnostic tests available • Information about risk based on empirical observations offered to families so familie s could avoid recurrence of a disorder that had already occurred
• By 1926, 23 of the 48 states in the United States had laws mandat ing sterilization of the "mentally defective" and over 6000 people had been sterilized
• Goal to prevent genetic disorder s by offering families information and the option to avoid childbearing
• Concept of eugenic sterilization found a receptive audience among the leadership of Nazi Germa ny
• Presumption that "rational" families would want to prevent recurrence
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Decision-Making Model • Human diploid complement of 46 chromosomes reported in 1956 • In late 1950s and early 1960s, findings led to an understanding of the cytogenetics of Down syndrome , Klinefelter syndrome, and Turner syndrome • Trisomies 13 and 18 discovered • Became possible to identify those heterozygous for ~-thalassemia and Tay-Sachs mutations • Amniocentesis first used in 1956 for prenatal diagnosis, initially for sex selection and later for karyotyping • Provided families with new options for assessing genetic risks and avoiding a genetic disorder • Legalization of abortion in 1972 meant that hard choices needed to be made regarding termination for genetic defects
• Educating families about choices was labor intensive and time consuming • Because of the almost exclusive focus on reproduction, the ideal of non-directive counseling was embraced with emphasis on patient autonomy in decision making • Goals of counseling shifted from providing information to a process in which individuals were not only educated but helped to make decisions that were consistent with their own values and needs
Psychotherapeutic Model • Recognition that families cannot process or act on information they have been given unless they have dealt with the powerful emotions evoked by such information • New emphasis on exploring client's experiences, emotional responses, goals, cultural expectations, religious beliefs, financial and social resources, family and interpersonal dynamics, and coping styles
COMMON GENETIC COUNSELING TERMS • Allele-One of the alternative versions of a gene that may occupy a given locus
• Karyotype-The chromosomes of an individual. For example , 46, XX or 46, XY
• Bayesian analysis-A mathematical method widely used in genetic counseling to calculate the risk of recurrence of a genetic disorder. This method combines information from a variety of sources including genetics, pedigree information, and test results to determine the probability that a specific individual may develop or transmit a specific disorder
• Penetrance-The probability that a mutant genotype will have any phenotypic expression
• Conditional probability-In Bayesian analysis, this is the chance of an observed outcome given the prior probability of the consultand's genotype • Consanguinity-Relationship by descent from a common ancestor • Consultand-The individual requesting genetic counseling • Dominant-A trait is dominant if it is phenotypically expressed in heterozygotes • Empiric risk-The probability that a trait will occur or recur in a family based on past experience rather than on knowledge of the causative mechanism • Expressivity-The extent to which a genetic defect is manifested • Pedigree-A diagram of a family history indicating the family members, their relationship to the proband, and their status with regard to a particular genetic condition (affected vs unaffected) • Joint probability-The product of the prior and conditional probabilities
• Phenotype-The observed biochemical, physiologic, and morphologic characteristics of an individual as determined by his or her genotype and the environment in which it is expressed • Proband-The family member through whom the family is ascertained . If affected, may be called the index case • Recessive-A trait that is expressed only in individuals who have inherited two copies of the gene • Recurrence risk-The probability that a genetic disorder present in one or more members of a family will recur in another member of the family of the same or subsequent generation • Teratogen-An environmental agent, medication , X-ray or pathogen that produces or raises the incidence of congenital malformation • Variable expressivity-When the manifestation of a phenotype differs in people who have the same genotype • Variable penetrance-When a condition is expressed in < I00% of individuals who carry the responsible allele • X-linked-Genes on the X chromosome, or traits determined by such genes • Y-Iinked-Genes on the Y chromosome, or traits determined by such genes
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Molecular Genetic Pathology
COMMON GENETIC COUNSELING PROBLEMS • Single gene disorder s known or suspected • Multi-factorial disorde rs known or suspected • Chromosomal disorder s diagnosed in the consultand or family member • An abnormal trait or carrier state identified by genetic screening
• Prenatal diagno sis for advanced maternal age or other indications • Consanguinity • Teratogen exposure • Repeated pregnancy loss or infertility
THE PROCESS OF CLINICAL GENETICS AND GENETIC COUNSELING Prior to Clinic Visit
- Nature and consequenc es of disorder - Recurrence risk
• Reason for referral • Collection of family history information and constru ction of a pedigree
- Means of modification of consequences
• Collection and review of medical record s and laboratory test results on con sultand and other family members
- Management plan
- Means of prevention of recurrence
Clinic Visit
Follow-Up Care
• Clinical examination • Diagno sis or ordering of further tests in order to make a diagno sis
• Referral to appropriate clinical specialists
• Recurrence risk estimation • Geneti c coun seling
• Further clinical assessment , if warranted
• Referral to appropriate health agencie s • Referral to appropriate support group s • Continued contact and support by genetic coun selor if needed
DETERMINING GENETIC RISKS Recurrence Risk Based on Known Genotype Autosomal Recessive Disorders (e.g., Tay-Sachs Disease) • Recurrence risk if both parents are known or obligate carriers is 25% for each future pregnancy (1/2 [chance that the mother passes on the mutation a] x 1/2 [chance that the father passes on the mutation a] = 1/4) (Figure 1)
Autosomal Dominant Disorders (e.g., Achondroplasia) • Recurrence risk if one parent is affected is 50% for each future pregnancy (chance that the parent passes on the mutation A) (Figure 2)
408
• Factors to consider when counseling about an autosomal dominant condition in the absence of a positive family history - It could be the result of a new mutation in the proband - The re may be decreased penetrance - There may be variable expressivity - Germline mosaicism may be present in a parent
X-Linked Recessive Disorders (e.g., Duchenne Muscular Dystrophy) • Recurrence risk if mother is a known or obligate carrier is 25% for each future pregnancy (1/2 [chance that she passes on the mutation x] x 1/2 [chance that she has a son] = 1/4) (Figure 3)
Genetic Counseling
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Aa
Aa
Aa
Aa
Xx
aa
Xx
Aa
Xx
XY
xY X-
X chromosome with the normal allele x- X chromosome with the mutant allele y- Y chromosome • - known carrier • -affected
A - normal allele a - mutant allele • - known carrier • -affected
aa
aa
Fig. 1. The recurrence risk for parents of a child with an autosomal recessive condition is 25% for each and every pregnancy.
aa
Fig. 3. The chance that a woman who is known to carry an X-linked recessive mutation will have an affected son is 25% for each and every pregnancy.
Aa
xx aa
Aa
aa
aa
Xx aa
Aa
aa
A - mutant allele a - normal allele .-affected
xx
X - X chromosome with the mutant allele x - X chromosome with the normal allele Y - Y chromosome • -affected
Fig. 4. Since X-linked dominant conditions are often lethal in males, affected sons are typically not seen.
• Half of the females will be unaffected Fig. 2. The recurrence risk for a parent who has an autosomal dominant condition is 50% for each and every pregnancy.
X-Linked Dominant Disorders (e.g., Incontinentia Pigmenti) • These conditions are often lethal in males • One-third of the children of an affected female will be affected (Xx females, Figure 4) • All of the liveborn males will be unaffected
• One may also see an increase in the number of spontaneous abortions (affected male fetuses)
Mitochondrial Disorders (Cause by a mtDNA Mutation; e.g., Leber Hereditary Optic Neuropathy) • Recurrence risk if mother is a known mtDNA mutation carrier in a homoplasmic form is 100% for each future pregnancy
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Recurrence Risks Using Empiric Data • Empiric recurrence risks are those in which the chance of having another affected individual in the family is based on observed data as opposed to mathematical calculations • Multi-factorial or polygenic conditions - Empiric recurrence risks are available for numerous conditions, such as congenital heart defects, cleft lip and palate, diabetes, psychiatric disorders, and cardiovascular disease - The risk is greatest among first degree relatives and decreases with the distance of the relationship - The recurrence risk depends on the incidence of the condition - An estimate of the recurrence risk, when specific risk figures are not available, is the square root of the incidence of the condition (i.e., the recurrence risk is approximately 1 in 100 for a condition with an incidence of 1110,000) - If there is an unequal sex incidence, the recurrence risk is greater for relatives of a proband of the sex in which the disorder is less common - The recurrence risk may increase if there are multiple affected family members and/or if the condition is more severe
Structural Chromosome Rearrangements • Empiric recurrence risks for those carrying common balanced chromosome rearrangements, such as a 14;21 Robertsonian translocation, are available - These risks may be dependent on sex of the transmitting parent (Table 1)
Autosomal Dominant Conditions With Germline Mosaicism • There are some disorders in which the risk of germline mosaicism has been determined • For example, the chance that a parent of a child with osteogenesis imperfecta type 2 will have another affected child due to germline mosaicism is approximately 7%
Molecular Genetic Pathology
Table 1. The Risk of Having an Abnormal Liveborn may Depend on the Sex of the Parent Who Carries a Robertsonian Translocation Transmitting parent
Risk of abnormal liveborn (%)
rob(l3;14)
Mother
Approximately 0.5
rob(l3;14)
Father
Approximately 0.5
rob(l4;21)
Mother
10-15
rob(l4;2l)
Father
1
Translocation
Table 2. The Risk of Having a Child with an Autosomal Recessive (AR) Disorder Increases the More Closely Related the Parents are Coefficient of inbreeding (F)
Risk of AR disease in offspring
Siblings
1/4
1/8
Half-siblings
1/8
1/16
Uncle-niecel aunt-nephew
1/8
1/16
First cousins
1/16
1/32
First cousins once removed
1/32
1/64
Second cousins
1/64
1/128
Relationship
Bayesian Analysis in Risk Estimation Bayesian analysis, which is based on Bayes' theorem of probability, is a method for modifying one's "prior" risk (i.e., risk based on Mendelian inheritance pattern or general population risk) using "conditional," or phenotypic, information.
Risk Assessment in Cases With Consanguinity
Autosomal Dominant Disorders
• Consanguinity refers to relationships involving persons who share a common ancestor (i.e., are blood relatives)
• Situations in which one may use Bayesian analysis include when the condition has reduced penetrance or variable expressivity, or when the disorder has a late age of onset • For example, a man, whose mother had Huntington disease (HD), is currently asymptomatic at age 60. Given that 3/4 of individuals with a HD gene mutation will have symptoms by age 60, what is the chance that this man inherited the gene mutation from his mother? • Prior probability is the chance that this man inherited the mutation based on Mendelian risks (he had a 1 in 2 chance of inheriting it, and a 1 in 2 chance of not inheriting it) (Table 3)
• The primary concern for children of these relationships is an increased risk of autosomal recessive disorders • The risk depends on the degree of relationship - The offspring of siblings have a 1 in 8 chance of having an autosomal recessive disorder while the risk for offspring of first cousins is 1 in 32 - The coefficient of inbreeding (F) indicates that chance that a child of a given relationship will be homozygous for an allele derived from a common ancestor (Table 2)
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Genetic Counseling
Table 3. The Posterior Probability the Man Inherited the Mutation, Given He is Asymptomatic at Age 60, is 1 in 5
Hypothesis Prior probability Conditional probability Asymptomatic at 60 Joint probability Posterior probability
A?
Aa
Man inherited Man did not inherit mutation mutation 1/2 1/4
1/2 I
1/2 x 1/4 = 1/8
1/2 x I = 1/2
(1/8) / (1/8 + 1/2)= 1/5
(1/2) / (1/8 + 1/2) = 4/5
A - normal allele a - mutantallele • - knowncarrier
• Conditional probability is the chance that this man is asymptomatic at age 60 if he did inherit the mutation (1/4 [the chance he is asymptomatic with the mutation]) or that he is asymptomatic at age 60 if he did not (there is essentially a 100% chance he would be unaffected if he did not inherit the mutation) (Table 3) • The joint probabilities are the product of the prior and conditional probabilities (Table 3) • The posterior probabilities are the joint probability for the particular hypothesi s divided by the sum of both joint probabilities (Table 3) • Therefore, this man has a I in 5 (or 20%) chance of having inherited the HD mutation from his mother given that he is asymptomatic at age 60 (compared with his prior risk of 50%) (Table 3)
Fig. 5. Using the results of the father's gene testing, Bayesian analysis may be used to calculate the recurrence risk for this couple.
Autosomal Recessive Disorders
•
• One may use Bayesian analysis when the genotypes of one or both parents are not known • For example, a Caucasian couple of Northern European ancestry is referred for a maternal family history of cystic fibrosis (Figure 5). Cystic fibrosi s is an autosomal rece ssive condition with a carrier frequency of approximately I in 25 in individuals with a Northern European ethnic background. The woman is a known mutation carrier. The man has also had CFTR gene testing. However, after being tested for the 87 most common mutations, no mutation was identified. The detection rate for this panel in individuals of his ethnicity is approximately 90% (I in 10 will have a mutation that is not identified). What is the chance that this couple will have a child with cystic fibrosi s given the man's gene test results? • Prior probability is the chance that this man has a mutation based on the carrier frequency in the population (he had a I in 25 chance of being a carrier, and a 24 in 25 chance of not being one) (Table 4) • Conditional probability is the chance that this man tests negative if he does have a mutation (1/25 [chance he has mutation] x IIlO [chance the test did not identify the
• •
•
mutation]) or that he tests negative if he is not a carrier (there is a 100% chance he would test negative if he does not have a mutation) (Table 4) The joint probabilities are the product of the prior and conditional probabilities (Table 4) The posterior probabilities are the joint probability for the particular hypothesis divided by the sum of both joint probabilities (Table 4) Therefore, this man has a 1 in 6001 (or a <1%) chance of being a carrier given that he tested negative (compared with his prior risk of I in 25, or 4%) (Table 4) The chance that this couple will have a child with cystic fibrosis is I in 24,004 (1/6001 [chance man is a carrier] x 1/4 [chance both parents pass on the mutation])
X-Linked Recessive Disorders • One may use Bayesian analysis when a possible carrier has had previously unaffected sons • For example, a 25-year-old woman is referred for prenatal genetic counseling because of a family history of hemophilia A (her sister is an obligate carrier of the factor VIII gene mutation) (Figure 6). Given that the client has already had two unaffected sons, what is the chance that the male fetus she is currently carrying will have hemophilia ? • Prior probab ility is the chance that this woman is a carrier based on the Mendelian risks (she had a I in 2 chance of being a carrier, and a 1 in 2 chance of not being one) (Table 5) • Conditional probability is the chance that this woman has two unaffected sons if she is a carrier (1/2 [chance that first son is unaffected] x 1/2 [chance that the second son is unaffected]) or that she has two unaffected sons if she is not a carrier (there is essentially a 100% chance
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Molecular Genetic Pathology
Table 4. The Probability that the Father is a Mutation Carrier, Given He had a Negative Gene Test, is 1 in 6001 Hypothesis
Man is a mutation carrier
Man is not a mutation carrier
1/25
24/25
1/25 x 1/10 = 1/250
I
1/25 x 1/250 = 1/6250
24/25 x I = 24/25
(1/6250)/(1/6250 + 24/25) = \/600\
(24/25)/(1/6250 + 24/25) = 6000/600\
Prior probability Conditional probability (negative gene test) Joint probability Posterior probability
Xx
X?
XY
xY
x-
Xchromosome with the normal allele
x - Xchromosome with the mutant allele p
XY
XY
?Y
y - Ychromosome
• •
- known carrier -affected
Fig. 6. Considering this couple has two unaffected sons, Bayesian analysis may be used to calculate the chance that the male fetus is affected with the X-linked condition in this family. she would have unaffected sons if she is not a carrier)
[lable 5. The Probability the Client is a Carrier,
Given She hasAlready had Two Unaffected Sons, is 1 in 5 a carrier
Client is
Client is not a carrier
1/2
1/2
Conditional probability (client has two unaffected sons)
1/2 x 1/2 = 1/4
\
Joint probability
\/2 x 1/4 = \/8
1/2 x 1=\/2
(1/8) / (1/8 +1/2) = \/5
(1/2) / (1/8 + 1/2) = 4/5
Hypothesis Prior probability
Posterior probability
412
(Table 5) • The joint probabilities are the product of the prior and conditional probabilities (Table 5) • The posterior probabilities are the joint probability for the particular hypothesis divided by the sum of both joint probabilities (Table 5) • Therefore, this woman has a I in 5 (or 20%) chance of being a carrier given that she has two unaffected sons (compared with her prior risk of I in 2, or 50%) (Table 5) • The chance that this woman's unborn son is affected is I in 10 (l/5 [chance that she is a carrier] x 1/2 [chance that she passes the mutation to him]) • Molecular genetic testing is currently available for hundreds of genetic conditions (www.genetests.org)
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Genetic Counseling
USE OF MOLECULAR GENETICS IN RISK ASSESSMENT • Many techniques exist for the identification of a particular gene mutation (gene sequencing, polymerase chain reaction analysis, SSCP(single-stranded conformation polymorphism), othermutation scanning techniques)
• Establishing a diagnosis or identifying a gene carrier by determining the gene mutation an individual carries, allows a genetic counselor to provide a precise recurrence risk
SUGGESTED READING Ad hoc Committee on GeneticCounseling. GeneticCounseling. Am J Hum Genet. 1975;27:240-241. Baker DL, Schuette JL, Uhlmann WR. eds. A Guide to Genetic Counseling. Wiley-Liss and Sons. NewYork, NY; 1998. Bennet RL, Steinhaus KA, Uhrich SB, et al, Recommendations for Standardized Human Pedigree Nomenclature. Am J Hum Genet. 1995;56:745-752.
Gardner RJM, Sutherland GR. Chromosome Abnormalities and Genetic Counseling. 3rd ed. Oxford: Oxford University Press; 2004. Harper PS. Practical Genetic Counselling. 6th ed. NewYork, NY: Arnold Publishers; 2005. Young 10. Introduction to Risk Calculation. 2nd ed. Oxford: Oxford University Press; 1999.
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Part II Disease-Based Sections
16 Molecular Medical Genetics Lisa Edelmann, PhD, Stuart Scott, PhD, and Ruth Kornreich, PhD
CONTENTS I. I ntroduction Inheritance of Single Gene Disorders
II. AR Disorders Cystic Fibrosis (CF) Clinical Prevalence Geneti cs Diagno sis Population Carrier Screening AJ Screening Tay-Sachs Disease Canavan Disease Familial Dysautonomia Type I Gaucher Disea se Fancon i Anemia, Group C Niemann-Pick Disease, Types A and B Bloom Syndrome Mucolipidosis, Type IV Hereditary Hemochromatosis (HH) Clinical Diagnosis and Prevalence HFE Mutations Spinal Muscular Atrophy (SMA) Clinical Inheritance and Prevalence Survival Motor Neuron (SMN) Gene s Diagnosis Medium Chain Acyl CoA Dehydrogenase Deficiency (MCAD) Clinical Inheritance and Prevalence
16-5 .16-5
16-5 16-5 16-5 16-5 16-5 16-6 16-6 16-6 16-7 16-8 16-8 16-8 16-8 16-8 16-9 16-9 16-9 16-9 16-9 16-9 16-10 16-10 16-10 16-10 16-10 16-11 16-11 16-11
Medium Chain Acyl CoA Dehydrogenase The ACADM Gene Genetic Testing Treatment
III. AD Disorders Nucleotide Repeat Expansion Disorders Myotonic Dystrophy Type 1 (DMl) Huntington Disease (HD) Spinocerebellar Ataxia (SCA) SCA2 SCA3/MJD SCA6 Friedreich Ataxia (FRDA)-AR Skeletal and Connective Tissue Disorders Achondroplasia FGFR-Related Cranio synostosis Syndromes Marfan Syndrome Osteogenesis Imperfecta
IV. X-Linked Inheritance Fragile X Syndrome Clinical Prevalence Inheritance The FMRJ Gene Diagnosis X-linked Muscular Dystrophy (DMD and BMD) Prevalence and Inheritance Clinical DMD Gene
16-11 16-11 16-11 16-12
16-12 16-12 16-12 16-12 16-13 16-14 16-14 16-16 16-16 16-17 16-17 16-17 16-18 16-19
16-20 16-20 16-20 16-20 16-20 16-20 16-21 16-22 16-22 16-22 16-22
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mtDNA Rearrangements 16-24 mtDNA Point Mutations 16-24 Diseases Resulting From Nuclear DNA Mutation 16-25 Structural Respiratory Chain Defects..16-25 Non-Structural Respiratory Chain Defects 16-25 Diagnostic Evaluation 16-25
Diagnosis 16-22 X-LinkedAdrenoleukodystrophy (X-ALD)..I6-23 Clinical 16-23 Prevalence 16-23 16-23 Inheritance The X-ALD Gene 16-23 Diagnosis 16-23
V. Mitochondrial Disorders 16-23 The Mitochondrion 16-23 Diseases Resulting FrommillNAMutation 16-24
418
VI.
Suggested Reading
16-26
16-3
Molecular Mediacal Genetics
INTRODUCTION Inheritance of Single Gene Disorders Mode of inheritance is important for predicting clinical status of individuals carrying mutations and for risk assessment of family members of a patient affected with a genetic disease. Modes include :
• Autosomal recessive (AR) • Autosomal domin ant (AD) • X-linked • Mitochondrial Examples will be given for each subgroup .
AR DISORDERS Cystic Fibrosis (CF) CF is one of the most common AR severe disorders in the Caucasian population with an incidence of 1 in 2500 to 1 in 3300 births in non-Hispanic Caucasians . Its pathophysiology results from the secretion of thick mucus by membrane epithelial cells, which interrupts the function of organs such as the lungs, pancreas, intestine, and male reproductive tract by defective chloride and sodium transport.
Clinical • Progressive, multi-system disorder that primarily affects the respiratory, digestive, and reproductive systems • Failure to thrive, meconium ileus in 10-20% of infants with CF • Recurrent respiratory infections lead to chronic pulmonary disease
- Spans 230 kb on the long arm of chromosome 7, encodes 6500 nucleotide mRNA, 27 exons -
1480-amino acid transmembrane protein predominately located at the apical membrane of epithelial cells of the lung, sinus, pancreas, intestine, sweat and bile ducts, and vas deferens
- Multi-functional protein • Chloride channel activated by cAMP • Transporter regulator of other channels including epithelial sodium channel - Five domains: two membrane spanning, two nucleotide binding that interact with ATP, one regulatory (Figure 1) • Over 1300 CFTR mutations have been reported ; most rare, but several recurring at a higher frequency (http://www.genet.sickkids.on.ca/cftr)
• Infertility in males due to congenital absence of vas deferens
• delF508 most common with frequencies ranging from 18 to 88% in different ethnic, demographic, and racial group s and with an overall world wide frequency of approximately 70%
• Treatment involves enzyme and dietary supplementation for pancreatic insufficiency and respiratory therapy with treatment of infections
• G542X is the next common mutation responsible for 2.4% of alleles and only another three mutations occur above 1% in United States CF patients
• Early death usually due to obstructive pulmonary disease
• Genotype-phenotype correlations are inexact and should not be used to predict survival or level of pulmonary disease - Some mutations (3849 + 10 kb C > T, A455E, 2789 + 5G > A, G85E, R334W) lead to milder CF phenotype with possibly pancreatic sufficiency - Variants reported which may lead to only limited lung involvement, sinusitis or male infertility due to congenital bilateral absence of the vas deferen s
• Exocrine pancreatic insufficiency in 85% of patients
• Median survival 30 years • Approximately 15% of CF individuals have a milder course with pancreatic sufficiency and a median survival of 56 years
Prevalence • Varies depending on the ethnic group - Non-Hispanic Caucasian-l in 2500 births - Ashkenazi Jewish (AJ)-l in 2300 births - Hispanic-l in 13,500 births - African American-l in 15,100 births - Asian Americans-l in 35,100 births
Genetics • Caused by mutations in CF transmembrane regulator (CFTR) gene - Cloned in 1989 by positional cloning
• Polypyrimidine tract in intron 8 variable-5TI7T/9T possible • 5T leads to low RNA splicing • 5T homozygosity or 5T with CFTR mutation on other chromosome not clinically significant in females, but can cause congenital bilateral absence of the vas deferen s in males • Rl17H without 5T on the same chromosome nonclassic CF allele (R 117H with 5T on same allele, in cis, associated with classic CF)
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MSD-1
MSD -2 Extracellular
Intracellular NH2
Fig. 1. Schematic representation of the proposed CFTR structure. CFTR is comprised of five domains : two membrane-spann ing domains (MSD-l and MSD-2) that form the chloride ion channel, two nucleotide-binding domains (NBD-l and NBD-2) that bind and hydrolyze ATP, and a regulatory domain (R) that is phosphorylated by protein kinase A and C. The location of the most common CF causing mutation , delF508, is noted.
Diagnosis • Sweat electrolyte levels • Immunoreactive trypsin • DNA analysis - Most testing methods initiate with multiplexed polymerase chain reaction (PCR) ; however, the technologies used to differentiate wild-type from mutant alleles following PCR vary Commercially available kits to detect common mutations including: • Reverse dot blot • Bead arrays • Amplification refractory mutation system • Oligonucleotide ligation assay • Invader technology Some laboratories develop testing in-house using homebrews, for example, forward allele-specific oligonucleotide hybridi zation , while others use commercially available analyte-specific reagents as well as specialized instrumentation
Population Carrier Screening • National Institutes of Health (NIH) recommended in 1997 that CF mutation screening be offered to adults with a positive family history of CF, partners of individuals with CF and couples who are pregnant or are planning a pregnancy - Implementation difficult due to the large number of mutations, varying distribution of mutations in different ethnic groups, and lack of sufficient clinical correlation and educational material • Guidelines established by joint American College of Medical Genetics (ACMG) , American College of Obstetricians and Gynecologists (ACOG), and NIH committee publi shed in 200 I
420
- Carrier screening should be offered to Caucasians and "made available" to other ethnic groups - Couple-based or sequential depending on circumstances - Patient literature be provided - Pan-ethnic mutation panel, which included 25 CF causing mutations with an allele frequency of ~O.l % in the general US population recommended (Table 1) - Advocated additional reflex testing of R 117H carriers for the 5/7/9T alleles in the polypyrimidine tract of intron 8 as well as reflex testing for interference from the benign variants F508C, 1506V, and 1507V in delF508 positive samples when indicated - Panel modified in 2005 (Table 2) • I078deiT removed because true frequency falls below the frequency threshold of ~O.l % • Il48T had a >lOO-fold increase in individuals having carrier screening compared with CF population • I148T deleted as it does not cause classic CF--exists as a complex allele with 3199de16, which is the true severe CF mutation
AJ Screening • AJ originated from Eastern Europe and represent approximately 90% of the 5.7 million Jews in the United States today • Over 40 disorders with a higher prevalence in this group have been described • Screening began with Tay-Sachs disease (TSD) in the 1970s by enzymatic analysis • ACOG and the ACMG now recommends screening for at least Tay-Sachs , Canavan, CF, and familial dysautonomia (FD) (other disorders may be offered) • Screening performed mostly by targeted mutation analysis as limited number of mutations are needed to detect >90% of carriers
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Molecul ar Med ical Genetics
Table 1. Recommended Core Panel of 25 Mutations for General Population CF Carrier Screening (2001) Standard mutation pan el delF508
delF507
R553X
621 +
R I 162X 2184deiA 3120+
IG~A
G542X
G55 1D
W I282X
NI303K
R II 7H
1717G~A
A455E
R560T
G85E
R334W
R347P
711 -IG~T
1898
1078deiT
3849 +
2789G~A
3659deiC
Il48T +
-
-
-
-
-
I G~T
IOkbC~T
IG~A
Reflex tests 1506V, 1507, F508C: non-CF causing variants test only if unexpected homozygosity for delF508 and/or delF507 (F508C has been associated with CBVAD) 5TI7T/9T-test only for R I 17H positives
Table 3. Carrier Screening in the AJ Population
Table 2. CF Mutation Detection and Carrier Rates Before and After Testing in Various Ethnic Groups Using the Recommended Core Panel of 23 Mutations Ethnic group
Carrier D etecti on frequency rate (%)
Number of A} mutations A} carrier co m monly D etectability (%) screened frequency
Carrier risk afte r negat ive result
D isease Gaucher Type I
1:15
4
95
CF
1:24
23°
94
Tay-Sachs
1:30
3
98
AI
I in 24
94
I in 384
Non-Hispanic Caucasian
I in 25
88
I in 206
Hispanic Caucasian
1 in 58
72
1 in 203
FD
1:32
2
>99
African American
1 in 62
65
I in 171
Canavan
1:40-1:57
3
98 99
49
1 in 183
I
1 in 94
Fanconi group C
1:89
Asian American
NiemannPick A and B
1:90
4
95
BS
1:107
I
99
MLIV
1:125
2
96
• Labs now offering testing for at least 8 "AJ" diseases and CF (Table 3) • Commercial kits are avai lable for AJ pane l predominately using bead array technol ogy
AJ, Ashkenazi Jewish; CF, Cystic fibrosis; FD, familial dysautonomia; BS, Bloom syndrome; M UV, mucol ipidosis, Type IV aM ost laboratories do not test for less than the 23 ACMG recommended panel, although only five mutations are common in AJ
Tay-Sachs Disease • A R lysosomal storage disorder ca used by defi cien cy of p-hexosaminidase A • Mostly infantile form , which is unifor mly fata l in childhood due to progressive neu rologic deterioration • M uc h less freq uent later-onset disorder known as chronic G M2 ga ng lios ido sis characterized by mu scle weakness, ataxia, dy sarthria, mild me nta l impairm en t, and psychosis • Mostl y AJ ( I in 30 ca rrier frequency), but also prevalent in Caj un or Fre nch-Canadian ances try
•
lO-fo ld lower (I in 300 carrier frequency) in non -Jewish ge neral population
• No treatment currently ava ilable • Diagn ostics
42 1
16-6
Molecular Genetic Pathology
Most labs use a combination of measurement of hexosaminidase levels and mutation analy sis: Screening can be done by targeted mutation analysis only if individual is of 100% AJ descent - Three mutations (1278 + TATC, IVS12 + IG > C, G269S) account for 98% of AI mutations, but <50% of non-Jewish alleles Measurement of hexosaminidase A enzyme levels in serum, plasma, leukocytes, platelet s, or other sources will pick up most carriers; however, 2-5% of individual s tested will be inconclu sive (because of overlap between high carrier and low normal ranges) Pseudodeficiency allele s exist such that individuals will have reduced enzyme activity using the in vitro screening assay, but not against the natural substrate , GM2 gangliosides (not true TSD carriers) • R247W-found in 2% of AJ and 32% of non-Jewish carriers by enzyme analy sis • R249W-found in 4% of non-Jewish carriers by enzyme analy sis All individuals found to be carrier s or who test inconclu sive by enzyme analysis should have follow up by molecular testing
Canavan Disease • AR neurodegenerative leukody strophy caused by deficien cy of the enzyme aspartoac ylase • Affected infants develop normally during first few month s of life and then experience a marked loss of early milestones and clinical feature s including megalocephaly, poor head control , and seizures • Death usually during first decade of life • Carrier frequencies reported to be I in 40 to I in 57 in AJ, significantly lower in non-Jews, but reported in many ethnic groups • Diagnostics - Measurement of the substrate N-acetylaspartic acid in fluids possible , but not reliable - E285A and Y231X account for approximately 97% of AJ Canavan disease (CD) mutation s - A305E accounts for approximately I% of AJ CD alleles, but 50% of non-Jewish mutation s
Familial Dysautonomia
- The missense mutation, R696P, has also been found in small number of AJ familie s - P914L was found in a non-Al individual
Type I GaucherDisease • AR lysosomal storage disorder caused by deficient activity of the enzyme , P-glucosidase • One of the most prevalent disorder s among AJ, with a carrier frequency of I in 15 • The glycolipid, glucocerebrosidase, accumulate s primarily in the cells of the macrophage-monocyte system • Extremely clinically heterogenous, ranging from early onset of severe disease to a mild course • Asymptomatic affected individual s have been picked up in routine carrier screening • Enzyme replacement therapy is available • Diagnostics - Testing for glucocerebrosidase activity in cell s is possible, but it is not accurate for carrier detection - Testing for four mutation s (N370S, 84GG , L444P, and IVS2) in the AJ, will detect about 95% of mutant Gaucher disease (GD) alleles - Care must be taken when designing assays as there is a nearby, processed pseudogene • Genot ype-phenotype correlations - Individuals homozygous for the most common AJ allele , N370S , will have a non-neurologic disorder with an average age of onset of about 30 years - Individual s with one copy of N370S and either 84GG , L444P, or IVS2 will have the non-neurologic form of Type I GD, but will have a severer course than N370S homozygotes - Affected individuals with two copies or any combination of 84GG, L444P, or IVS2 will have severe disease with a neurodegenerative course
Fanconi Anemia, Group C • AR disease characterized by short stature, bone marrow failure, congenital malformations, and a predisposition to acute myelogenou s leukemia • A single mutation in the FACC gene (IVS4 + 4A > T) accounts for nearly all AJ mutant alleles • AJ carrier frequenc y of I in 89
• AR neuropathy that almost exclu sively affects AJ infants
Niemann-Pick Disease, Types A and B
• Disorder of sensory and autonomic function characterized by absence of tearing, absence of papillae on the tongue , protracted vomiting , decrea sed discrimination to pain and temperature, and cardiovascular instability
• AR disorder s resulting from the deficient activity of the lysosomal enzyme, acid sphingomyelinase
• Carrier frequency of I in 32 in AJ and found almost exclusively in AJ • Diagno stics - One mutation in the IKBKAP gene, IVS20 + 6T > C, accounts for >99% of mutant AJ alleles
422
• Type A disease - Characterized by a rapid progressive neurodegenerative course and hepato splenomegaly in infancy with death usually occurring by 3 years of age - Carrier frequency of I in 90 in AJ • Type B disease - Pan-ethnic
16-7
Molecular Medical Genetics
- Milder than Type A with primarily hematologic and pulmonary symptomology and little if any neurologic involvement • Diagnostics - Three common AJ Type A mutations (R496L, L302P, and fsP330) account for approximately 95% of AJ alleles - One recurrent Type B mutation, delR608
Bloom Syndrome • AR neuropathy condition that is characterized clinically by severe prenatal and postnatal growth deficiency, a sun-sensitive telangiectatic rash, and a predisposition for different malignant and benign tumors that develop in early childhood and adolescence • Results from mutations in the BLM gene, a RecQ helicase • AJ carrier frequency 1 in 107 • Diagnostics - A single mutation (BLMASh, 2281delATCTGAins TAGATTC, a 6-bp deletion, and a 7-bp insertion) accounts for most AJ BLM mutations
Mucolipidosis, Type IV • AR, neurodegenerative lysosomal storage disease characterized by a variable degree of growth and psychomotor retardation, and ophthalmologic abnormalities, which include corneal clouding and progressive retinal degeneration • Life span may be normal, but most patients remain at a developmental level of 1-2 years • Accumulation of cytoplasmic storage bodies but normal levels of lysosomal hydrolases present • 80% of individuals with mucolipidosis, Type IV (MLIV) areAJ • Mutations in MCOLNJ gene are responsible • Diagnostics - Two mutations, IVS3-2A > G and de16.4 kb account for 95-96% of AI MLIV alleles
Hereditary Hemochromatosis (HH) HH consist of a group AR genetic disorders that can lead to tissue injury from accumulation of excess iron in the body. The most common cause of HH is mutations in the hemochromatosis (HFE) gene, but defects have been reported in transferrin receptor 2, ferroportin and hepcidin, and haemojuvelin genes. Must be differentiated from iron storage as a secondary complication of thalassemias, anemias, transfusions, and so on.
Clinical • Increased absorption of iron from gastrointestinal tract resulting in iron deposits particularly in liver, heart, pancreas, and skin • More males than females affected • Onset in fourth decade for men and fifth for women
• Treatment by routine phlebotomies • If untreated , organ damage possible such as: - Cirrhosis of the liver, hepatocellular cancer, liver failure - Type 2 diabetes Congestive heart failure or arrhythmia - Arthritis - Hypogonadism in males - Skin bronzing
Diagnosis and Prevalence Typically diagnosed by combination of genetic and phenotypic findings . • Measurement of transferrin saturation (>50% for women and >60% for men) and serum ferritin levels (>200 ug/ml.) • Liver biopsy to measure iron concentration is the gold standard • Mutation analysis (see HFE Mutations section) • Estimated that 3-5 individuals per 1000 in general population are clinically affected • Penetrance in males is higher than females before menopause
HFE Mutations • Most common cause of HH
• HFE gene - Located on chromosome 6 - Class I HLA gene - Binds ~2-microglobulin and is associated with transferrin receptor • C282Y (c.845G > A) - Major disease causing mutation found in 80-90% of affected individuals - 1 in 11 Europeans are C282Y heterozygotes - Arose relatively recently (in the last 1000-3000 years) - Mostly found in Northern Europeans - 0.44% of non-Hispanic Caucasian individuals are C282Y homozygotes-most are asymptomatic (reduced penetrance) • H63D (287C > G) - Older, more common, world wide distribution - 2% of Europeans H63D homozygotes - Extremely low penetrance such that homozygotes and compound heterozygotes with C282Y slightly increased risk of iron overload • DNA Diagnosis - Targeted mutation analysis by any number of methods including PCR followed by restriction digestion (Figure 2), hybridization, fluorescence resonance energy transfer (FRET,) capillary electrophoresis - Polymorphisms have been reported-one is in the binding site of one of the primers used in an original report therefore must be careful when designing primers
423
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Molecular Genetic Pathology
- Type II SMA onset of weakness by 18 months and survival beyond 4 years of age
A Normal:
--
Rsa I
I
248bp
C282Y:
-B
140 bp
-
Rsa I Rsa I
l~
248 bp
.•
•
-
111 bp
.
~
--- -- -- --Fig. 2. Molecular genetic testing of HH. (A) The C282Y HFE mutation results from an 845G > A transition, which creates a novel Rsal restriction site. PCR primers are depicted with arrows . (B) The image depicts gel electrophoresis of PCR products, which encompass the C282Y mutation site that is digested with RsaI. In this illustration, sample A is negative for the C282Y mutation given the absence of the smaller Rsal restriction fragment (111 bp; arrow). The 29-bp fragment in C282Y hetero- and homozygotes is typically undetectable.
Spinal Muscular Atrophy (SMA) SMA is one of the most common AR diseases and is characterized by symmetric proximal muscle weakness due to the degeneration of anterior hom cells of the spinal cord .
Clinical • Progressive degeneration and loss of anterior hom cells (lower motor neurons) in the spinal cord and brain stem nuclei causing symmetric muscle weakness and atrophy • Diagnosis is made based on poor muscle tone, symmetric muscle weakne ss that spares the face, and ocular muscles • Other signs include tongue fasciculations and absence of deep tendon reflexes • Normal reaction to sensory stimuli and normal intellect • Onset of weakness varies from before birth to adolescence and adulthood, but is always progressive - Type I SMA (Werdnig-Hoffman disease) onset of weakness and hypotonia in first few months with fatal respiratory failure before 2 years of age (most common [60%])
424
- Type IlIa and I1Ib have age of onset before and after 3 years , respectively
Inheritance and Prevalence • AR disorder • Disease frequency of I in 10,000 • Carrier frequency of 1 in 50 in Caucasians
SurvivalMotor Neuron (SMN) Genes • SMN genes (SMN1 and SMN2) on 5q 13 (Figure 3) - Located within low copy repeat with Neuronal Apopto sis Inhibitory Protein (NAlP) and p44 genes (500 Kb)
SMN1 telomeric to SMN2 High sequence similarity between genes, no amino acid differences Nine exons-l , 2a, 2b, 3-8, and span 20 kb Single base differences between SMN1 and SMN2 in exons 7 and 8 are exploited in diagnosi s Single-coding sequence difference in exon 7 (840C > T) important for splicing differences between SMN1 and SMN2 (silent change) SMNI encodes protein of 38 kDa whereas SMN2 encodes a protein that is lacking exon 7 Required for pre-mRNA splicing role in snRNP biogenesis and function Loss of SMN1 function is responsible for SMA phenotype Mutation analysis • 95% of patients have homozygous loss of SMN1 by deletion or gene conversion • Sequence differences of exons 7 and 8 are exploited in diagnosis (Figure 3) • 5% have intragenic mutations • No genotype/phenotype correlations with SMNI mutations, but additional copies of SMN2 are associated with less severe phenotype
Diagnosis • Carrier testing detects the SMA deletion by dosage sensitive techniques, such as quantitative PCR or multiplex ligationdependent probe amplification (MLPA) - Can be problematic because some individuals have duplications on one chromosome and deletion of the other • 2% of mutations are de novo • 4% of the population carries a duplication of SMN1 • Not possible to detect the duplication carriers/deletion carriers, referred to as 2 + 0 carriers, from normal individuals using dosage sensitive PCR techniques
Molecular Medical Genetics
Exons
16-9
2b
2a
3
4
5
6
7
8
I 840C >T
Centro meric
Telomeric
SMN2
SMN1
Large inverted dup lications on chromosome 5q13 - 500 kb in size
Fig. 3. Schematic of SMA genomic locus showing the inverted duplications and the positions of the SMN1 and SMN2 genes . The only coding sequence change between the two genes is shown in exon 7-840C > T SMNl :SMN2.
Medium Chain Acyl CoA Dehydrogenase Deficiency (MCAD) MCAD is the most common disorder of fatty acid oxidation. During periods of fasting or prolonged aerobic exercise when glycogen stores are depleted, fatty acids become a main energy source by ~-oxidation in the liver, skeletal, and cardiac muscles.
- Defect leads to the accumulation of metabolites of the medium-chain fatty acids, mainly dicarboxylic acids, acylglycine in urine, and acylcarnitine in plasma • Metabolites are at their highest concentration in the bloodstream in the first few days of life so newborn period is the ideal time for detection
Clinical
• Specificity of this testing is 100% as no falsenegatives have been reported
• Symptoms appear after periods of prolonged fasting or intercurrent infections
• MCAD enzymatic activity can also be assayed in several different cell types
• Hypoketotic hypoglycemia • Lethargy, seizures, coma and death without treatment. • Complications include hepatomegaly, acute liver disease , and brain damage • Disease typically presents before 2 years of age but after the newborn period • Variable onset in some patients-first few days of life to adults • Screening for MCAD included in tandem mass spectrometry of newborn screening program of most states
Inheritance and Prevalence • AR disorder • Prevalent in individuals of Northwestern Europe - Highest overall frequency of 1 in 4900 in Northern Germany - Incidence in the United States is estimated at 1 in 15,700
The Acyl-CoA Dehydrogenase, Medium Chain
(ACADM) Gene • Located on chromo some 1p31 and spans 44 kb • Contains 12 exons • Encodes a protein of 421 amino acids • Mutations - Founder mutation in exon 11, 985A-;G, (K304E), represents 90% of all alleles in the Northern European population - Studies of the US population indicate that this mutation accounts for 79 % of the total mutant alleles (greater ethnic diversity) - Additional mutations have been spread throughout the gene with no obvious mutation hotspot • Affect overall stability of the protein, improper folding (mostly missense mutations located away from the active center)
Medium Chain Acyl CoA Dehydrogenase
Genetic Testing
• Enzyme that is intra-mitochondrial, but is encoded by a nuclear gene
• DNA testing for MCAD mutations offered as confirmatory testing after the initial diagnosis by biochemical testing
- Normal function is the initial dehydrogenation of acyI-CoAs with carbon chain lengths 4-12
- K304E allele is performed initially by PCR amplification followed by restriction enzyme digestion
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Molecular Genetic Pathology
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- Other methods that can discriminate between single nucleotide changes, such as allele-specific oligonucleotide hybridization or ligation chain reaction amplification - Affected individuals heterozygous or negative for the K304E mutation have whole gene sequencing performed on all 12 exons - The majority of mutation s identified in ACADM are
thought to affect the folding of the protein and located away from the active center
Treatment • With prompt postnatal diagnosis MCAD can be treated Precautions such as avoidance of fasting and saturated fats and ingestion of carbohydrates prior to bedtime can eliminate the symptoms and related complications of the disease
AD DISORDERS Nucleotide Repeat Expansion Disorders Myotonic Dystrophy Type 1 (DMl) An AD multi-system disorder involving progressive muscle weakness, myotonia, cataracts, electrocardiogram (ECG) abnormalities, hypersomnia, and endocrine dysfunction.
Clinical • Categorized into overlapping phenotypes: - Mild : cataract and mild myotonia; normal life span - Classic: muscle weakness, myotonia, cataract, cardiac arrhythmias, balding; adults may have shortened life span - Congenital: neonatal hypotonia, motor and mental retardation, respiratory deficits , and early death
• DMPK protein shares homology to a cyclic AMP-dependent serine-threonine protein kinase but its in vivo substrate remains unknown; expressed in specialized cells of the heart and skeletal muscle • As the CTG repeat occurs in the 3' UTR of the DMPK transcript, allelic expansion does not alter DMPK protein structure • Exact mechanism as to how CTG expansion results in decreased DMPK protein production is not fully resolved; however, evidence exists implicating abnormalities in DMPK pre-mRNA processing and transport
Molecular Genetic Testing
• Exhibits disease anticipation • Caused by expansion of a CTG trinucleotide repeat in the 3' UTR of the dystrophia myotonica-protein kinase (DMPK) gene , located at 19q13.3 (Figure 4 and Table 4)
• PCR amplification of the DMPK trinucleotide repeat region followed by gel electrophoresis • Alleles with> 100 repeats may not be detectable by PCR • Individual s with a single allele size as detected by PCR should also be analyzed by Southern blot
Prevalence
• Combination of PCR and Southern blot detect s nearly 100% of DMI cases
• Estimated worldwide prevalence is 1120,000
Diagnosis
Huntington Disease (HD)
• Based on a positive family history, characteristic clinical findings, and positive molecular testing • Molecular testing is clinically useful for diagnosis, prenatal diagnosis, and predictive testing
An AD neurodegenerative disordercharacterizedby involuntary movements, cognitive impairment,and emotional disturbance.
Dystrophia Myotonica-Protein Kinase • Normal alleles are polymorphic and contain 5-35 repeats • Phenotypically normal individuals can have intermediate alleles that contain 35-49 repeats; can result in pathologic expansion in subsequent generations • Affected individuals have at least one allele with 50 or more CTG repeats and severity generally correlates directly with repeat size • Mutant DMPK alleles display con siderable somatic instability, resulting in mosaicism for the repeat in affected individuals • The largest alleles (>2000 repeat s) that produce the most severe form of the disorder, congenital DM 1, are almost always maternally transmitted
426
Clinical • Age of onset is typically in the third to fifth decade; however, symptoms may begin in childhood or after age 60 • Juvenile form is defined by onset before 21 years of age • Early degenerative changes are marked in the striatum ; advanced stage neuronal loss is widespread involving the cortex and cerebellum • Exhibits disease anticipation • Is caused by expansion of a CAG trinucleotide repeat in the coding region of the HD gene, located at 4p16 .3 (Figure 4 and Table 4)
Prevalence • More common in populations of Western European descent (prevalence of -1120,000) and less common in Asian and African populations
Molecular Medical Genetics
A
16-11
Huntington Disease
-ll-ETG DMPK (3184 bp)
5-35
3 5-49 ~50
B
HD (13,482 bp)
- Full penetrance alleles : 40 or more repeats
~2 6
- Reduced penetrance alleles : 36-39 repeats • An inverse correlation exists between the number of CAG repeats and the age of onset ; individuals with juvenile onset usually have >60 repeats • Wild-type HD is widely expressed as two different-sized transcripts and is required for normal development
b
AG
27-35 ~36
C
ATXN2 (4702 bp) ~3 1
n=
L~32~ D
ATXN3 (1776 bp) ~4 7
b
AG
48-51 53-86
E
• Normal alleles have up to 26 repeats • Phenotypically normal individuals can have intermediate alleles that contain 27-35 repeats • Intermediate alleles can pathologically expand in subsequent generations, occurring almost exclusively by paternal transmission • Affected individual s have at least one allele with 36 or more CAG repeats
CACNA1A (7827 bp)
• Normal function of the HD protein is not well understood • Abnormal CAG expansion results in polyglut amine expansion in the HD protein and likely confers an RNA or protein gain-of-function • Polyglutamine expansion may affect HD function by influencing protein-protein interaction or may result in abnormal accumulation of toxic substance through the activity of transglutaminases • Polyglutamine expansion also increases affinity for Huntington-associated protein 1, which may playa role in aberrant neuronal cell death
Molecular Genetic Testing • PCR amplification of the HD trinucleotide repeat region followed by gel electrophoresis (Figure 5) • Alleles with> 100 repeats may not be detectable by PCR • Individuals with a single allele size as detected by PCR should also be analyzed by Southern blot • Combination of PCR and Southern blot detects nearly 100% of HD cases
Spinocerebellar Ataxia (SeA)
Fig. 4. Schematic of nucleotide expansion disorder genes . Depicted are the mRNA sequences for the genes involved in myotonic dystrophy (A), HD (B), SCA2 (C), MJD (D), and SCA6 (E). Sequences highlight the location of the particular trinucleotides involved in expansion. Coding regions are displayed by horizontal gray bars and the normal number of repeats are noted above the particular mRNA. Intermediate range alleles, if they occur, and disea se-causing alleles are displayed below the specific trinucleotide sequence s.
Diagnosis • Based on a positive family history, characteristic clinical findings , and positive molecular test ing • Molecular testing is clinically useful for diagnosis, prenatal diagnosis, and predictive testing
SCA is a group of AD neurodegenerative disorders , which share many clinical and neuropathologic features , most notably progres sive cerebellar ataxia • Characterized by poor coordination of movement and a wide-based, uncoordinated, unsteady gait; poor coordination of the limbs and speech are often present • Categorized by causative gene or chromosomal locus • The underlying genetic abnormalities have been identified in many subtypes and include trinucleotide repeat expansions (CAG and CTG) in coding and non-coding regions, pentanucleotide repeat expansions (ATTCT), and point mutations • Pathogenic mutations remain elusive in 20-40% of SCA families • More than twenty forms of SCA exist; however, molecular testing is only available for well-characterized subtypes. Causative genes and location :
427
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Molecular Genetic Pathology
Table 4. Representative AD Nucleotide Repeat Expansion Disorders Number of repeats
Disease
Nucleotide repeat
Gene
Repeat location
Normal
Unstable intermediate
Affected
OMl
CTG
DMPK
3'UTR
5-35
35-49
;?:50
HD
CAG
HD
Codingregion
:'>26
27-35
;?:36
SCA2
CAG
ATXN2
Coding region
:'>31
-
;?:32
SCA3/MJD
CAG
ATXN3
Coding region
:::;47
48-51
53-86
SCA6
CAG
CACNA1A
Coding region
:::;18
19
20-32
OM I, myotonic dystrophy type 1; Hl), Huntington disease; SCA, spinocerebellar ataxia; MJo, Machado-Joseph disease
- SCA1: ATXNl, 6p23
Diagnosis
- SCA2: ATXN2, 12q24.1
• Very difficult to distinguish from other hereditary ataxias; requires molecular genetic testing to detect an abnormal CAG trinucleotide repeat expansion in the coding region of the ataxin 2 (ATXN2) gene (Figure 4 and Table 4)
- SCA3/Machado-Joseph disease (MJD): ATXN3, 14q24.3-q32.2 - SCA6: CACNAIA, 19pI3.2-pI3.I - SCA7 : ATXN7, 3p2U-p12
ATXN2
- SCA8 : KLHLlAS, 13q21
• Normal alleles have up to 31 repeats • Affected individuals have at least one allele with 32 or more CAG repeats • Most common disease-causing alleles contain 37-39 repeats • The CAG repeat is normally interrupted by CAA trinucleotides, which may enhance the meiotic stability of the repeat; expanded alleles that lack the CAA interruption have increased risk of disease anticipation to subsequent generations • CAG trinucleotide expansion results in polyglutamine expansion in the ATXN2 protein; however, the normal function of the ATXN2 protein is unknown
- SCAIO: ATXNlO, 22q13.31 - SCAI2: PPP2R2B, 5q31-q33 - SCAI4: PRKCG, 19q13.4 - SCAI7: TBP,6q27 • Majority of CAG trinucleotide repeat ataxias exhibit disease anticipation • Molecular testing is clinically useful for diagnosis, prenatal diagnosis, and predictive testing
Prevalence • Estimated world wide prevalence is 11100,000 • Prevalence of individual subtypes varies by geographical area • SCA2, SCA3/MJD, and SCA6 are the most common forms of AD ataxia:
SCA2 Distinctive Clinical Attributes • Age of onset is typically in the fourth decade • Variable findings include nystagmus, slow saccadic eye movements, and occasionally ophthalmoparesis and dementia
Prevalence • Accounts for approximately 15% of all SCA
428
Molecular Genetic Testing • PCR amplification of the ATXN2 trinucleotide repeat region followed by gel electrophoresis • Alleles with> 100 repeats may not be detectable by PCR • Individuals with a single allele size as detected by PCR should also be analyzed by Southern blot • Combination of PCR and Southern blot detects nearly 100% of SCA2 cases
SCA3/M]D Distinctive Clinical Attributes • Age of onset varies but is typically in the second to fourth decade
Molecular Medical Genetics
A
16-13
M13 pUC18 sequencing ladder
CAG Repeats: 16/16 24/41 20/42 15/18 16/19 I'¢.: ~~.,
'176' "'7
B ~=Il-
.,
.._' '"--'
CAG Repeats:
. '":-,... ,
16/19
'~~~. --------_r....
16/19
,00
21/39 o
"
.... ft o_·,_IV~~
.~o ~ 16/28 .....v,...>r-,---- ----z·oV.~-
~~,T--------~~Q;~----__zl,~
,..,
......
21/39
o~g~a----~"-":Ao-~--- -z,~ , ...., 26/40 o- - 1tIr--- -JJ.Ir,----~~~---__z,Gtr_, ..... 26/40
"'"
"'" o---4,b--- - - - - - - - -- - - - - - -i"
L "'"
Size markets
Fig. 5. Molecular genetic testing of HD. (A) An autoradiograph of radiolabeled PCR products that encompass the HD CAG repeat following acrylamide gel electrophoresis. The number of individual CAG repeats is calculated by comparison to an M13 sequencing ladder and are noted below the gel. (B) Acrylamide electrophoresis of fluorescently labeled PCR products that encompass the HD CAG repeat detected by an automated sequencer. The number of individual CAG repeats is calculated by compari son with standard size markers and are noted to the right of the graphical output. (Images courtesy of M Galvez, P Scott, and D Rosenblatt, McGill University Health Centre, Division of Medical Genetics, Montreal , Canada .)
• Variable findings include pyramidal and extrapyramidal signs, nystagmus, amyotrophy fasciculations, and sensory loss
Prevalence • Accounts for approximately 20% of all SCA
Diagnosis • Very difficult to distinguish from other hereditary ataxias ; requires molecular genetic testing to detect an abnormal CAG trinucleotide repeat expansion in the coding region of the ataxin 3 (ATXN3) gene (Figure 4 and Table 4)
ATXN3 • Normal alleles have up to 47 repeats • Unlike SCA2 , phenotypically normal individuals can have intermediate allele s that contain 48-51 repeats; can result in pathologic expansion in subsequent generations • Affected individuals have at least one allele with 53-86 CAG repeats • Increase in disease severity has been observed in individuals homozygous for expanded ATXN3
429
Molecular Genetic Pathology
16-14
• CAG trinucleotide expansion results in polyglutamine expansion in the ATXN3 protein; however, the normal function of the ATXN3 protein is unknown
Molecular Genetic Testing • PCR amplification of the ATXN3 trinucleotide repeat region followed by gel electrophoresis • Detects nearly 100% of SCA3 cases
SCA6 Distinctive Clinical Attributes • Characterized by adult onset, typically in the fifth to sixth decade • Variable findings include very slow progression, dysarthria, nystagmus, and occasionally diplopia • Can present with episodic ataxia
Prevalence • Accounts for approximately 15% of all SCA
Diagnosis • Very difficult to distinguish from other hereditary ataxias ; requires molecular genetic testing to detect an abnormal CAG trinucleotide repeat expansion in the coding region of the calcium channel, voltage-dependent, o-I A subunit (CACNAJA) gene (Figure 4 and Table 4)
• The combination of PCR and mutation scanning detects nearly 100% of SCA6 cases • CACNAJA mutation disorders (AD)
- G293R causes a disorder similar to SCA6 but with a more severe clinical presentation - Episodic ataxia Type 2 - Familial hemiplegic migraine
Friedreich Ataxia (FRDA)-AR • Very unique among the nucleotide expansion ataxias as is most commonly caused by an unstable expansion of a GAA trinucleotide repeat, inherited in an AR fashion • Is not associated with disease anticipation
Distinctive Clinical Attributes • Characterized by onset in the first to second decade; however, atypical cases presenting beyond 25 years of age have been observed • Associated with depressed tendon reflexes, dysarthria, Babinski responses, and loss of position and vibration senses
Prevalence • It is the most common hereditary ataxia with an estimated prevalence of 1-2/50,000 • Carrierfrequency is 1/60-1/100
CACNAIA
Diagnosis
• Has multiple transcript variants • Short form variants: the CAG repeat is located within the 3' UTR and is not associated with any disease
• Requires molecular genetic testing to detect an abnormal GAA trinucleotide repeat expansion in the first intron of the frataxin (FXN) gene, located at 9q 13-q21.1
• Long form variant:
• >96% of FRDA individuals have FXN GAA expansion; approximately 4% of FRDA individuals are compound heterozygous for the GAA expansion and another deleterious FXN gene mutation
- Normal alleles have up to 18 repeats - Intermediate alleles with 19 repeats have unclear clinical significance - Affected individuals have at least one allele with 20-32 CAG repeats - Unlike many other AD ataxias , anticipation of SCA6 is not observed, as expansions of CACNAJA from parent to child rarely occur
FXN • Normal alleles have 5-33 repeats • Phenotypically normal individuals can have intermediate alleles that contain 34-65 repeats; can result in pathologic expansion in subsequent generations
• CAG trinucleotide expansion results in polyglutamine expansion in the CACNAIA protein
• Full penetrance alleles contain 66-1700 GAA repeats • Other inactivating mutations of FXN include nonsense and missense mutations • Encodes a mitochondrial protein, which belongs to the frataxin family; regulates mitochondrial iron transport and respiration
Molecular Genetic Testing
Molecular Genetic Testing
• PCR amplification of the CACNAJA trinucleotide repeat region followed by gel electrophoresis • As missense mutations in the CACNAIA gene cause disorders with phenotypic overlap to SCA6, mutation scanning is available • Mutation scanning typically involves the entire coding region
• PCR amplification of the FXN trinucleotide repeat region followed by gel electrophoresis • Alleles with> 100 repeats may not be detectable by PCR • Individuals with a single allele size as detected by PCR should also be analyzed by Southern blot • Individuals who fulfill the clinical diagnostic criteria of FRDA but who are heterozygous for a pathogenic
• CACNAJA encodes for the n-IA subunit of voltage-
dependent calcium ion channels, which are involved in muscle contraction and hormone/neurotransmitter release; expressed predominantly in neuronal tissue
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Molecular Medical Genetics
expanded allele should be tested for inactivating mutations by sequencing of the FXN coding region
• G380R has been shown to result in constitutively activated FGFR3
• Combination of PCR, Southern blot, and mutation scanning detects nearly 100% of FRDA cases
• As the FGFR3 pathway normally exerts a negative growth control, FGFR3 mutations result in gain-of-function
Skeletal and Connective Tissue Disorders
Molecular Genetic Testing
Achondroplasia
• Targeted G380R mutation analysis (Figure 6) and/or DNA sequencing of select exons
An AD disorder characterized by short-limb dwarfism.
Clinical • Affected individuals exhibit short stature with disproportionate arms and legs, characteristic faces with frontal bossing and midface hypoplasia, exaggerated lumbar lordosis, limitation of elbow extension, genu varum, and trident hand • Intelligence and life span are usually normal; however, spinal cord and upper airway abnormalities increase risk of infant death • In the majority of individuals it is caused by a sporadic G380R mutation in the fibroblast growth factor receptor-S (FGFR3) gene, located at 4p16.3 • Rare homozygous achondroplasia (ACH) has distinct radiologic findings and results in neurologic abnormalities and early death
• Sequence analysis of remaining exons is recommended when the two common G380R mutations are not found and ACH is suspected based on clinical and radiographic findings • Combination of G380R mutation analysis and FGFR3 exon sequencing detects nearly 100% of ACH cases • Other phenotypes associated with FGFR3 mutations: - Hypochondroplasia - Thanatophoric dysplasia - Severe ACH with developmental delay and acanthosis nigricans dysplasia - FGFR-related craniosynostosis
FGFR-Related Craniosynostosis Syndromes
• Is the most common form of inherited disproportionate short stature , occurring in approximately 1126,000
An AD spectrum of disorders comprised of Pfeiffer syndrome, Apert syndrome, Crouzon syndrome, BeareStevenson syndrome, FGFR2-related isolated coronal synostosis, Jackson-Weiss syndrome, Crouzon syndrome with acanthosis nigricans, and Muenke syndrome.
Diagnosis
Clinical
• Based on characteristic clinical and radiographic findings; molecular testing is available for atypical cases and those too young to diagnose with certainty
• Majority of syndromes are characterized by bicoronal craniosynostosis or cloverleaf skull, distinctive facial features, and variable hand and foot findings
• Molecular testing is clinically useful for prenatal diagnosis and confirmatory diagnostic testing
• Muenke syndrome and FGFR2-related isolated coronal synostosis are characterized only by uni- or bicoronal craniosynostosis
Prevalence
FGFR3 • Penetrance of mutated FGFR3 is 100% • Majority of affected individuals have one of two point mutations resulting in the same amino acid substitution (G380R) - Major mutation (-98% of affected individuals): ll38G > A - Minor mutation (-1 % of affected individuals): 1138G>C • Affected individuals with G375C and G346E mutations have been reported • Mature FGFR3 protein is a receptor tyrosine kinase • FGFRs have highly conserved amino acid sequences, differing from one another in their ligand affinities and tissue distribution • Interacts with fibroblast growth hormone resulting in receptor dimerization, autophosphorylation, and signal transduction, ultimately modulating bone development and maintenance
• Abnormal skull may be detected by ultrasound prenatally or not until later infancy • Each syndrome has specific clinical features; however, most share common characteristics: hypertelorism, midfacial hypoplasia with proptosis, down-slanting palpebral fissures, high-arched palate , developmental delay/mental retardation, hydrocephalus, hearing loss, and visual impairment • Caused by mutations in the FGFRJ (8pll.2-plLl), FGFR2 (lOq26), and FGFR3 (4pI6.3) genes
Prevalence • Overall incidence for all forms of craniosynostosis is 112,000-112,500 live births
Diagnosis • Typically diagnosed based on clinical findings • Molecular testing of FGFRJ, FGFR2, and FGFR3 assists the diagnosis of suspected craniosynostosis
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Molecular Genetic Pathology
- S252W and P253R account for 71 and 26% of Apert syndrome individuals, respectively
----
- Identical FGFR2 mutations have been reported in Pfeiffer, Crouzon, and Jackson-Weiss syndromes Cysteine residues 278 and 342 are common mutation sights for Pfeiffer and Crouzon syndromes
-----
-
Sic I (G>A)
--
Mspl (G>C)
Fig. 6. Molecular genetic testing of ACH. The G380R FGFR3 mutation results from either the 1138G > A or the 1138G > C nucleotide substitution, which create Sid or MspI restriction sites, respectively. The image depicts gel electrophoresis of PCR products that encompass the G380R mutation site and which are digested with either Sid or MspI. In this illustration, the pro-band is heterozygous for the more common 1138G > A mutation given the presence of the Sid restriction fragments (arrows).
• FGFR3 - P252R: is diagnostic for Muenke syndrome - A391E: majority of individuals with Crouzon syndrome with acanthosis nigricans
- FGFR3 mutations also cause ACH, hypochondroplasia, thanatophoric dysplasia, and severe ACH with developmental delay and acanthosis nigricans • Molecular Genetic Testing - Involves initial analysis of recurrent mutations followed by selective gene sequencing
- FGFRJ- and FGFR3-targeted mutation analysis and sequencing of select exons
- FGFR2-targeted mutation analysis, sequencing of select exons, and mutation scanning of entire coding region
Mar/an Syndrome An AD systemic disorder characterized by ocular, skeletal, and cardiovascular abnormalities.
• Molecular testing is available for prenatal diagnosis, yet does not yield informative prognosis
FGFRs • A family of four tyrosine kinase receptors that nonspecifically bind FGFs-a family of signaling molecules that regulate cell proliferation, differentiation, and migration • FGFR sequence differences effect ligand binding specificity
• FGFR4 is not involved in craniosynostosis syndromes • Normal function is likely to restrain limb growth • Mutations cause excessive activity • FGFR amino acids 252-253 are located within an extracellular "linker region" and are common mutation sites believed to alter ligand binding
• FGFRJ - Approximately 5% of individuals with Pfeiffer syndrome Type 1 (mild form) have a P252R mutation
• FGFR2 - Most mutations are missense ; however, deletions, insertions, and splice site mutations have been reported - Mutations identified in Pfeiffer, Apert, Crouzon, Beare-Stevenson, and Jackson-Weiss syndromes
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Clinical • Affected individuals show a very broad phenotypic spectrum • Symptoms may be present at birth or appear in childhood or adulthood • The four major diagnostic findings include dilation or dissection of the aorta at the level of the sinuses of Valsalva, ectopia lentis, dural ectasia, and four of eight typical skeletal features • Typical skeletal features : bone overgrowth, joint laxity, long extremities, pectus excavatum or carinatum, scoliosis, high arched palate, positive wrist and thumb signs, reduced upper to lower segment, arm span to height ratio> 1.05, and flat feet • Primarily caused by a mutation in the fibrillin -l (FBNl) gene, located at 15q21.1 • Approximately 75% of affected individuals have an affected parent; remaining have a de novo FBN J mutation
Prevalence • One of the most common connective tissue disorders , occurring in approximately 1-2/1 0,000
Diagnosis • Diagnosis of Marfan syndrome (MFS) is based on family history and characteristic clinical findings in multiple organ systems
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Molecular Medical Genetics
• Requires "major" manifestations in at least two body systems, with "minor" involvement of a third body system; if a positive family history is established, diagnosis requires "major" manifestation in one body system with "minor" involvement of a second • Molecular testing is clinically useful for prenatal diagnosis, predictive testing, and confirmatory diagnostic testing
FBN] • Penetrance of mutated FBN] is 100% with variable expressivity • >500 FBN] mutations have been reported in MFS individuals • No definitive genotype-phenotype correlations have been observed; however, mutations associated with severe and rapidly progressive MFS intermittently cluster between exons 24 and 32 • No common mutation exists in any population • Is found in elastic and non-elastic connective tissues of the body • Wild-type FBNI protein is an important component of extra-cellular microfibrils • Participates in the formation and homeostasis of elastic matrix and matrix-cell attachments
- Familial ectopia lentis - Shprintzen-Goldberg syndrome
Osteogenesis Imperfecta A primarily AD group of bone formation disorders characterized by low bone mass and propensity to fracture.
ClinicaL • Affected individuals also may exhibit blue sclera, dentinogenesis imperfecta, skin hyperlaxity, joint hypermob ility, and hearing loss • Fractures are most common in extremities but can occur in any bone • Is a broad clinical entity but is artificially classified into seven types (I-VII) based on clinical presentation, radiographic findings, mode of inheritance, and molecular genetics • Severity: - Type I: mild - Type II: perinatal lethal - Type III: severe - Type IV: moderate-to-mild - Types V-VII: moderate • In the majority of individuals is caused by a mutation in the collagen, Type I, n-I (COLlAl), or COLlA2 genes, located at 17q21.33 and 7q22.1, respectively
• Mutant FBN] is believed to be dominant-negative as MFS individuals typically have reduced FBNI protein expression below that which would be expected from the remaining wild-type allele
• COLlA] and COLlA2 mutations are found in Types I-IV; loci for Types V-VII have not been accurately mapped
MoLecuLar Genetic Testing
PrevaLence
• Mutation scanning of the FBN] gene is available ; includes denaturing high-performance liquid chromatography (DHPLC) and direct sequencing of all exons using genomic DNA
• Overall incidence for all forms of osteogenesis imperfecta (01) is 6-71100,000; Types I and IV account for over half of all 01
• If a specific mutation is known within a family, targeted mutation analysis by bidirectional DNA sequencing is recommended • Mutation scanning of the FBN] gene by cDNA sequencing is available ; given the large size of FBN] , it is more efficient than genomic DNA sequencing • Linkage analysis may be used to determine if an individual has inherited an FBNI allele associated with MFS in multiple family members - Markers are highly informative and are within the FBN] gene - Not independently conclusive, as locus heterogeneity has not been definitively excluded in MFS • Mutations are detected in 70-93% of MFS individuals • Other phenotypes associated with FBN] mutations: - Mitral valve prolapse, Aortic root diameter at upper limits , stretch marks of the skin, skeletal conditions similar to MFS (MASS) phenotype - Mitral valve prolapse syndrome
Diagnosis • Based on characteristic clinical and radiographic findings, family history , and biochemical and molecular testing • Molecular testing is clinically useful for prenatal diagnosis and confirmatory diagnostic testing
eOLlAI and eOLlA2 • >200 structural mutations have been identified • Encode Type I pro-collagen chains containing repeating sequences of uninterrupted Gly-X- Y that are essential for proper chain folding • Two pro-a-l(I) chains and one pro-a-2(I) chain form a triple helix from carboxy to amino terminus • Pro-collagen is secreted and terminal peptides removed forming Type I collagen molecules, which are then assembled into collagen fibrils • Major protein in bone, connective tissue, and cartilage • 01 Type I is associated with heterozygous truncating COLlA] mutations resulting in haploinsufficiency
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Molecular Genetic Pathology
• 01 Types II-IV are associated with heterozygous triple helix domain COLlAl and COLlA2 mutations, resulting in glycine substitution and abnormal procollagen folding • Biochemical analysis is typically performed in vitro on cultured dermal fibroblast s by assaying the structure and quantity of synthesized Type I collagen
Molecular Genetic Testing • Mutation scanning of the COLlAl and COLlA2 genes is available using both genomic DNA and cDNA for sequence analysis
• If a specific mutation is known within a family, targeted mutation analysis by bidirectional DNA sequencing is recommended • Combination of targeted mutation and COLlA I and COLlA2 coding region sequence analysis detects nearly 100% of 01 Types I and II, and approximately 60-80% Types III and IV • Other phenotypes associated with COLlAl and COLlA2 mutations: - Ehlers-Danlos syndrome (classic and arthrochalasia types) - Osteoporosis - Arterial dissection
X-LINKED INHERITANCE
Fragile X Syndrome Fragile X syndrome (FGLX) is the leading cause of Xlinked mental retardation among males. The disorder was named for the cytogenetically visible fragile site (FRAXA) at band Xq27.3 that in some cases was heritable.
Clinical • Mental impairment, ranging from learning disabilities to mental retardation with delay of milestones in infancy • Attention deficit and hyperactivity • Anxiety and unstable mood • Autistic-like behaviors • Large head, long face, large ears, and flat feet • Macro-orchidism • Hyperextensible joints, especially fingers • Seizures (epilepsy) affect about 25% of people with fragile X • Sex-specific differences - Boys are typically more severely affected than girls - While most boys have mental retardation, only onethird to one-half of girls have significant intellectual impairment; the rest have either normal IQ or learning disabilities
Prevalence • Prevalence of 1 in 4000 affected males with 1/2 as many affected females • Prevalence of female pre-mutation carriers was estimated at I in 259 in one study (possibly higher in specific populations)
Inheritance • Pre-mutation carrier females, but not males, are at risk for transmitting full mutation alleles to both male and female offspring
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• Many families transmit pre-mutation fragile X mental retardation-l (FMRI) alleles for generations with little or no presentation of clinical symptoms until a full mutation is produced , resulting in an affected individual
The FMRI Gene • The FMRl gene is located at Xq27.3, contains 17 exons, and spans 38 kb • FMRl encodes an mRNA-binding protein of 632 amino acids-fragile X mental retardation protein (FMRP) • FMRP is thought to shuttle select mRNAs between the cytosol and nucleus and playa role in synaptic maturation and function • FMRl is highly expressed in the brain, testes, ovaries, esophageal epithelium, thymus, eye, and spleen • Mechanism of expansion (>99% of cases) - Expansion of CGG repeat located in 5' UTR region of FMRl gene (Figure 7) - Expansion of CGG leads to methylation of promoter CpG of FMRl gene, silencing the gene and resulting in lack of protein product-FMRP - In full mutation females, methylation of FMRl full mutation is independent of X-inactivation • Characterization of repeat size - Normal alleles : ~4 repeat s-with 29 and 30 repeats most common - Normal "gray zone" alleles: 45-54 repeat s-these alleles can exhibit instability, but have never been observed to expand to full mutation - Pre-mutation alleles: 55-230 repeats-these alleles exhibit instability and are at risk for expansion to full mutation • 59 repeats is the smallest pre-mutation allele observed to expand to a full mutation. The ACMG recommends using 55 repeats as smallest premutation to account for inter-laboratory differences in size standards
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Molecular Medical Genetics
A
FMR1 (4362 bp)
5 - 40
B Premutation
Gray zone
c Normal
5.2 kb
2.8 kb 2.4 kb 123 456
2
3
4
5
6
Fig. 7. (A) Schematic of FMRI gene trinucleotide repeat in the 5' UTR. Ranges for normal, gray zone, pre-mutation, and full mutations are shown. 54 repeats is the ACMG cut off for gray zone alleles and 55 repeats is now considered a pre-mutation (see text). (B) Example of fragile X PCR products run on a 6% denaturing polyacrylamide gel. (Lane 1) Full mutation male with no apparent PCR product; (lane 2) normal male with 24 repeats; (lane 3) pre-mutation male with 59 repeats, and (lane 4) normal female with 29 and 30 repeats. (Lane 5) female with one normal allele of 19 repeats and a gray zone allele of 47 repeats , (lane 6) female with one normal allele of 30 repeats and a gray zone allele of 46 repeats . (C) Example of a fragile X Southern blot with genomic DNA that was digested with both EcoRI and XhoI (methylation sensitive). The 5.2 kb band represents the methylated alleles, unable to digest with XhoI, whereas the 2.8 kb and 2.4 kb bands represent unmethylated alleles that were cut with Xhol. (Lane 1) normal female, (lane 2) pre-mutation male, (lane 3) normal male, (lane 4) normal female, (lane 5) pre-mutation female , and (lane 6) full mutation male . • Pre-mutation carriers are at risk for additional adult onset disorders • Premature ovarian failure in 20% of premature carriers • Fragile X tremor ataxia syndrome-higher penetrance in males Full mutation alleles: >230-2000 repeats-these alleles cause FGLX - Mosaicism for full mutation can complicate the analysis and may not be detectable if low level
Diagnosis • Cytogenetic analysis of the fragile site (FRAXA) is not an acceptable method of diagnosis • PCR of CGG repeat is performed to determine allele sizes with use of appropriate control samples or size ladder (Figure 7B) - PCR cannot distinguish a homozygous female from one with a non-amplifiable second allele - PCR is not adequate for the detection of mosaic individuals with both pre-and full mutations
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• Southern blot used with double enzyme digestion with a second enzyme that is internal to the first and is methylation sensitive (Figure 7C) - Females will have an undigested methylated (inactive X or full mutation) allele and digested unmethylated allele (active X) - Males will have digested allele only unless full mutation is present - Southern blot can detect pre-mutation/full mutation mosaics - Prenatal analysis of chorionic villus sampling can be problematic • Methylation is absent or incomplete in this tissue at time of procedure • Full mutations, which are unstable and may have mosaic repeat size, are difficult to interpret on Southern blot • Follow up amniocentesis may be necessary
X-Linked Muscular Dystrophy (DMD and BMD) Dystrophinopathies are a spectrum of X-linked muscle diseases that include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and DMD-related dilated cardiomyopathy (DCM) .
Prevalence and Inheritance • 1 in 5600 live male births • 2/3 are inherited by mother and 1/3 are new mutations • 75% of female carriers have no signs or symptoms • Risk for female carrier to have an affected male child is 25% with each pregnancy • Germline mosaicism is present in approximately 15% of carrier mothers, complicating risks to siblings
Clinical • Characterized by a spectrum of muscle disease that ranges from mild to severe • DMD is most severe and rapidly progressive and presents in early childhood - Presents with delayed milestones (I8 months-8 years) - Progressive muscle weakness that is symmetrical (proximal> distal) - Generalized motor delay, delay in sitting, standing , and walking -
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Gait problems including flat footedness Wheelchair bound by 13 years old Cardiac involvement in 90% of patients Approximately 80% of females show no signs or symptoms, but when present usually milder than males, later onset muscle weakness and cramps
Molecular Genetic Pathology
• BMD is characterized by later-onset skeletal muscle weakness - Progressive muscle weakness (proximal> distal) If wheelch air bound, after 18 years old - Activity induced cramping - Cardiac involvement in 90% of patients Approximately 80% of females show no signs or symptoms, but when present usually milder than males, later onset muscle weakness and cramps, dilated cardiomyopathy in some • DCM shows no evidence of skeletal muscle disease - Dilated cardiomyopathy with congestive heart failure - Males present at ages death
2~0
with rapid progression to
- Females present later in life with slower disease progression
DMDGene • The DMD gene spans 2.4 Mb of DNA on Xp21.2 and contains 79 exons (largest human gene known) • Encodes a membrane associated protein, dystrophin, present in muscle cells and some neurons - Part of a complex that links the cytoskeleton with cell membrane and bridges the cytoskeleton with the extracellular matrix - Full length protein is 427 kDa, but many different isoforms identified • Mutations - 6-10% of males with DMD or BMD have duplication of one or more exons - 25% of males with DMD and 5-10% of males with BMD have small insertions/deletions, point mutations, or splicing mutations - Mutations that obliterate or severely disrupt dystrophin function cause DMD, whereas mutations that affect the quantity of dystrophin or truncate the protein in frame, result in BMD - DCM results from mutations that effect the expression or function of dystrophin in cardiac muscle (exon 1 and muscle specific promoter mutations)
Diagnosis • Serum creatine phosphokinase levels are elevated to > lOX in all DMD males and >5X in all BMD males • Creatine phosphokinase levels are also elevated from 2X to lOX in approximately 50% of female carriers • Muscle biopsy with immunohistochemistry for dystrophin is informative in males and some females • Molecular analysis of DMD mutation status - Mutations found in 100% of DMD patients and 85% of BMD patients
Molecular Medical Genetics
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- Multiple x PCR, Southern blotting , and MLPA are used to detect deletion s - Quantitati ve PCR and MLPA can be used to detect duplications - Mutation scanning method s, such as DHPLC and sequencing are used to screen for small insertion s/deletion and point mutation s
X-linked Adrenoleukodystrophy (X-ALD) X-ALD is the most common of the peroxisomal disorders. It is a severe, often fatal disease that manifests in a progressive demyelination of the central nervous system, dysfunction of the adrenal cortex, and testicular dysfunction in hemizygous males.
Clinical • Most common form has early onset that appears at 4-8 years of age resulting in a progre ssive irreversible dementia and death • Less severe presentations include adrenomyeloneuropathy with a later age of onset, adrenal insufficiency, and neurologic complications limited to spinal cord and peripheral nerves
Prevalence • Incidence of all variant forms is I in 15,DOO-most common genetic determinant of peroxi somal disease
The X-ALD Gene • ATP-binding cassette , subfamily 0 , memberl (ABe D I) is located on Xq28 , contains 10 exons, and spans 21 kb • Encodes a protein of 745 amino acids-ALD protein (ALDP) • Mutation analysis - Whole gene sequencing of exons and exon/intron boundaries performed as well as other mutation scanning techniques, such as DHPLC. - Potential compl ications with PCR amplification because of paralogous gene segments exons 7-10 on chromosomes 2pll, 10pll, l6pll , and 22qll - Over 250 different lesions have been found in the ABCDI gene - Mutations in all 10 exons have been reported - Vast majority are point mutation s (58.4%), although frameshift s and nonsense, and exon deletion s have also been identified (http://www.x-ald.nV) - Two base pair AG deletion in exon 5 found in 10.3% of families with X-ALD (most common mutation identified) - No genotype-phenotype correlations are apparent and wide phenotypic variation has been reported within familie s - 70% of missense mutation s result in absent or reduced ALDP, indicating that most mutation s in ABCD I result in complete loss of protein function
Diagnosis
Inheritance • X-linked with males affected and up to 20% of carrier female s with late onset neurologic symptoms similar to adrenomyeloneuropathy • >93% of X-ALD patients inherit mutation s from their mothers with remaining 7% carrying de novo mutation s
• Primary biochemical defect-impaired peroxisomal ~-oxidation with accumulation of very long chain fatty acids-mostly C26 in plasma and tissues • In hemizygous males (99%) and 85% of carrier female s, plasma concentration of very long chain fatty acids are elevated-used as a diagno stic marker for the disease
MITOCHONDRIAL DISORDERS A clinically heterogeneous group of disorders that arise from mitochondrial respiratory chain dysfunction. Caused by mutations of mitochondrial (mtD NA) or nuclear DNA (nONA). Clinical symptoms are first seen in tissues with high energy demand s or low thresholds for energy deficiency ; central nervous system and muscles often involved. • Prevalence - Including both mtDNA and nONA mutations in children and adults, prevalence is approximately 1/5000
The Mitochondrion • An essential cytoplasmic organelle present in all eukaryotic cells that provides majority of cell energy • Typical human cells have several hundred mitochondria; 1000-2000 in a single liver cell
• Energy-generating apparatu s is the oxidati ve phosphorylation pathway (OXPHOS), compo sed of electron transport chain and ATPase (Figure 8): - Is located in the inner membrane and employs five multi-polypeptide enzyme complexes and two electron carriers - Main function is coordinated transport of electron s and protons and production of ATP - Majority of OXPHOS complex proteins are nONA encoded and imported from cytosol • Has its own 16.5-kb double- stranded circular genome that contains two rRNA genes, 22 tRNA genes, and 13 structural genes, which encode OXPHOS subunits (for illustration, see http://www.mitomap.org/)
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Molecular Genetic Pathology
Intermembrane space Compl ex
V Mitochondrial matrix
Succinate
Fumarate
H+
Fig. 8. The respiratory chain system in the inner membrane of the mammalian mitochondria. Electrons (e) are transferred from complex I and II to coenzyme Q (CoQ). There they are transferred via complex III and cytochrome c (Cyt c) to complex IV, where oxygen is reduced to water. The movement of H+ from the matrix to the inter-membrane space is coupled with energy release from the electrons. The proton gradient is used for the production of ATP by complex V. Mutations in nDNA-which encodes for OXPHOS subunits or proteins involved in respiratory chain homeostasis-and in mtDNA leads to mitochondrial disease. • mtDNA genome: - Both strands are transcribed from a single promoter - Does not contain introns - Some differences in genetic code between mtDNA and nDNA - l G-I? times faster mutation rate than nDNA - Is maternally inherited - Each cell has 103-104 mtDNA molecules • Homoplasmy-the state in which all mtDNA molecules are identical • Heteroplasmy-the presence of more than one type of mtDNA molecule within a cell • New mtDNA mutations are multiplied by replication and randomly divided into daughter mitochondria during cell division ; leads to differences in level of heteroplasmy between tissues • Penetrance of pathogenic mutation is increased with the degree of mutant heteroplasmy • Tissue phenotype is normal until threshold level of mutant heteroplasmy is exceeded • Organs with greatest ATP requirements are most sensitive to mtDNA mutations • Mitochondrial disorders grouped into two major categories, those due to defects of mtDNA and those due to defects in nDNA • All inheritance models are possible in connection with mitochondrial disorders • Mitochondria dysfunction is also observed in late-onset neurodegenerative disorders and aging • Majority of patients with enzymatically verified mitochondrial deficiency have an unidentified mutation
Diseases Resulting From mtDNA Mutation • Phenotypes of diseases vary between mtDNA mutations and between individuals with same mutation
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• Probability of disease increases with age and diseases are often progressive
mtDNA Rearrangements • Deletions vary in size and location but a 5-kb common deletion has been observed in some sporadic disorders: • Pearson syndrome-typically early onset; sideroblastic anemia, and exocrine pancreatic failure ; often fatal • Kearns-Sayre syndrome (KSS)-onset <20 years of age; progressive external ophthalmoplegia (PEa), pigmentary retinopathy, plus one of heart block, elevated cerebrospinal fluid (CSF) protein, or cerebellar ataxia • Chronic PEO-onset >20 years of age; similar to KSS, bilateral ptosis • Duplications of mtDNA have been observed in patients with KSS and diabetes mellitus with deafness • Deletions/duplications usually encompass several essential coding and/or tRNA genes, which impairs mitochondrial protein synthesis
mtDNA PointMutations • Most are transition mutations occurring in tRNNrRNA genes or respiratory chain subunit genes • Are maternally inherited
tRNA Mutation Disorders • Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes-characterized by recurrent vomiting, headache, and stroke-like episodes causing cortical blindness, hemiparesis, or hemianopia - Usually present in children or young adults after normal early development - Most common mutation: A3243G in tRNALeu(UUR) - Other less common mutations have been reported • Myoclonus epilepsy with ragged redfiberscharacterized by mycoclonus, seizures, mitochondrial myopathy, and cerebellar ataxia
Molecular Medical Genetics
- Less common signs include hearing loss and dementia - Most common mutations: A8344G, T8356C, G8363A in tRNALys
• Non-syndromic sensorineural deafness-most common mutation: A7445G in tRNASer(UCN)
rRNA Mutation Disorders • Aminoglycoside-induced non-syndromic deafness-most common mutation: AI555G in mitochondrial 12S rRNA gene
Protein-Encoding Mutation Disorders • Neurogenic weakness, ataxia, and retinitis pigmentosacharacterized by late childhood or adult onset, ataxia, pigmentary retinopathy, and dementia - Less common signs include sensorimotor neuropathy - Most common mutation : T8993G/C (heteroplasmy -70%) • Maternally inherited leigh syndrome-characterized by early onset, devastating encephalopathy, hypotonia, cerebellar, and brain-stem signs - Less common signs include ophthalmoplegia and respiratory depression - Most common mutation: T8993G/C (heteroplasmy -90%) • Leber's hereditary optic neuropathy-characterized by young adult visual loss due to bilateral optic atrophy with a bias toward males - Less common signs include cardiac dysrhythmia and dystonia - Most common mutations: G3460A, G 11778A, Tl4484C, and A 14495G - Other less common mutations have been reported
Diseases Resulting From Nuclear DNA Mutation • Most of the respiratory chain subunits are encoded by the nDNA • Structure and function of the respiratory chain requires many steps, which are largely encoded by the nDNA • nDNA mutation disorders can follow AR, AD, and X-linked reces sive (XLR)-inheritance patterns
Structural Respiratory Chain Defects • Complex 1 deficiency:
- Leigh and Leigh-like syndrome-(AR)-NDUFS4 (5ql1.1), NDUFS7 (l9pI3 .3) , and NDUFS8 (llqI3) - Hypertrophic cardiomyopathy and encephalomyopathy -(AR)-NDUFS2 (lq23) - Macrocephaly, leukodystrophy, and myoclonic epilepsy-(AR)-NDUFVl (llq13)
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• Complex II deficiency:
- Leigh and Leigh-likesyndrome-(AR)-SDHA (5pI5)
Non-Structural Respiratory Chain Defects • Intergenomic communication defects:
- Mitochondrial neurogastrointestinal encephalomyopathy-(AR)-TP (22q 13.33) - Dominant PEO-(AD)-ANTl (4q35) • Complex I assembly defects :
- Early onset progressive encephalopathy(AR)-BJ7.2L (5qI2.l) • Complex III assembly defects:
- Metabolic acidosis, tubulopathy. encephalopathy, and liver failure-(AR)-BCSlL (2q33) • Complex IV assembly defects:
- Leigh syndrome-(AR)-SURFJ (9q34.2) - Cardioencephalomyopathy-(AR)-SC02 (22q13.33) - Neonatal-onset hepaticfailure and encephalopathy(AR)-SCOJ (I7pI2-p13) Leigh and de Toni-Fanconi-Debre syndrome-(AR)COXlO (I7pI2-17pl1.2) Early-onset hypertrophic cardiomyopathy-(AR)COXJ5 (lOq24) - French-Canadian Leigh syndrome-(AR)-LRPPRC (2p2l) • Complex V assembly defects:
- Early-onset encephalopathy, lactic acidosis-(AR)ATPAF2 (17p 11.2) • Homeostasis and import:
- Freidreich's ataxia-(AR)-FXN (9q13-q21.1) - Hereditary spastic paraplegia-(AR)-SPG7 (I6q24.3) - mtDNA depletion myopathy-(AR)-TK2 (16q 22-q23.1) - Hepatocerebral mtDNA depletion-(AR)-DGUOK (2p13) - Wilson disease-(AR)-ATP7B (I6q24.3) - PEO-(AD or AR)-ANTl (4q35), POLG (I5q25) - Dominant optic atrophy-(AD)-OPAI (3q28-q29) - Deafness-dystonia syndrome-(XLR)-TIMM8A (Xq22.1) - Anemia, sideroblastic, and SCA-(XLR)-ABCB7 (Xq12-q13) - Barth syndrome-(XLR)-TAZ (Xq28)
Diagnostic Evaluation • Some individuals have a clear characteristic phenotype of a specific disorder; can be confirmed by biochemical and molecular genetic testing
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Molecular Genetic Pathology
• Metabolic testing and muscle biopsy (respiratory chain activity) are useful for diagnosis • Family history and inheritance evaluation is essential in directing molecular genetic testing • Prenatal diagnosis is available for AR nDNA mutations • Genetic counseling is complex based on the dual contribution of mtDNA and nDNA to the respiratory chain and the general characteristics of mitochondrial genetics • Molecular genetic testing
Performed on DNA from blood (suspected nDNA mutations) or skeletal muscle (suspected mtDNA mutations) Targeted mutation analysis of a panel of genes Southern blot analysis may detect mtDNA rearrangements If no recognized point mutation is identified, entire mtDNA sequencing and/or mutation scanning is available
SUGGESTED READING Boehm CD, Cutting GR, Lachtermacher MB, Moser HW, Chong 55. Accurate DNA-based diagnostic and carrier testing for X-linked adrenoleukodystrophy. Mol Genet Metab . 1999;66:128-136. Boileau C, Iondeau G, Mizuguchi T, Matsumoto N. Molecular genetics of Marfan syndrome . Curr Opin Cardiol. 2005;20:94-200. Beutler E. Hemochromatosis: genetics and pathophysiology . Annu Rev Med.2oo6;57:331-347. Brandon MC, Lott MT, Nguyen KC, et al. MITOMAP: a human mitochondrial genome database-2004 update. Nucleic Acids Res. 2oo5;33(Database Issue):D611--613. URL: http://www.mitomap.org. Chace DH, Kalas TA, Naylor EW. The application of tandem mass spectrometry to neonatal screening for inherited disorders of intermediary metabolism . Annu Rev Genomics Hum Genet. 2002;3:17-45 . Cohen MM, Jr. Some chondrodysplasias with short limbs: molecular perspectives. Am J Med Genet. 2002;112:304-313 . DiMauro 5. Mitochondrial diseases. Biochim Biophys Acta. 2004;1658:80-88.
Maddalena A, Richards CS, McGinniss MI, et al. Technical standards and guidelines for fragile X: the first of a series of disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics. Quality Assurance Subcommittee of the Laboratory Practice Committee . Genet Med. 2001;3:200-205 . Muntoni F,Torelli S, Ferlini A. Dystrophin and mutations : one gene, several proteins, multiple phenotypes. Lancet Neurol. 2003;2:731-740. Ogino S, Wilson RB. Spinal muscular atrophy : molecular genetics and diagnostics . Expert Rev Mol Diagn. 2004;4:15-29. Paulson HL, Fischbeck, KH. Trinucleotide repeats in neurogenetic disorders . Annu Rev Neurosci. 1996;19:79-107. Qaseem A, Aronson M, Fitterman N, Snow V, Weiss KB, Owens OK. Screening for hereditary hemochromotosis: a clinical practice guideline from the American College of Physicians . Ann Intern Med. 2005;143:517-521 .
Graff C, Bui TH, Larsson NG . Mitochondrial diseases. Best Pract Res Clin Obstet Gynaecol. 2002;16:715-728.
Sherman 5, Pletcher BA, Driscoll DA. Fragile X syndrome: diagnostic and carrier testing Genet Med. 2005;7:584-587 .
Gregersen N, Andresen BS, Corydon MJ, et al. Mutation analysis in mitochondrial fatty acid oxidation defects : exemplifed by acyl-CoA dehydrogenase deficiencies, with special focus on genotype phenotype relationship . Hum Mutat, 2001;18:169-189.
Smeitink J, van den Heuvel L, DiMauro S. The genetics and pathology of oxidative phosphorylation . Nat Rev Genet. 2001;2:342-352.
Grody WW, Cutting GR, Klinger KW, Richards CS, Watson M5, Desnick RI. Subcommittee on Cystic Fibrosis Screening, Accreditation of Genetic Services Committee, ACMG. Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med. 2001;3:149-154. Kemp 5, Pujol A, Waterham HR, et al. ABCDI mutations and the X-linked adrenoleukodystrophy mutation database : role in diagnosis and clinical correlations. Hum Mutat. 2001;18:499-515.
440
Timchenko LT, Caskey CT. Trinucleotide repeat disorders in humans: discussions of mechanisms and medical issues. FASEB J. 1996;10:1589-1597. Watson MS, Cutting GR, Desnick RI, et al. Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel. Genet Med. 2004;6:387-391. Zinberg RE, Kornreich R, Edelmann L, Desnick RI. Prenatal genetic screening in the Ashkenazi Jewish population . Clin Perinatol. 2001;28:367- 382.
17 Prenatal Diagnosis Nataline Kardon, MD and Lisa Edelmann, PhD
CONTENTS
I. General II.
Prenatal Testing Modalities Amniocentesis Methodology Benefits Limitations Laboratory Methodology In Situ Clonal Analysis Chorionic Villus Sampling (CVS) Methodology Benefits Limitations Laboratory Methodology Percutaneous Umbilical Blood Sampling Methodology Benefits Limitations Laboratory Methodology Fetal Skin Biopsy Methodology Benefits Limitations
III. Indications for Prenatal Testing Advancing Maternal Age Ultrasound Findings Abnormal Screening Results
Parental Chromosome Abnormality Previous Pregnan cy with Cytogenetic Abnormality
17-2 17-2 17-2 17-2 17-2 17-2 17-2 17-2 17-2 17-2 17-2 17-3 17-3 17-3 17-3 17-3 17-3 17-3 17-3 17-3 17-3
17-3 17-3 17-3 .17-3
17-4 17-4
IV. Fetal Abnormalities and Outcome
17-4
Aneuploidy Structural Rearrangements Supernumerary Marker Chromosomes Uniparental Disomy
17-4 17-5 .17-5 17-6
V. Fluorescence In Situ Hybridization in Prenatal Testing
17-6
FISH on Direct Specimens to Screen for Common Aneuploidies 17-6 Subtelomere FISH Probes can be Used to Examine Fetal Chromosomes When Deletion or Duplication is Suspected ..........17-6 Locus Specific FISH Probes can be Used to Examine Fetal Chromosomes 17-6
VI.
Chromosomal Microarray Comparative Genomic Hybridization in Prenatal
Diagnosis Constitutional Arrays are Used for Specific Genomic Gains and Losses
VII. Suggested Reading
17-7 17-7
17-7
441
17-2
Molecular Genetic Pathology
GENERAL • Cytogenetic prenatal diagnosis involves examination of the fetal chromosome complement • Cells are obtained by various modalities during the first or second trimester and are subsequently established in tissue culture
• Short term in situ tissue culture methods produce sufficient cells for metaphase analysis • Chromosome preparations are banded and analyzed microscopically • Digitized images are submitted for diagnosis
PRENATAL TESTING MODALITIES Amniocentesis Methodology • Performed during second trimester between 16 and 18 weeks gestation • 20 cc of amniotic fluid is withdrawn from the amniotic sac transabdominally under ultrasound guidance - The first 1-3 cc are discarded to remove maternal cells • Amniocytes, which are similar to fibroblasts grow in tissue culture
to a harvesting procedure utilizing a robotic harvester - Cell division is arrested by Colcemid - Chromosomes are swollen by hypotonic treatment - Preparation is fixed on cover slip by acetic acid/ methanol fixative • Cover slips are transferred and fixed on slides • Metaphase preparations are stained by a Giemsa-Trypsin protocol and are then analyzed under the microscope
• Primary care obstetrician can perform procedure
Chroionic Villus Sampling (CVS) Methodology
• Outpatient procedure in physician's office
• Performed during first trimester at 10-12 weeks gestation
• Procedure risk is 1 in 200 or 0.5%
• Chorionic villi are gently aspirated by catheter transvaginally or by needle aspiration transabdominally
Limitations
• Placental tissue is dissected and enzymatically digested with a mixture containing Trypsin and Collagenase.
Benefits
• Chromosome preparations cannot be synchronized so high-resolution chromosome analysis is not possible • Tum around time is 6-10 days • Results not available until end of second trimester • Interpretation is dependent upon the degree of chromosome band resolution
Benefits • Results are available by the end of the first trimester • Tum around time is 4-7 days with adequate specimens • Direct analysis produces preliminary results in 8-24 hours
Laboratory Methodology In Situ ClonalAnalysis • Amniotic fluid specimen is centrifuged in order to obtain the fibroblast cells
Limitations
• The cell pellet is suspended in tissue culture medium
• Tum around time is dependent upon the size of the initial sample
• The suspension is placed on a sterile cover slip that is inside a small Petri dish
• Long technically proficient learning curve requiring specialist to perform procedure
• The culture is placed in a 5% CO 2 incubator at 37°C
• Procedure risk is 1-2% • Interpretation is dependent upon the degree of chromosome band resolution and high-resolution analysis is not possible
• Culture medium is added after 24 hours and then on a specified culture regimen • After 5 days in culture, the cover slip is examined to determine if there is sufficient clonal activity for harvest, which occurs at 7 days • When there is sufficient activity to produce adequate metaphases for analysis, the culture is subjected
442
• Separation of villi from maternal decidua is critical for analysis -
1-2% risk for maternal cell contamination
• 1-2% incidence of confined placental mosaicism
Prenatal Diagnosis
17-3
Laboratory Methodology
• Procedure risk comparable with CVS
• Cell pellet is obtained after above digestion
• Late second trimester diagnosis
• Cells are suspended in media
Laboratory Methodology
• The same methodology as amniotic fluid cells (see Amniotic Fluid Laboratory Methodology section) is employed to produce adequate metaphases for analysis
• Note: the tissue does not produce individual clones and generally the cultures are ready to be processed after 3-5 days
See Chapter 2 Red Blood Chromosomes Analysis section.
Fetal Skin Biopsy Methodology • A full thickness skin biopsy is obtained from the fetus under ultrasound guidance or fetoscopy
Percutaneous Umbilical Blood Sampling Methodology
• Performed during second or third trimester depending upon diagnosis
• Needle is inserted into fetal umbilical vessel under ultrasound guidance
• Requires specialist to do procedure
• Performed at 18-20 weeks gestation • Requires maternal fetal medicine specialist to do procedure
• Specific diagnoses are made by electron microscopy or immunohistochemical analysis of the tissue
Benefits • No other methods can be used to make the diagnosis of rare dermatologic genetic disorders
Benefits • Chromosome analysis results in 48-72 hours
- Some examples are erythemolysis bullosa, congenital icthyosis, and oculocutaneous albinism
• Direct fetal lymphocyte diagnosis • High-resolution chromosome analysis may be possible
Limitations • Most invasive procedure
Limitations
• Useful for specialized diagnoses only
• Technically proficient specialist performs procedure • Risk of maternal blood contamination
• Less invasive procedures are utilized for cytogenetic and molecular genetic diagnoses
INDICATIONS FOR PRENATAL TESTING
Advancing Maternal Age
Abnormal Screening Results
• ACOG recommendation over the age of 35 - Some practitioners present option as a consideration over the age of 30
• First trimester screening - Pregnancy-associated plasma protein-A
• Risk of having a chromosome abnormality at 35 is I in 200
-
Free ~-human chorionic gonadotropin
-
Nuchal fold translucency measurements with crown rump length to establish gestational age
- Gradually increasing risk as age advances • Risklbenefit analysis determines patient preference methodology
• Combined screen -
Above three values plus maternal age
Ultrasound Findings
- False-positive rate is 5%
• Structural abnormalities - Single or multiple congenital abnormalities
-
- 25% of structural defects are associated with a chromosome abnormality • Nuchal fold translucency Cystic hygroma - Increased measurements of fetal neck
Detection rate 80-90%
• Second trimester screening -
n-fetoprotein
-
Unconjugated estriol
- Inhibin-A - Chorionic gonadotropin
443
17-4
Molecular Genetic Pathology
• Integrated screen - Results from first and second trimester screen plus maternal age - False-positive rate 2- 3% - Detection rate 80-90%
Parental Chromosome Abnormality • Structural abnormality
• Viability is possible depending upon what chromosomes are involved • Small pericentric inversions have been noted as population variants, particularly involving chromosomes 2 and 9 • No clinical consequences in these cases - Paracentric inversion carrier • During meiosis acentric and dicentric chromosomes may result • Associated with early pregnancy loss
- Balanced translocation carrier • Can produce unbalanced offspring • Viability determined by nature of translocation and reproductive history • Risks dependent on mechanism of meiotic separation - Robert sonian translocation carrier • Involves chromosomes 13, 14, 15,21, and 22 • Most common transloc ation i.e., 13114 associated with multiple miscarriages • Combinations with chromosome 21 results in translocation or familial Down syndrome • May be at risk for uniparental disomy (see Fetal Abnormal ities and Outcome section) if translocation involves chromosomes 14 and 15
• No risk for an abnormal live born • Mosaicism - Constitutional mosaicism • Different percent of normal vs aneuploid cell lines in different tissues • Phenotypic effects determined by percentage of aneuploid cells - Gonadal mosaici sm • Mosaic cell line is present only in gonadal tissue • Gamete production affected • Risk of abnormal offspring is based on percentage of mosaicism in gonad s • All other tissues are normal • No phenot ypic consequen ces
- Pericentric inversion carrier • Unbalanced recombinant chromo somes may result from crossing over in a recombinant loop during meiosis
Previous Pregnancy with Cytogenetic Abnormality
• More likely to occur if the loop is large due to greater chromosome distance
• Previou s trisomy 21, 18, or sex chromosome aneuploid y - Recurrence risk 1-2%
• Duplication or deficiency of chromosomal material will produce phenotype effects
• Balanced de novo rearrangement - Sporadic recurrence unless gonadal mosaici sm
FETAL ABNORMALITIES AND OUTCOME
Aneuploidy • Most common trisomie s identified, which may survive to term delivery Trisomy 2 I-Down syndrome • Mental retardation • Clinical history of hypoton ia • Prominent occiput • Characteristic facies consisting of oblique palpebral fissures, epicanthal folds, low set ears, flat nose bridge , and large protruding tongue • Congenital heart disease, i.e., A-V canal • Duodenal atresia • Bilateral simian creases
444
- Trisomy 18-Edward syndrome • Intrauterine growth retardation • Mental retardation • Micrognathia • Low set ears • Congenital heart disease, i.e., ventricular septal defect (VSD). • Contractures with characteristic hand position , i.e.. 2nd digit over 3rd and 5th over 4th • Rockerbottom feet - Trisomy 13-Patau syndrome • Mental retardation
Prenatal Diagnosis
• Characteristic craniofacial abnormalities consisting of bilateral cleft lip and palate and holoprosencephaly
17-5
• May be picked up at time of CVS • Survival similar to double aneuploidy
• Polydactyly • Polycystic kidneys
Structural Rearrangements
• Congental heart disease, i.e., ASD, VSD
• Known familial rearrangement - Parental balanced translocation (see Parental Chromosome Abnormality section)
• Sex chromosome aneuploidies - Klinefelter syndrome-XXY Tall stature • Small sclerotic post-pubertal testes • Azoospermia • Gynecomastia • Clinical history of learning disabilities - Triple-X syndrome-XXX • Variable clinical history of spontaneous abortions • Normal phenotype • Clinical history of severe learning disabilities - XYY
- Parental pericentric inversion (see Parental Chromosome Abnormality section) - Parental paracentric inversion • May have history of reproductive loss • Balanced inversion progeny can survive and have similar reproductive history as parent • De novo rearrangement - May be associated with Multiple Congenital Anomaly/Mental Retardation (MCAlMR) syndromes. - Risk reported as high as 10% - Cannot determine if completely balanced
• Tall stature
• Limitation dependent upon microscopic resolution
• Prominent metopic suture
• Molecular techniques may define possibility of balanced rearrangement
• Clinical history of learning disabilities • Clinical history of behavior problems • Monosomy - Turner syndrome-45, X • Believed to be due to a paternal meiotic error • High incidence in spontaneous abortions • Of those that survive to term, phenotype consists of: • Short stature • • • •
Webbed neck Triangular facies Coarctation of the aorta (20%) Structural kidney abnormalities
• Cafe au lait spots • Clinical history of learning disabilities (spatial) • Ovarian dysgenesis • Double aneuploidy - Identified in very early pregnancies May be picked up at time of Chorionic villus Sampling (CVS) . - Very few survive to term - Frequent finding in spontaneous abortions - Involves both autosome and sex chromosomes • Triploidy/tetraploidy - Three or four sets of chromosomes • Chromosome number 69 or 92 • Results from dispermy event or reabsorption of one polar body • Identified in very early pregnancies
• Time limitations • Molecular limitations
Supernumerary Marker Chromosomes • Variable size - Some may be "dot"-like • Banding patterns cannot be determined by routine staining - Nucleolar organizing region (NOR) staining or molecular probes to determine whether satellites present - C-banding to determine amount of heterochromatin • Origin usually dependent on molecular studies - Fluorescence in situ hybridization (FISH) panel for most common derivatives • Prognosis for chromosome 15 markers • 50% of all markers • Known syndrome of mental retardation when Prader-Willi Syndrome (PWS) region is present - Comparative genomic hybridization useful in nonmosaic cases • Even if origin is determined may not be able to predict outcome and prognosis since there is a limited pool of data - Unknown phenotype for the majority of cases except for PWS with chromosome 15. • Familial markers Parental marker may seem to be the same by routine staining and if so, then the risk for abnormality is reduced However, molecular subtelomere studies recently have shown that parental marker is balanced and proband marker is not
445
17-6
Molecular Genetic Pathology
Uniparental Disomy (UPD) • Arises when an individual inherits both copies of a chromosome from one parent - Loss of a chromosome in a trisomic zygote (trisomy rescue) - Duplication of a chromosome in a monosomic gamete (monosomy rescue) - Fertilization with two copies of a chromosome in one gamete and no copies of the chromosome in the other gamete (nullisomic and disomic gametes) • Associated with clinical phenotypes only for chromosomes that contain imprinted genes, which are genes that are expressed from either the maternally or the paternally inherited chromosome but not both • One copy of an imprinted gene is silenced whereas the other is active • A maternally imprinted gene is not expressed from the maternally inherited chromosome and vice versa - Chromosome 15 • Paternal UPD-Angelman syndrome • Maternal UPD-Prader-Willi syndrome
- Chromosome 7 • Maternal UPD-Russell-Silver syndrome and IUGR - Chromosome II • Maternal UPD-Beckwith-Wiedemann syndrome - Chromosome 14 • Maternal UPD 14-Intrauterine Growth Retardation (IUGR) and mild dysmorphic features • Paternal UPD I4-hypotonia, thoracic dystrophy, and developmental delay - Chromosome 6 • Paternal UPD 6-transient neonatal diabetes mellitus - Chromosome 16 • Maternal UPD I6-IUGR and congenital anomalies • UPD testing should be offered - When mosaic or non-mosaic trisomy for any of the above chromosomes is observed on CVS specimen, if follow-up amniocentesis is chromosomally normal, UPD testing should be offered - When Robertsonian translocation involving chromosomes 14 or 15 is observed in the fetal karyotype
FISH IN PRENATAL TESTING FISH on Direct Specimens to Screen for Common Aneuploidies • Rapid analysis on interphase nuclei of direct amniocytes or chorionic villi (8-24 hours) - Follow-up for abnormal ultrasound findings • Cystic hygroma-abnormality most commonly associated with aneuploidy - Follow-up for abnormal first or second trimester biochemical screen - In cases of advanced maternal age or previous aneuploid pregnancy • Enumeration of most common autosomal (13, 18, 21) and sex chromosome (X, Y) aneuploidies (Figure 1) • Used in conjunction with standard karyotype analysis • Limitations - Difficulty interpreting mosaic findings - Maternal blood in specimens may confound results - Direct CVS specimen (trophoblast) represents different population of cells than cultured cells (villus stroma); therefore, placental mosaicism may yield FISH results that conflict with karyotype - Does not detect all aneuploidies
446
Subtelomere FISH Probes can be Used to Examine Fetal Chromosomes When Deletion or Duplication is Suspected • When parent is a carrier of a cryptic balanced translocation - Risk for partial monosomy and partial trisomy in unbalanced fetuses due to adjacent -I segregation • When de novo translocation involving at least one telomere is identified in fetal karyotype to assess whether it is balanced
Locus Specific FISH Probes can be Used to Examine Fetal Chromosomes • Ultrasound abnormality indicative of a specific microdeletionlduplication syndrome - Cardiac defects and 22qll FISH probe (e.g., Tuple I or N25) - Lissencephaly and 17p13.3 FISH probe (e.g., Lis 1) • Parent is a carrier of a microdeletion (50% chance of transmission) • For couples with a previous child with a microdeletion/duplication syndrome in case of gonadal mosaicism in one parent (rare)
Prenatal Diagnosis
17-7
X(green)
Y(red)
Disomy 21 female
21 (orange) Trisomy 21 male
Fig. 1. FISH on interphase nuclei from amniotic fluid FISH results using a probe mix containing the X centromere sequences fluorescentIy labeled in green, the Y centromere sequences fluorescentiy labeled in red, and the chromosome 21 locus-specific probe at 21q22 fluorescentIy labeled in orange. The left side is a FISH image from a female fetus with a normal hybridization pattern for chromosome 21. The right side is a FISH image from a male fetus with a hybridization pattern for chromosome 21 consistent with trisomy 21.
CHROMOSOMAL MICROARRAY COMPARATIVE GENOMIC HYBRIDIZATION IN PRENATAL DIAGNOSIS Constitutional Arrays are Used For Specific Genomic Gains and Losses
+
+ +
BAC and PAC clones are printed onto glass slides to create array More than 40 genomic disorders and all subtelomeric regions are represented Detects rearrangements not visible by standard karyotype analysis
+ Limitations - Not a whole chromosome array-targeted regions of the genome Will not detect balanced rearrangements - Copy number polymorphisms can confound results so parents may need to be tested Positives must be confirmed by FISH
SUGGESTED READING Cheung SW, Shaw CA, Yu W, et al, Development and validation of a CGH microanray for clinical cytogenetic diagnosis. Genet Med. 2005;7:422-432 . Crolla JA. FISH and molecular studies of autosomal supernumerary marker chromosomes excluding those derived from chromosome 15: n. Review of the literature . Am J Med Genet. 1998;75(4):367-381. Evans MI, Wapner RJ. Invasive prenatal diagnostic procedures 2005. Semin Perinatol. 2005 ;29(4):215-218. Gardner RJM, Sutherland GR. Chromosome Abnormalities and Genetic Counseling. 3rd ed. New York, NY: Oxford University Press; 2004; p. 311-335,p. 392-432.
Knight SJ, Lese CM, Precht KS, et al, An optimized set of human telomere clones for studying telomere integrity and architecture . Am J Hum Genet. 2000;67:320-332.
Ledbetter DH, Engel E. Uniparental disomy in humans: development of an imprinting map and its implications for prenatal diagnosis. Hum Mol Genet. 1995;4:1757-1764. Randolph LM . Prenatal Cytogenetics. In: Gersen SL, Keagle MB, eds. The Principles of Clinical Cytogenetics. Totowa: Human a Press ; 2005 ;267-321 .
447
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ShafTer LG. Risk estimates for uniparental disomy following prenatal detection of a nonhomologous Robertsonian translocation. Prenat Diagn. 2006;26:303-307. Tepperberg J, Pettenati MJ, Rao PN. Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH), 2-yearmulti-center retrospective studyand review of the literature. Prenat Diagn. 2001 ;21:293-301.
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Molecular Genetic Pathology
Warburton D. De novobalanced chromosome rearrangements and extra markerchromosomes identified at prenatal diagnosis: clinical significance and distribution of breakpoints. Am J Hum Genet. 1991 ;49(5):995-1013. Wenstrom KD. Evaluation of Downsyndrome screening strategies. Semin Perinatol. 2005;29(4):219-224.
18 Familial Cancer Syndromes Michelle P. Elieff, MO, Antonio Lopez-Beltran, MO, PhD, Rodolfo Montironi, MD, FRCPath, and Liang Cheng, MO
CONTENTS
I. Overview II. Retinoblastoma
18-2
VI.
Syndromes
18-2
III. Li-Fraumeni Syndrome
18-5
IV. Hereditary Breast and Ovarian Cancers BRCAI BRCA2
18-6 18-6 18-7
V. Hereditary Gastrointestinal Cancers Overview Hereditary Non-Polyposis Colorectal Cancer (HPNC, Lynch Syndrome) Familial Adenomatous Polypo sis (FAP) MYH-Associated Polyposi s Syndrome Peutz-Jegher Syndrome Juvenile Polyposis Syndrome Hereditary Diffuse Gastric Cancer
VII. Von Hippel-Lindau Syndrome VIII. Genodermatoses Hereditary Melanoma Birt-Hogg-Dube Syndrome Carney Complex PTEN-Associated Multiple Hamartoma Syndrome (Cowden Syndrome) Nevoid Basal Cell Carcinoma Syndrome (Gorlin Syndrome)
18-7 18-7 18-7
IX. .18-7 18-9 18-10 18-10 18-10 18-11
18-11
MEN Type 1 18-11 MEN Type 2A and 2B Familial Medullary Thyroid Cancer (MTC) 18-12
Cell Cycle Checkpoint Kinase 2
(CHEK2)
Hereditary Endocrine Tumor
18-13 18-13 18-14 18-15 18-15 18-15
Neurofibromatosis Type 1 (Von Recklinghausen Syndrome) ..18-16
X. Neurofibromatosis Type 2 XI.
18-12
Tuberous Sclerosis Complex
XII. Suggested Reading
18-17 18-17 18-17
449
Molecular Genetic Pathology
18-2
OVERVIEW In 2006, about 1.4 million new cancers were diagnosed in the United States. This includes 1 million non-melanomatous skin cancers. Almost 10 million Americans are alive today with a diagnosis of cancer. The likelihood that any given person has a family history of cancer is, therefore, quite high. The majority of these cases are due to complex environmental factors or chance . However, in 5-10% of cases this is due to a heritable familial cancer syndrome • Clues that suggest familial cancer syndrome - Cancer diagnosed at significantly earlier age than is epidemiologically typical - Multiple different non-skin primary cancers in the same person - Two or more first- or second-degree relatives with the same cancer - An extremely high number of cancers within genetically related individuals in a family, not due to chance or environmental causes - Pedigree that suggests a specific inheritance pattern (autosomal dominant, autosomal recessive) - Membership in certain ethnic groups • Caveats in exploring a suspected inherited cancer syndrome: - Sometimes a family history will appear negative due to early death or failure to diagnose the disease in a parent. - Patient information can be inaccurate and confirmation of important cancer diagnoses from the medical
record, if possible, should be attempted to accurately assess risk and need for genetic counseling. A proband reporting that three close relatives had "throat cancer" is vague, learning that all three had medullary thyroid carcinoma, however, can provide a first step towards risk assessment and guide further medical interventions -
Assessment of a patient's or family's risk of cancer from a suspected inherited syndrome is dynamic. The clinician must evaluate each case mindfully, with an understanding of how the accuracy of provided medical information, new diagnoses of cancers within a family, new research findings, and the limits of molecular testing will impact the patient Many sporadic human carcinomas share the same mutations as hereditary forms. A positive test result for a mutation must be interpreted in the context of accurate clinical information and a thorough genetic work-up
• When to offer molecular testing The patient (or client) can give full, informed consent - There is a significant family history or personal risk of disease - Test results are accurate and can be meaningfully interpreted in the context of the particular patient - Results impact the patient's management Education, support, and appropriate follow-up are adequate
RETINOBLASTOMA • Overview - Most common intraocular malignancy in children, affecting 1 in 20,000 Usually diagnosed before age four 60% of patients have unilateral and 40% bilateral disease Bilateral disease occurs earlier (mean age 5 months) than unilateral disease (mean age 2 years) and is more likely to be due to a germline mutation The disease is the model for carcinoma due to loss of a tumor suppressor gene • Knudson's "two-hit" hypothesis • Retinoblastoma occurs in cells that have diseasecausing mutations in both copies of the gene • In retinoblastoma due to somatic mutations in both alleles, retinoblastoma is unilateral and not hereditary • If a first mutation occurs in germline, retinoblastoma is heritable and tumors are multifocal and bilateral
450
• Clinical features - Children present early (sometimes at birth) with eukocoria (white eye) on indirect ophthalmoscopy - Bilateral and "trilateral" (with intracranial neuroblastoma or pineoblastoma) - 400% increase risk of developing mesenchymal tumors (oesteosarcoma, fibrosarcoma, and so on) - Second primary tumors in 25% (up to 50% after external beam radiation) • Brain tumors • Melanoma • Leukemia • Osteosarcoma • Fibrosarcoma • Adrenocortical carcinoma • Lung, breast, and prostate • Genetics (Table 1) - Large gene (RBi) with 27 exons, located on chromosome l3q 14; codes for retinoblastoma-associated protein
Familial Cancer Syndromes
18-3
Table 1. Genes Associated with Common Syndromes Syndrome
Gene
Chromosome
Retinoblastoma
Rbl
13ql4
Retinoblastoma Pineoblastoma
Li-Fraumen i
TP53
17pl3.1
Adrenal Breast Brain Soft tissue sarcomas Bone sarcomas
Hereditary breast
BRCA l
17pI2-21
BRCA2
I3q12 -13
CHEK2
13q21
Breast cancer Ovarian cancer Prostate cancer Breast cancer in men and women Prostate cancer Ovarian cancer Breast cancer Colon cancer Brain tumors Sarcomas
Hereditary non-Polyp osis colon cancer
MLHl MSH2 MHS6 PMS2
3p21-23 2p21 2pl6 7p22
Colorectal cancer with MSI Gastric cancer Small intestinal cancer Ovarian and endometrial cancers
Familial adenomatous polyposis (FAP)
APC
5q21-22
Multiple polyps with high risk of colorectal cancer Duodenal or ampullary adenomas/carcinomas Desmoid Congenital retinal epithelial hypertrophy
Peutz-Jegher
LKBl/ STKll
19p13.3
Multiple GI hamartomas Pigmented oraUlabial macules Breast, colon, gastric, and ovarian cancer Sex cord tumors with annular tubules
Hered itary diffuse gastric carcinoma
CDH-l
16q22.1
Diffuse signet ring gastric cancer Lobular breast cancer in women
Wermer syndrome
MENl
llql3
Pituitary adenoma Parathyroid hyperpla sia Pancreatic neuroendo crine tumors
Multiple endocrine neoplasin, MEN 2A
RET
IOql1.2
Medullary thyroid cancer (MTC) Hyperparathyroidism Pheochromocytoma
Multiple endocrine neoplasin, MEN 2B
RET
IOql1.2
Medullary thyroid cancer (MTC) Marfanoid habitus GangliomaslNeurom as Pheochrom ocytoma
Von Hippel Lindau
VHL
3p25
Endolymphatic sac papillary adenocarc inoma CNS hemang ioblastomas Renal and pancreat ic cysts Pancreatic endocrine tumors Pheochromocytomas
Hereditary papillary renal cell Carcinoma
MET
7q31
Mutations cause the transmembrane receptor to function as an activate tyrosine kinase in the absence of hepatocyte growth factor
Key clin ical features
(Continued)
451
18-4
Molecu lar Genetic Pathology
Table 1. (Continued) Syndrome
Gene
Chromosome
Key clinical features
Hereditary prostate cancer
HPC I HPCX BRCAI BRCA2
Iq24-25 Xq27-2 8 17pl2 13qI2-13
Early onset prostate cancer
Carney complex
PRKARIA
7q23-24
Cutaneous pigmented lesions Cutaneous, breast and cardiac myxomas Psammomatous melanotic schwannomas Large cell calcifying Sertoli cell tumors of the testis Primary pigmented nodule adrenocortical disease Thyroid carcinoma Breast adenomas
Cowden syndrome
PTEN
lOq22-23.3
Macroceph aly Mental retardation Multiple gastrointestinal hamartom as Hyperkeratotic oral papules, facial trichilemmomas, acral keratosis Cerebell ar gangliocytic tumors Endometrial cancer Thyroid follicular tumors Breast fibroadenom as and carcinomas
Gorlin syndrome
PTCH
9q22.3
Multiple basal cell carcinomas Basal cell nevi Palmar or plantar pits Odontogenic keratocyst Ectopic calcifications Skeletal abnormalities including bifid, fused or absent ribs or vertebrae Macrocephaly (>97%ile) Ovarian fibroma Medulloblastoma Cleft lip or palate Polydactyly
Von Recklingh ausen syndrome
NFl
2p22-21
Plexiform neurofibroma Multiple neurofibromas Cafe au-Iait spots Melanocytic iris hamartomas (Lisch nodules) Axillary or inguinal freckling Optic nerve glioma Specific bone abnormalities
Neurofibromatosis Type 2
NF2
22q 12.2
Acoustic schwannomas Meningiomas Ependymomas Astrocytomas
Tuberous sclerosis
TSCI TSC2
9q34 16p13.3
Cortical tubers Subepend ymal glial nodules Cardiac rhabdomyomas Subependymal giant cell astrocytomas Renal angiomyolipomas Periungual fibromas, Hypopigmented macules, Shagreen patch Retinal hamartomas, Iymphan giomatosis
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Famil ial Cancer Syndromes
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- Acts as cell cycle (entry into S phase) and transcriptional regulator - Activated by phosphorylation during cell cycle progres sion - Complexes with E2F and prevents it from stimulating the transcription of DNA replication genes - Most germline mutations are single nucleotide substitutions - New mutations typically affect paternal allele • Diagnos is Depends on clinical presentation • In patients with family history of bilateral retinoblastoma • DNA sequencing analysis is performed on peripheral blood to identify single nucleotide substitutions (detect s 75% of mutations) • Fluorescence in situ hybridization (FISH) to detect partial or complete deletion of RB gene (detect about 10% of mutations) • In patients with bilateral retinoblastoma, without family history • DNA sequencing analysi s and/or FISH is performed using peripheral blood
• If no mutation identified from peripheral blood DNA, tumor DNA can be tested by DNA sequencing analysis , loss of heterozygosity, and aberrant DNA methylation. Once mutations are detected in tumor DNA samples, peripheral blood DNA should be checked again for verification. • In patients with unilateral disease, but without family history • Tests are initially performed on tumor samples to identify specific mutations, followed by testing on peripheral blood • Management - Goal is to preserve life and sight through early diagnosis and treatment - In a patient with a family history of retinoblastoma, testing for germline mutation can be performed even prenatally - In patients with germline mutations in retinoblastoma gene, eye examination every 4 weeks for the first year of life and then less frequently thereafter - Photocoagulation, cryotherapy, and enucleation used in treatment
LI-FRAUMENI SYNDROME
• Overview - Autosomal dominant syndrome in which affected individuals are at great risk of developing multiple primary cancers at a young age - Rare , if classic syndrome criteria used for definition - Highly penetrant with a 90% lifetime risk of cancer • Clinical and pathologic feature s - Half will develop cancer by age 30 and 90% by age 60 - Tumors include soft tissue and bone sarcomas, adrenal cortical tumors , brain tumors, hematopoietic malignancies, colon , and breast cancer • Genetics - TP53 on chromosome l7p13.I most common (70%) - Gene product p53 is gatekeeper • Regulates cell cycle • Halts replication for repair • Induces apoptosis - Mutations • Mostly missen se • Exons 4-9 most common region
• Diagnosis - Classic syndrome criteria: • Proband younger than 45 diagnosed with sarcoma, plus • First-degree relative with a Li-Fraumeni tumor (breast, brain, sarcoma, leukemia, adrenal) diagnosed before age 45, plus • Another first or second degree relative with any cancer before age 45 or a sarcoma at any age - Diagnosis suspected on the basis of clinical criteria is then confirmed by molecular testing. Direct DNA sequencing of the gene is the most accurate (detects -98%) but time consuming. DNA sequence analysis of exons 4-9 is more common and can detect 95% of mutations in the 70% of kindreds known to have a p53 mutation • Management - Genetic testing offered to those at risk - Ethical consideration is that minors are at risk for many of the malignancies-should testing be offered? - Increased surveillance for those at risk, including physical examination, earlier mammography, colonoscopy
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Molecular Genetic Pathology
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HEREDITARY BREAST AND OVARIAN CANCERS • Overview Breast cancer • Breast cancer is most common cancer in women (11% lifetime risk) with an average age at diagnosis of 60 years • Breast cancer incidence is 200,000 cases per year • Over 15,000 cases associated with hereditary predisposition (5-7%) • Often younger age • Higher grade • Multifocal and bilateral • Clustering in certain ethnic groups (Eastern European Jewish ancestry) • 80-90% of familial breast cancers due to BRCAI or BRCA2 mutations • Other syndromes associated with increased risk of breast cancer:
• 40% lifetime risk of ovarian carcinoma, usually papillary serous carcinoma (reported to have a possible better prognosis than for sporadic ovarian cancer) - Men have increased risk of prostate cancer (both BRCAI and BRCA2) - Highest level of expression seen in thymus and testis (both BRCAI and BRCA2) • Clinical and pathologic features High-grade invasive ductal carcinomas of breast with associated Iymphoplasmacytic inflammation - Less likely to have associated in situ components (ductal carcinoma in situ) - Triple negative tumors (unlike BRCA2) • Estrogen receptor negative • Progesterone receptor negative • HER2/neu negative
• Cowden syndromes • Li-Fraumeni syndrome
Overexpression of p53 , MIB-l (high proliferation), and cyclin E
• Ataxia-telangioectasia
- Medullary carcinomas more common
• Hereditary non-polyposis colorectal cancer (Lynch Syndrome) (with MLHI gennline mutations)
- Controlled for stage and grade, prognosis is similar to sporadic breast cancer
• Hereditary melanoma • Peutz-Jeghers syndrome • Others Ovarian cancer • Fifth most common cancer in women • Frequently presents late at high stage • 50% 5-years survival • 10% of ovarian cancer due to familial predi spos ition
• Genetics
- BRCAI is a tumor suppressor gene on chromosome 17p12-21 - Large gene with 24 exons (22 encode the mRNA gene product), 1863 amino acids
-
BRCAI
-
• Overview - Autosomal dominant hereditary breast cancer
-
Gene frequency varies depending on population (1/150-1/1000) Accounts for about 50% of hereditary breast cancers and 80% of hereditary ovarian cancers Carriers have 50-80% lifetime risk of breast cancer (risk depends on particular mutationlkindred), in comparison to 10% lifetime risk in general population Presence of a BRCAI mutation • 18-20% risk of breast cancer before age 40 • 60% risk of developing breast cancer before age 50 • 20% risk of ovarian cancer before age 50
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• Exon II is the largest region, encodes 60% of the protein, accounts for 555 of all mutations BRCAI gene product involved in DNA repair and transcription regulation, acts as a "caretaker gene" Complexes with RAD51, a protein involved in the repair of dsDNA breaks N-tenninal region contains a zinc-biding RING finger domain Gennline mutations usually truncate protein and lead to loss-of-function of the BRCAI carboxy-terminus domain
- Over 500 mutations; most mutations unique to a kindred, some common in a particular group • Management - Earlier screening mammography - Prophylactic mastectom y Prophylactic oophorectomy yields 50% reduction in breast cancer risk Annual or semiannual transvaginal ultrasound and CA-125
Familial Cancer Syndromes
BRCA2 • Overview - Autosomal dominant with an incidence between 8/100 and 1/1000 - Account s for about 25% of hereditary breast cancers - Carriers have 40-70% lifetime risk of breast cancer 20% lifetime risk of ovarian carcinoma • Usually papillary serous carcinoma - Men have increased risk of breast cancer (6%) , over 100 increases over general population (not seen with BRCAl carrier) - Increased risk of prostate cancer (seen in both BRCAl and BRCA2 carrier)
18-7 • Test results most meaningful for patient risk assessment and treatment - Population specific mutation analysis • Ashkenazi Jewish ancestry • BRCAI 185deiAG (1/100 carriers) and 5382insC • BRCA2 6 I74deiT
• Mutation analysis identifie s 90% of mutation s in this population • Dutch populations • Specific deletion in exon 13 of BRCAl • Specific mutation in exon 22 of BRCA 1
• Clinical and pathologic feature s - Histology and prognosis similar to sporadic breast cancer
• Europeans • BRCAl duplication in exon 13, deletion in exons 8-9 and 14-20 - Full gene sequencing
• Genetic s - Large tumor suppressor gene on chromosome 13q12-13, 27 exons
• Costly • Difficult to interpret, may detect non-pathogenic variations
- BRCA2 gene product involved in DNA repair and transcription regulation
- Germline mutation s usually truncate protein and lead to loss of function - Common mutations • 6174deiT has 8/100 carrier frequency in Ashkenazi Jews • 999del5 common in Iceland - Sequencing can be done to detect carriers • Management - Similar to BRCAl with added attention to screening in males (prostate and breast) • Strategies for the molecular diagnosi s of BRCAl and BRCA2 mutations - Test for specific mutation in kindred , if known • Most cost effective
Cell Cycle Checkpoint Kinase 2 (CHEK2) • Overview and clinical feature s - Breast cancer, colon cancer, brain tumors, and mesenchymal tumors - About 5% of inherited breast cancers - Carriers of mutation have I in 5 lifetime risk of developing breast cancer • Genetic s - Tumor suppressor gene on chromosome 13q21 - Possibly dominant inheritance with other factors involved in expression - Gene product is a kina se involved in DNA damage repair Gene acts in association with other proteins including p53 to halt cell division during damage
HEREDITARY GASTROINTESTINAL CANCERS
Overview • Colon cancer is most common internal organ malignancy in developed countries • About 5% of colorectal cancers due to single gene mutations - Most are autosomal dominant Often earlier age of onset Risk of other systemic malignancies
Hereditary Non-Polyposis Colorectal Cancer (HPNCC, Lynch Syndrome) • Overview - Autosomal dominant syndrome involving cancers of many organs, especially the gastrointestinal tract - Responsible for up to 3.5% of colorectal cancers - Incidence is about I in 1000
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Molecular Genetic Pathology
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• Clinical features - Carriers have 80% risk of colorectal cancer Diagnosis around age 4()...45 Increased risk of upper GI (small bowel and gastric) , biliary, gynecologic (ovary and endometrium), and upper urinary tract (urether and renal pelvis) transitional cell carcinoma Patients with MLHI germline mutation especially at risk of developing breast cancer No increased risk of lung cancer Muir-Torre syndrome • Colorectal carcinomas • Multiple sebaceous tumors • Gastric , small intestinal, gynecologic, and kidney cancers • MSH2 and MLH1 mutations - MMR (mismatch repair gene)-associated Turcot Syndrome
• MSH6 at 2p16 (10%) • PMSI at 2q • PMS2 at 7p22 • Others - Mutation in DNA mismatch repair (replication error repair) gene • Results in incorrect base-base pairing • Results in insertion deletion loops in microsatellite regions • Diagnosis - Microsatellite instability (MSI) (also see Chapter 1) • Tumor available • Tumor testing for MSI using microsatellite markers o
Microsatellites have different number of repeats in tumor vs patient DNA
o
A 1997 National Cancer Institute consensus workshop recommended a 5 microsatellite marker panel for the detection of MSI including BAT25, BAT26, D2S123, D5S346, and D17S250 (Bethesda panel)
• Colorectal carcinoma • Glioblastoma • MLHI and PMSI mutations • Pathologic features - 2/3 of cancers in proximal colon (right-sided) - Mucinous or poorly differentiated carcinomas more frequent - Prominent Crohn's-like lymphocytic inflammatory response - Risk of synchronous or metachronous tumors (tumor multifocality) - Stage for stage have better prognosis than patients with sporadic colorectal cancer with fewer lymph node metastasis • Diagnostic screening criteria - Amsterdam I criteria • Three relatives with histologically verified colorectal cancer, one of whom is a first-degree relative of the other two • Two generations affected • One person diagnosed before age 50 • Likelihood of mutation in patients meeting criteria is 40-60% - Amsterdam II criteria • Less restrictive • Includes HPNCC-associated cancers (GI, GYN, skin, and urothelial carcinomas) • Likelihood of mutation in patients is about 20% • Genetics - Due to mutations in one of several DNA mismatch repair genes (MMR) • MLHI at 3p21-23 (3()...40%) • MSH2 at 2p21 (3()...40%)
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BAT25
Mononucleotide repeats
BAT26
Mononucleotide repeats
D2S123
Dinucleotide repeats
D5S346
Dinucleotide repeats
DI7S250
Dinucleotide repeats
o
If ~2/5 loci show instability, then-tumor is considered MSI-H and gene sequencing is done
o
MSI-Low (MSI-L): cancers show instability in only one of the five microsatellite markers
o
MSI-Stable (MSI-S): cancers show no microsatellite instability in any of the five markers
• Immunohistochemistry for mismatch repair proteins o
Better for identification of MSH6 mutation , which does not cause MSI
o
Guides which gene to sequence, therefore reducing cost for work-up
• Tumor not available • Direct gene sequencing • Start with MSH2 and MLHI • Caveat: microsatellite instability is identified in 15% of all colon cancers. A positive test of MSI must be interpreted in the appropriate clinical context • Management - Earlier and more intense surveillance - Prophylactic colectomy
Familial Cancer Syndromes
18-9
Fig. 1. Familial adenomatous polyposis (FAP). Numerous tubular adenomas are present in the colon resection specimen.
Familial Adenomatous Polyposis (FAP) • Overview - Autosomal dominant syndrome conferring increased risk of gastrointestinal and other carcinomas - Near 100% penetrance Incidence of 1 in 5000 to 1 in 12,000 - One fourth of cases due to new germline mutation • Clinical features - Diagnosis clinically defined as > 100 polyps in the colon, or <100 polyps and a first-degree relative with FAP (Figure 1) - May also have upper GI polyps - Risk of colorectal cancer is near 100% - Average age at diagnosis is 40 years - Patients at high risk of developing carcinoma of the ampulla of Vater - 80% have duodenal or ampullary adenomas, with 5-10% risk of duodenal carcinoma - Congenital hypertrophy of the retinal epithelium (up to 90% in some groups) • Eye examination in infants can aid in diagnosis of FAP - Other tumors: gastric, pancreaticobiliary, thyroid, hepatoblastoma, adrenal, brain, desmoids tumor (l 0%), osteoma, hepatoblastoma
- May be associated with dental abnormalities including supernumerary or missing teeth - APC-associated turcot syndrome • Colonic polyposis • Medulloblastoma and glioblastoma - Gardner syndrome • Colonic polyposis • Osteomas of mandible and long bones • Epidermoid cysts • Desmoid tumor • Genetics - Due to mutation in APC tumor suppressor gene on 5q21-22 - Most germline mutations (about one-third) lead to premature chain termination, result in a non-functional truncated protein APC protein halts cell proliferation • Normally complexes with axin and glycogen synthase kinase 3P • Part of the Wingless (Wg)/Wnt signaling pathway • Protein complex binds to and phosphorylates pcatenin and allows for its degradation • p-catenin functions as an activator of transcription factors
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Molecular Genetic Pathology
• Defective APC product prevents complexing with p-catenin • Excess p-catenin enters nucleus, activates transcription of cellular proliferation and anti-apoptotic proteins like c-myc and cyclin D I - APC protein may also stabilize mitotic spindle - Mutations in APC • Present in 80% of sporadic colon cancers • Classic FAP involves mutations between codons 169 and 1393 • Desmoids seen with mutation in 1444 codon's 3' end • 30% of mutations occur in codons 1061 and 1309 • Il307K mutation in 6-8% of Ashkenazi Jews • Not a direct cancer-causing mutation itself • Creates unstable adenine series susceptible to additional somatic mutations • Diagnosis Diagnosis can be made on clinical grounds or by molecular methods in young patients who do not exhibit the phenotype. - Linkage analysis • Very accurate (98%) • Requires two affected family members - Full gene sequencing of APC gene (detects 90%) - Mutational scanning (detects 80-90%) - Protein truncation assay (detects up to 80%) • Management Molecular testing of at-risk individuals around age 10 years - Prophylactic colectomy - Surveillance for other tumors
MYH-Associated Polyposis Syndrome • Overview - Autosomal recessive disease with an incidence up to l/100 • Clinical features - Patients present early in life with 20-100 polyps - Increased risk of upper gastrointestinal and extraintestinal tumors as in FAP • Genetics - Due to mutation of MYH gene on chromosome Ip34.3 - MYH is part of base excision repair complex in DNA oxidation damage repair - Retards G:C => T:A transversions - Y165C and G382D mutations seen in 85% of Europeans affected • Diagnosis
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- Direct mutation analysis - Gene sequencing • Management - Similar to FAP
Peutz-Jegher Syndrome • Overview Autosomal dominant, rare syndrome with an incidence of up to 1 in 25,000 - Half of cases are due to new mutations • Clinical and pathologic features - Early onset of multiple hamartomatous gastrointestinal polyps - Increased risk of GI bleed and intussusception - Pigmented macules of the lips - Increased risk of cancer (90% lifetime risk) • Including : breast (50%), colon (40%), gastric (30%), and ovarian (20%) cancer • Sex cord tumors with annular tubules • Genetics - Due to a mutation in LKBI (STKII) tumor suppressor gene at chromosome 19p13.3 in over half of patients
LKBI (or STKIl) gene product is a serine-threonine kinase , functions as a tumor suppressor gene • Diagnosis - Clinical criteria • Characteristic freckling and two or more hamartomatous polyps • Family history of Peutz-Jegher syndrome and either freckles or hamartomatous polyps - Molecular testing • Sequencing of LKBI gene • Pathologic mutations detected in only 70% of patients with clinical diagnosis • If kindred mutation is one of the 70% that are detectible, absence of mutation can spare family members from costly screening • Management - Earlier screening schedules for gastrointestinal, breast, and testicular cancer
Juvenile Polyposis Syndrome • Overview - Juvenile polyps (retention, hamartomatous, cystic) are the most common type of gastrointestinal polyp in children - The presence of more than three polyps may be a clue to polyposis syndrome
18-11
Familial Cancer Syndromes
• Clinical and pathologic features - Rare autosomal dominant syndrome with increased risk of gastrointestinal cancers Triad of diarrhea, GI bleeding, and protein-losing enteropathy Increase risk of colon, stomach, small intestinal, and pancreatic cancers SMAD4 mutations especially linked to pancreatic cancer - Up to 70% risk of colon cancer by age 60 - Multiple hamartomatous polyps of the GI tract • Genetics - Two gene mutations characterized
• SMAD4IDPC4 at chromosome lSq21 • BMPRJAIALK3 at chromosome IOq22 - 25% of cases are due to a new mutation
- SMAD4 gene is involved in signaling related to TGF~II receptor, the gene mutations is also involved in sporadic colon cancer
• Diagnosis - Diagnosis can be made on clinical grounds in patients exhibiting features of the classic syndrome or by molecular methods - Genetic testing via sequence analysis for both genes available
Hereditary Diffuse Gastric Cancer
- Rare autosomal dominant disorder characterized by diffuse, poorly differentiated gastric carcinoma • Clinical and pathologic features - Gastric cancer around age 40 years -
10% 5-years survival Increased risk of lobular breast cancer in women
- Diffuse signet ring histology - Difficult to detect the cancer endoscopically • Genetics
- CDH-J at chromosome 16q22.l (50%) - Has 16 exons - Mutation in e-cadherin gene, a cell-cell adhesion molecule • Diagnosis - Clinical • Two or more first- or second-degree relatives, one diagnosed before age 50 • Three or more first- or second-degree relatives with diffuse gastric carcinoma, any age - Molecular • Sequence analysis of CDH-J gene • Mutations detected in 30% of patients who fit the clinical criteria • Management - Close endoscopic surveillance - Prophylactic gastrectomy
• Overview
HEREDITARY ENDOCRINE TUMOR SYNDROMES
Multiple Endocrine Neoplasia, Type 1 (Wermer Syndrome) • Overview - Rare autosomal dominant syndrome with incidence between 1110,000 and 1/50,000 - Characterized by multiple endocrine tumors • Clinical features Pituitary adenomas (mainly prolactinoma) (-50%) Parathyroid • Adenomas (90%) • Hyperparathyroidism (hyperfunction) - Pancreaticoduodenal neuroendocrine tumors (gastrinomas and insulinomas) (50-75 %) • Genetics - MEN1 tumor suppressor on chromosome 11q13
- Gene product menin interacts with transcription factors (e.g., lunD), functions as tumor suppressor gene • Diagnosis - Direct DNA sequencing • Mutations seen in S0-90% • Very labor intensive - Test selectively for mutation, if known
- If no mutation identified, can do linkage analysis • Management - Screen with special clinical attention to endocrine system - Lab tests for hormone production, gluco se, and calcium - Imaging of pituitary
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Molecular Genetic Pathology
Multiple Endocrine Neoplasia, Type 2 MEN2A (Sipple Syndrome), MEN2B, and Familial Medullary Thyroid Cancer (MTC) • Overview - Autosomal dominant-related group of syndromes with an incidence of 1130,000 - Chara cterized by medullary thyroid carcinoma and other features
- Binding of ligands leads to dimerization of the receptors and activation of tyrosine kinase - Oncogene-activating point mutations lead to gain-offunction by cell membrane receptor tyrosine kinase (able to phosphoryl ate tyrosine in the absence of binding by GDNF or neurturin ) - Interestingly, 25% of patient s with Hirschesprung disease have germline mutation s in RET gene, leading to loss of protein function • Diagno sis - Genetic Testing by direct gene sequencing
• Clinical and pathologic feature s MEN2A
- Targeted mutation analysis
MTC
• MEN2A mutation
• Hyperparathyroidism • Pheochromocytoma • Phenotype seen at young age (- 30 years) - MEN2B
• Usually located around I of 5 cysteine residues in the extracellular domain between amino acids 609-634 • Exon s 10, II often involved
• MTC • Marfanoid habitus
• MEN2B mutation • M918T substitution in tyrosine kinase region of exon 16 (ATG to ACG at codon 918 ) (95%)
• GangliomaslNeuromas
• A883F in exon 15 recently characterized
• Pheochromocytoma • Phenotype seen at young age (30 years), maybe even younger than MEN 2A patients - Familial MTC • MTC • Tumor occurs at slightly older age than MEN2 • Genetic s - Due to mutation in RET protooncogene on chromosome IOq11.2 - RET product is a cell membrane receptor tyrosine kinase Ligands include glial cell lined-derived neurotrophic factor (GDNF) and neurturin
• MTC mutation • Usually around one of 5 cysteine residues in the intracellular regions at amino acids 768 and 804 in exons 10, II, similar to MEN2A - Linkage analy sis • Used when RET mutation not identified • Required at least two affected family members • Accuracy of over 95% • Management - Prophylactic thyroidectomy before age 5 - Urine or plasma catecholamine screening - Abdominal imaging
VON HIPPEL-LiNDAU SYNDROME • Overview - Tumor syndrome characterized by multiple vascularrich tumors - Autosomal dominant with nearly 100% penetrance - Incidence is about 1135,000 - Decreased life-expectancy (50 years ), with renal cell carcinoma most common cause of death • Clinical and pathologic features - Endolymphatic sac papillary adenocarcinoma (10%) (it may cause deafness) - Central nervou s system (CNS) hemangioblastomas (cerebellum, spinal cord, and retina), most common presenting symptom (40%)
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- Clear cell renal cell carcinoma - Renal and pancreatic cysts - Pancreatic endo crine tumors Pheochromocytomas Papillary cystadenoma of the epididymi s or broad ligament of the uterus - Retinal angiomas • Genetics - 98% of cases due to inherited mutation in VHL gene ; 2% due to new mutation - VHL tumor suppressor gene at chromosome 3p25, consisting of 3 exons
Familial Cancer Syndromes
18-13
- VHL gene product has two form s, each produced by alternative splicing, which block elongation during transcription; both function as tumor suppressor gene - VHL gene product forms complex with elongin Band C to get rid of hypoxia induced factor-I a - With mutation in VHL, hypoxia induced factor-la builds up and induces production of vascular endothelial growth factor - Missense mutations are often associated with pheochromocytoma (VHL type 2) • Diagnosis - Von Hippel-Lindau can be diagnosed clinically or by molecular methods
Clinical Diagnostic Criteria
• For a diagnosis of vHL, patient must have: - One feature from category A and one from category B - Two features from category A - A family history of vHL and one feature from category A or B When the clinical criteria are not met and there is strong suspicion for the disease, genetic sequencing may be used . This is extremely accurate and detects small mutations. • Management In individuals at risk, molecular testing is recommended to identify the presence of mutations. Those found to be negative could avoid costly screening . - Annual physical, including eye examination - CNS magnetic resonance imaging
Category A
Category B
-
Abdominal ultrasound, beginning in the mid-teens
Retinal hemangioblastomas
Pheochromocytomas
- Urine or plasma catecholamines
Cerebellar hemangioblastomas
Pancreatic cysts
- Annual imaging of pancreas and kidneys
Spinal hemangioblastomas
Epididymal cystadenoma Renal cysts Renalcarcinoma (clear cell)
GENODERMATOSES Hereditary Melanoma • Significance - Genetic predisposition is very heterogeneous - Accounts for 10% of melanoma - About 3000 of the 30,000 diagnosed cases each year are due to increased susceptibility within families (i.e., environment plus multiple genetic risk factors) - Smaller percentage due to familial melanoma! dysplastic nevus syndromes - The majority of these familial melanoma syndromes are autosomal dominant • Clinical and pathologic features - Up to 2/3 may develop melanoma by age 80 - Some kindreds have increa sed risk of other cancers • Breast • Pancreas - Multiple dysplastic nevi - Melanoma • Genetics - CDKN2A gene (pI6, INK4A) on chromosome 9p21
• Mutations in CDKN2A are found in about 50% of cases • Gene products are p 14 and p 16 proteins • p 16 tumor suppressor inhibits cyelin D lIcdk4 and stops cell cycle at G /S • pl4 complexes with p53 or pRb to lead to cell cycle arrest at G I or G, • pl4 also binds to mdm2 to prevent p53 destruction • mdm2 facilitates transport of p53 from nucleus to the cytoplasm where p53 was degraded - CDK4 on chromosome 12ql3 • CDK4 phosphorylates pRB, which leads to release of the pRB-bound transcription factor, E2F • Diagnosis - Mainly on clinical grounds - Genetic testing • Limited predictive value • Available but difficult to assess due to genetic heterogeneity • Management - Increased surveillance - Avoid sun exposure
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Molecular Genetic Pathology
Birt-Hogg-Dube Syndrome • Overview - The first description of an affected family was provided by Birt, Hogg, and Dube in 1977 - Birt-Hogg-Dube (BHD) syndrome is a rare genetic disorder characterized by inherited predisposition to hamartomatous skin lesions (cutaneous fibrofolliculomas), spontaneous pulmonary cysts, and renal carcinoma, and transmitted in an autosomal dominant fashion • Clinical - BHD is a rare autosomal dominant syndrome characterized by a triad: • Cutaneous lesion tend to appear in the third or fourth decade of life as a group of three skin tumors-the fibrofolliculoma, trichodiscoma, and acrochordon • Fibrofolliculoma is characterized by multiple perifollicular fibromas on the face, neck, and trunk . It is consisted of a circumscribed proliferation of collagen and fibroblasts surrounding distorted hair follicles from which basaloid cells protrude into the surrounding fibromucinous stroma. Whether these lesions are neoplastic or a result of hair follicle malformation is uncertain • Trichodiscoma is consisted of interwoven fascicles of fine fibrillar collagens embedded in alcian blue-positive mucinous ground substance. It is generally considered a hamartoma • Acrochordon (skin tag) is a benign skin lesion which consists of a benign skin that projects from the surrounding skin. The lining epidermis may display hyperkeratosis, acanthosis, and papillomatosis. The stroma may be edematous, vascular or fibrotic. • Renal tumors • Kidney tumors in BHD syndrome occur earlier than sporadic tumors, and are usually multiple and bilateral • Most commonly oncocytomas and chromophobe renal cell carcinomas (39%) • Clear cell and type I papillary renal cell carcinoma also common (10%) • Hybrid oncocytic tumors which are characterized by histologic features similar to both chromophobe renal cell carcinoma and oncocytoma (50%) • Pneumothorax • The incidence of pulmonary cysts or spontaneous pneumothorax in BHD syndromeaffected individuals is about 90% but only a fifth will have spontaneous pneumothorax
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• The typical morphology of pulmonary cyst is characterized by thin walled cystic dilatation of alveolar spaces lined by cuboidal epithelium, ranging from microscopic foci to a few millimeters in diameter with no obvious fibrous or smooth muscle tissue in the wall • Pneumothorax may develop when cysts rupture under pressure • Patients may also suffer from colonic polyps and colorectal cancer - Clinical manifestations usually begin in 20-30's The male-to-female ratio is 2: I • Genetics - BHD gene (also known as FLCN) was located to chromosome 17p12q11.2 in 2001. A the 579-amino acid BHD gene product, folliculin was cloned later - All FLCN/BHD germline mutations identified in BirtHogg-Dube (BHD) patients are predicted to truncate the mutant protein, including frameshift (insertions/deletions), nonsense and splice-site mutations - Cytosine insertions or deletions in a mononucleotide repeat tract containing 8 cytosines within exon II , which result in an abnormally small, non-functional folliculin protein, are the most frequent constitutional mutations found, being detected in approximately 50% of BHDS families - Other mutations located elsewhere in the coding sequence are heterogeneous. Overall , point mutations in th BHD gene are found in approximately 80% of BHD cases • Diagnosis - Clinical criteria using classic features included the skin fibrofolliculomas, trichodiscomas, and acrochordons together with an increased risk of renal tumors and spontaneous pneumothoraces - Skin biopsy is necessary to confirm the diagnosis of characteristic skin lesions seen in BHD patients (trichodiscomas, fibrofolliculomas, and perifollicular fibromas) . Adults with a positive skin biopsy result should also undergo renal ultrasound, abdominal CTIMRI, chest x-ray and colonoscopy to determine if there are additional diseases - FLCN (BHD) is the only gene known to be associated with Birt-Hogg-Dube syndrome. Sequence analysis detects mutations in the FLCN gene in 84% of affected individuals and is available in few laboratories • Management - There is no specific treatment for this syndrome - Laser ablation of tissue lessions results in substantial improvement, but relapse usually occurs
18-15
Familial Cancer Syndromes - Nephron-sparing surgery is the treatment of choice when possible for renal tumors but nephrectomy may be necessary in some cases
PTEN-Associated Multiple Hamartoma Syndrome (Cowden Syndrome)
- Surveillance for renal cell carcinoma is especially important for at-risk individuals and relatives
• Overview - Autosomal dominant multiple hamartomatous syndrome with almost 100% penetrance
- Use of molecular genetic testing for early identification of at-risk family members improves diagnostic accuracy and reduces costly screening procedures
- Incidence of 11200,000 • Clinical and pathologic features - Phenotypic expression both within and between kindreds is extremely variable
Carney Complex
- Majority of those affected show features by age 20
• Overview Very phenotypically heterogeneous
- Macrocephaly in 30%
Autosomal dominant syndrome with early age of onset, often infancy - The majority of the neoplasms associated with the syndrome are benign, but life-expectancy is decreased
- Mental retardation in 10% - Multiple gastrointestinal hamartomas - Skin lesions • Include hyperkeratotic oral papules, facial trichilemmomas, acral keratosis - Risk for certain tumors
- Cardiac complications most common cause of death and may result in sudden death
• Cerebellar gangliocytic tumors • Endometrial cancer
• Clinical and pathologic features - Cutaneous pigmented lesions
• Thyroid follicular tumors • Breast
• Blue nevi
• Fibroadenomas
• Lentigines
• Carcinomas (25-50%)
- Cutaneous, breast, and cardiac myxomas • 5-10% of cardiac myxomas may be due to Carney complex - Psammomatous melanotic schwannomas (benign or malignant) • Gastrointestinal • Paraspinal - Large cell calcifying Sertoli cell tumors of the testis - Endocrine disorders • Primary pigmented nodule adrenocortical disease • Thyroid carcinoma • Pituitary tumors - Breast adenomas • Genetics - Mutation in PRKARIA gene on chromosome 7q23-24 seen in 50% - RKAR IA binds cAMP and regulates protein kinase A • Diagnosis - Clinical features - Mutational analysis of PRKAR IA gene • Management - Cardiac echo starting in infancy - Endocrine tests including cortisol, growth hormone - Thyroid and testicular ultrasound
• Genetics - Phosphatase and tensin homolog gene (PTEN) is a tumor suppressor gene on chromosome IOq22-23.3 - PTEN protein acts as a dual-specificity protein and lipid phosphatase • Converts PIP3 to PIP2 • Dephosphorylates tyrosine and serine/threonine - Mutation results in unchecked cell cycling - Most common area for mutation is in exon 5 • Diagnosis - Clinical criteria -
PTEN gene sequencing
• Management - Increased surveillance
Nevoid Basal Cell Carcinoma Syndrome (Gorlin Syndrome) • Overview Autosomal Dominant syndrome with almost 100% penetrance and variable expression - Incidence of about 1150,000 • Clinical features Diagnostic criteria: two major criteria or one major and two minor criteria
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Molecular Genetic Pathology
• Major criteria • Family history of the disease • Multiple basal cell carcinomas • Basal cell nevi • Palmar or plantar pits • Odontogenic keratocyst • Ectopic calcifications • Minor criteria • Skeletal abnormalities including bifid, fused or absent ribs or vertebrae • Macrocephaly (>97%ile) • Ovarian fibroma • Medulloblastoma • Cleft lip or palate • Polydactyly
• Genetics - PTCH gene on chromosome 9q22.3 - Gene product is a transmembrane receptor for signaling b the hedgedog molecule
- Involved in cell growth, gene also has role in DNA maintenance and mutations lead to chromosomal instability Mutations are usually truncations • Diagnosi s - Based mainly on clinical features above - Molecular testing via linkage or sequencing • Management Increased and earlier surveillance Avoid radiation therapy in treatment of associated tumors due to increased risk of secondary tumors (controversial)
NEUROFIBROMATOSIS TYPE 1 (VON RECKLINGHAUSEN SYNDROME) • Overview - Relatively common autosomal dominant inherited disorder with a high degree of penetrance - Half of all cases are due to a new mutation Incidence is I in 3000 -
I in 200 mentally handicapped patients have the disorder
• Clinical and pathologic features The phenotypic expression is variable. Clinical features include the diagnostic criteria below and are often accompanied by learning difficulties or mental retardation. - Clinical diagnosis criterias, at least 2 of the following : • One plexiform neurofibroma or 2 neurofibromas • Six or more cafe au-lait spots • Two or more melanocytic iris hamartomas (Lisch nodules) • Axillary or inguinal freckling • Optic nerve glioma • Specific bone abnormalities • Family history of NF • Up to 5% can develop malignant peripheral nerve sheath tumors
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• Slightly increased risk of childhood myelodysplastic syndromes, chronic myelogenous leukemia (CML) • Genetics - Mutation in the NFl tumor suppressor gene on chromosome 2p22-p21 - Gene product is neurofibromin, a GTPase-activating protein • Normally regulate RAS-like G protein • Loss-of-function leads to accumulation of ras-GTP • Diagnosi s - Mainly based on clinical criteria - Molecular methods • Sequence analysis • Targeted mutation analysis • Linkage analysis • FISH - Prenatal testing available • Management - Supportive - Surgery when needed - Monitoring for malignant transformation
Familial Cancer Syndromes
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NEUROFIBROMATOSIS TYPE 2 • Clinical - Autosomal dominant condition affecting I in 40,000 - Characterized by acoustic schwannomas, meningiomas, ependymomas, and (rarely) astrocytoma s • Genetics - Due to mutations of NF2 gene on chromosome 22ql2 - Gene product is neurofibromin 2 (merlin) , related to a family of cytokeratin-membrane protein and tyrosine kinase
- Functions as tumor suppressor gene in maintaining cellular integrity • Diagnosis Clinical - Mutation analysis • Treatment - Close surveillance and surgery
TUBEROUS SCLEROSIS COMPLEX • Overview - Autosomal dominant complex with markedly variable expression resulting in tumors of multiple organs including the CNS , skin, kidney, and heart - Relatively common with an incidence of up to I in 6000 - 2/3 of cases of TS are due to new mutations in one of two genes - CNS disease is the leading cause of death - 80% of patients have seizures and 50% have developmental delay • Clinical - Major criteria • • • • • •
Cortical tubers (70% of patients) Subependymal glial nodules (90%) Cardiac rhabdomyomas (50%) Subependymal giant cell astrocytomas (10%) Renal angiomyolipomas Other findings include periungual fibromas, hypopigmented macules, Shagreen patch , multiple retinal hamartomas, Iymphangiomatosis
- Minor criteria • Non-renal hamartomas • Renal cysts
• Dental enamel pits • Gingival fibromas • Genetics
- TSCI on chromosome 9q34 produces protein hamartin
TSC2 on chromosome 16p13.3 produces protein tuberin - Both TSCI and TSC2 are large genes with a high mutation frequency Hamartin and tuberin form a dimer that functions in b-catenin, calmodulin, mitogen-activated protein kinase (MAPK), and other signaling pathways • Diagnosis Based on clinical criteria - Due to heterogeneity of expression of disease, molecular diagnosis via sequence analysis or FISH can be attempted • Very complicated • Large genes • Multiple small mutations • Management CNS imaging in diagnosed patients Anticonvulsants Abdominal imaging for renal tumors
SUGGESTED READING GeneTests-www.genetests.org. Online Mendelian Inheritance in man (OMIN)-www.ncbi.nlm.nih.gov. August 15, 2007
Abdel-Rahman WM, Mecklin JP, Peltomaki P. The genetics of HNPCC: application to diagnosis and screening. Crit Rev OneolHematol. 2006;58: 208-220.
Abdel-Rahman WM, Peltomaki P. Molecular basis and diagnosis of hereditary colorectal cancers. Ann Med. 2004;36:379-388.
Bornstein SR, Glmenez-Roqueplo AP. Genetic testing in pheochromocytoma: increasing importance for clinical decision making. Ann NY Aead Sci. 2006;1073:94-103 .
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Carney JA. Familial multiple endocrine neoplasia : the first 100 years . Am J Surg Pathol. 2005;29:254--274.
Lackner C, Hoefler G. Critical issues in the identificationand management of patients with hereditary non-polyposiscolorectal cancer. Eur J Gastroenterol
Frank TS, Deffenbaugh AM, Reid JE, et al. Clinical characteristics of individuals with germline mutations in BRCA I and BRCA2 : analysis of 10,000 individuals. J Clin Oneol. 2002;20:1480-1490.
Lindor NM, Greene MH. The concise handbook of family cancer syndromes. Mayo Familial Cancer Program. J Natl CancerInst. 1998;90:1039-1071.
Garber J, Offit K. Hereditary cancer predisposition syndromes. J ClinOneol. 2005;23:276-292. Hemminki K, Eng C. Clinical genetic counselling for familial cancers requires reliable data on familial cancer risks and general action plans . J Med Genet. 2004;41 :80 1- 807. Kaz AM, Brentnall TA. Genetic testing for colon cancer. Nat Clin Pract
Gastroenterol Hepatol. 2006;3:670-679. Khoury-Collado F, Bombard AT. Hereditary breast and ovarian cancer: what the primary care physician should know. Obstet GynecolSurv. 2004; 59:537-542. Lenz HJ. First Amsterdam, then Bethesda, now Melbourne? J Clin Oncol. 2005;23:6445- 6449.
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Hepatol. 2005;17:317-322.
Lux MP, Fasching PA, Beckmann MW. Hereditary breast and ovarian cancer: review and future perspectives. J Mol Med. 2006;84:16-28 . Nagy R, Sweet K, Eng C. Highly penetrant hereditary cancer syndromes.
Oncogene 2004;23:6445-6470. Roach ES, Sparagana SP. Diagnosis of tuberous sclerosis complex. J Child
Neurol. 2004;19:643-649. Woodward ER, Maher ER. Von Hippel-Lindau disease and endocrine tumour susceptibility. Endocr Relat Cancer 2006;13:415-425. Zbar B, Glenn G, Merino M, et aI. Familial renal carcinoma: clinical evaluation,clinical subtypes and risk of renal carcinoma development.J Urol. 2007;177:461-465.
19 Molecular Testing for Solid Tumors Neal I. Lindeman,
MD and
Paola Dal Cin,
PhD
CONTENTS
I. General Concepts Clinical Features Molecular Genetic Pathology Molecular Diagnostics Test Indications Gener al Technical Considerations Basic Methodologies
II . Specific Sarcomas Alveolar Rhabdomyosarcoma Alveolar Soft Part Sarcoma Clear Cell Sarcoma (Melanoma of Soft Parts) Congenital (Infantile) Fibrosarcoma Dermatofibrosarcoma Protuberans Desmoplastic Round Cell Tumor Endometrial Stromal Tumors Ewing Sarcoma Inflammatory Myofibroblastic Tumors Low-Grade Fibromyxoid Sarcoma
19-2 19-2 19-3 19-4 19-4 19-4 19-5
19-8 19-8 19-10 19-10 19-11 19-11 19-12 19-13 19-14 19-15 19-16
Extraske1etal Myxoid Chondrosarcoma Myxoid Liposarcoma Synovial Sarcoma
III. Specific Carcinomas
19-17 19-18 19-18
19-20
Breast Cancer Bladder Cancer Cervical Cancer Colorectal Cancer Lung Cancer Other Carcinomas Renal Cancer Thyroid Cancer
19-20 19-21 19-22 19-23 19-25 19-27 19-27 19-27
IV. Other Solid Tumors
19-27
Gastrointestinal Stromal Tumor (GIST) Oligodendroglioma
V. Suggested Reading
19-27 19-28
19- 28
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Molecular Genetic Pathology
GENERAL CONCEPTS
Table 1. Specific Chromosomal Translocations in Sarcomas Tumors
Translocation
Molecular event"
Alveolar rhabdomyosarcoma
t(2;13)(q35;q 14) t(l; 13)(p36;q 14)
PAX3-FKHRb PAX7-FKHRb
Alveolar soft part sarcoma
t(X;17)(p11 .2;q25)
ASPL-TFE3
Clear cell sarcoma
t(l2;22)(q 13;q12) t(2;22)(q34;q12)
CREBI-EWSb
Congenital fibrosarcoma
t(l2 ;15)(p13;q25)
bETV6-NTRK3
Dermatofibrosarcoma protuberans
t(l7;22)(q22;q13)
COLlAI-PDGFB
Desmoplastic round cell tumor
t(ll ;22)(p13;q12)
WTI-EWSb
Endometrial stromal sarcoma
t(7;l7)(p15;q2l) t(6;7)(p21;pI5) t(6;1O)(p21;pll)
JAZFI-JJAZI PHFI-JAZFI PHFI-EPCI
Ewing sarcoma/PNETc
t(ll ;22)(q24;q12) t(21 ;22)(q22;q12)
bEWS-FLIl bEWS-ERG
Inflammatory myofibroblastic tumor"
t(2;19)(p23;pI3.1) t(l ;2)(q22-23;p23)
bALK-TPM4 TPM3-ALKb
Low-gradefibromyxoid sarcoma
t(7;16)(q33;pll) t(lI ;16)(pll ;pll)
bFUS-CREBL2 CREB3Ll-FUSb
Myxoid chondrosarcoma, extraskeletal
t(9;22)(q22;q12) t(9;17)(q22;qII) t(9;15)(q22;q21)
CHN-RBP56 CHN-TCFI2
Myxoid liposarcoma
t(12;16)(q13;pll) t(l2 ;22)(q13;q12)
bFUS-CHOpb bEWS-CHOpb
Synovial sarcoma
t(X;18)(pll ;qll)
bSYT-SSXI bSYT-SSX2
ATFI-EWSb
CHN-EWSb
"Gene nomenclature changes more rapidly than pathological terminology bOual color, break apart probes available commercially
Solid Thmors
Clinical Features
Solid tumors are neoplasms ansmg from nonhematopoietic tissues of the body, which may be either benign or malignant, although most solid tumors for which molecular diagnosis is applied are malignant. Malignant solid tumors are divided into two broad categories, sarcoma and carcinoma, based upon their tissue of origin.
Presentation, treatment, and outcome are very heterogeneous, not only between different tumor types, but also within each tumor type. Some very coarse generalizations can be made, but exceptions are very common
• Sarcoma: malignant mesenchymal neoplasm, classified primarily by the type of tissue (e.g., liposarcoma [adipose], rhabdomyosarcoma [skeletal muscle]), rather than by anatomic site • Carcinoma: malignant epithelial neoplasm, classified by site of origin and cell type (e.g., squamous cell carcinoma of the lung, colonic adenocarcinoma, hepatocellular carcinoma)
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• Presentation, treatment, and prognosis are dependent on disease stage (function of tumor size, local extension, and distant spread), and grade (function of the microscopic features of the tumor cells and the architecture/pattern of their growth) • Carcinomas tend to spread via lymphatics and sarcomas tend to spread via blood vessels • Sarcomas are much less common than carcinomas, accounting for approximately I % of all cancers
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Molecular Testing for Solid Tumors
Table 2. Specific Translocations in Epithelial Carcinomas Chromosomal rearrangement
Molecular event
Papillary thyroid cancer
inv(10)(q11.2q21.2) t(1O;17)(qI1.2;q23) inv(1 0)(q11.2q11.2) inv( 1)(q22q22) inv(1)(q22q25) t(1 ;3)(q22;qll-12)
RET-H4 RET-PRKARIA RET-ELEI NTRKI-TPM3 NTRKI-TPR NTRKI-TFG
Follicular thyroid
t(2;3)(qI3;p25)
PAX8-PPARyl
Renal cell tumor
t(X; 1)(p11.2;q2l) t(X;1)(p11.2;p36) inv(X)(p 11.2q12) t(X; 17)(p11.2;q25) t(X; 17)(p11.2;q25) t(X;17)(p11 .2;q25) t(6;1l)(p21.1;q 13)
TFE3-PRCC TFE3-PFS
Tumors
TFE3/NonO
TFE3-ASPL TFE3-CLTC TFE3-CLTC Alpha-TFEB
Lethal midline cancer
t(15;19)(q13;p13.I)
NUT-BRD4
Mucoepidermoid cancerlWarthin/ clear cell hidradenoma
t(1I;19)(q21 ;p13)
MECTI-MAML2
Prostate cancer
t(21 ;21)(q22.2;q22.3) TMPRSS2-ERG t(7;21 )(p21.2;q22.2) TMPRSS2-ETVI
• Carcinomas have more complex molecular/genetic abnormalities and are more heterogeneous than sarcomas
Molecular Genetic Pathology Molecular pathology of solid tumors is also very heterogeneous,but generallyinvolves alteration(s) in genes encoding proteins criticalfor regulating cellulargrowthand proliferation, programmed cell death (apoptosis), differentiation, and/or motility. • Tumor suppressor genes (TSG) inhibit growth/proliferation/motility and/or promote adhesion/apoptosis/differentiationlDNA repair TSGs are inactivated in cancer, by diverse mechanisms including deletion, point mutation, promoter methylation, or chromosomal rearrangement - Generally, both tumor suppressor alleles are inactivated in a cancer (recessive), although cancer may arise in association with haploinsufficiency of some TSGs as a result of a single mutant allele - Many hereditary cancer syndromes involve an inherited dysfunctional TSG (e.g., RBl in Retinoblastoma, TP53 in Li-Fraumeni Syndrome), and neoplasia result when
the second allele is affected by a sporadic mutation (second hit) in a given tissue. These syndromes have dominant inheritance patterns, even though both alleles are mutated in the tumors • Oncogenes promote growth/proliferation/motility, and/or inhibit differentiation/adhesion/DNA repair/apoptosis - Oncogenes are activated in cancer, by diverse mechanisms including polyploidy, polysomy, gene amplification, chromosomal rearrangement, and point mutation - Generally, one oncogene allele is altered in a cancer, unless the mechanism of alteration is amplification, polysomy, or polyploidy, in which case multiple copies are present • Sarcomas usually have characteristic, recurrent chromosomal translocations thathave, in some instances, become criteria for the diagnosis of these tumors (see Table 1) - Most are balanced translocations that lead to in-frame fusions of two genes, resulting in a hybrid protein with altered function and/or expression. Usually the fusion gene has oncogenic properties • For example,t(11 ;22)(q24;q12) in Ewing sarcoma results in a hybrid protein containingthe C-terminal DNA-binding domain of FLll (11q24) and the N-terminal transactivating domain of EWS (22q12), under the regulation of the constitutively expressed EWS promoter. The result is increased transcriptional activation of genes with FLll binding sites, leading to increasedcell growth and proliferation - Many translocations have chromosomal variants, where one chromosomeband is consistently rearranged, but may be translocated to different chromosome partner regions • 22q12 (EWS) may be translocated to l1q24 (FLll), 21ql2 (ERG), 7p22 (ETVl), 2q33 (FEV), or 17ql2 (E1AF) in Ewing sarcoma - Many translocations have molecular variants, where the breakpoints may vary within the involved gene(s), leading to fusion between different exons • EWS-FUl fusions in Ewing sarcoma commonly fuse exon7 of EWSto eitherexon5 or 6 of FU1, although many otherconfigurations havebeendescribed - Some genes are involved in different translocations in different type of lesions • In addition to the Ewing sarcoma fusions, EWS is fused to WTl in desmoplastic small round cell tumor, to CHN in extraskeletal myxoid chondrosarcoma, and to ATFl in clear cell sarcoma Although most translocations are unique to a specific sarcoma, some translocations can be seen in different types of lesions
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Molecular Genetic Pathology
• E7V6-NTRK3 fusions are seen in both congenital
fibrosarcoma and in congenital mesoblastic nephroma - Numerical abnormalities of whole chromosome s (e.g., trisomy, mono somy ) or subchromosomal region s (e.g., amplification) tend to be less specific, second ary alterations in sarcomas • Carcinomas usually have complex and multiple chromosomal and molecular abnormalities, very few of which are recurrent translocations (see Table 2), and the alterations are sufficiently heterogeneou s within each type
of carcinoma such that they are generally not used as criteria for diagnosis - Most molecular diagno stic testing of carcinomas is done for prognosis or for selection of a specific therapy in a narrowly defined setting
• ERBB2 amplification testing is done in breast carcinoma to select patient s for therap y specifically tailored to inhibit the amplified gene product Different techniques are required for molecular studies of different carcinomas, such that few labs offer a comprehensive menu for carcinoma testing
MOLECULAR DIAGNOSTICS Test Indications • Diagnosi s, usually as an adjunct to morphology and immunohistochemistry • Prognosis • Theranostics, selection of therapies targeted to specific molecular genetic abnormalities • Risk assessment for hereditary cancer syndromes • Minimal disease testing , either for monitoring success/failure of therapy or for screening
- Tissue additi ves are available that report to preserve nucleic acids if applied at the time of fixation ; however, they have yet to gamer widespread use • Heterogeneity: most tumors contain an admixture of the cancer cells with benign cells (see Figure 1), including residual norma l tissue infiltrated by the cancer, reactive elements (e.g., lymphocytes, macrophages, and fibroblasts) recruited and/or stimulated to try to contain the cancer, and foci of necrosis - The cancer cell s them selves are often heterogeneous, especially in carcinomas, and
General Technical Considerations • Sampling: solid tumors are somatic diseases, and the lesion itself must be analyzed, which is likely to require an invasive procedure Less invasive techniques, including small biopsies , fine needle aspiration , brushing, and fluid collection , tend to yield small amount s of cancer cells admixed with benign cells that can interfere with some kinds of analysis • Fixation : most archi ved tumor samples are embedded in paraffin after fixation in formalin (FFPE), which cross-links DNA-RNA-proteins, inactivating the protein s and protecting the nucleic acids from digestion DNA isolated from FFPE tissues breaks into pieces roughly 500 bp or smaller during isolation, so techniques that require larger stretches of DNA are unreli able for these samples - Some other fixatives (e.g., Zenker's, B5) contain heavy metal s that inhibit enzymes (e.g., Taq polymerase) used for molecular diagno sis, and other tissue treatments (Bouin's, bone decalcifying ) contain acids that damage nucleic acids and/or inhibit testing (e.g., Fluorescence in situ hybridi zation) - Samples that are not fixed promptly undergo nucleic acid degradation from ubiquitous nuclea ses, which impairs analysis, especially for RNA
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Fig. I. Adenocarcinoma of the lung (x400 magnification, Hematoxylin and Eosin [H&E] stain), showing intratumor heterogeneity. The malignant epithelial cells form acinar structures that are intimately intermingled with reactive, benign element s, including inflammatory cells and fibroblasts. A DNA sample from this typical section of a carcinoma would contain admixed malignant and benign DNA, which may make certain types of DNA analysis challengin g.
Molecular Testing for Solid Tumors
genetic changes in one part of the tumor may not be seen in another part - Some techniques are particularly unreliable for heterogeneous samples, and partial puri
