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Breakthroughs in Molecular Diagnostics and Monitoring Domnita Crisan, MD, PhD. Clinical Pathology William Beaumont Hospital “Everything in medicine is genetic-or influenced by genetics – with the possible exception of trauma, and I’m not entirely sure about that”. Francis Collins, MD, PhD., Head of the National Center for Human Genome Research 1 The role of B-catenin and c-myc in the inhibition of epithelialization and wound healing. Stojadinovic O, et. at. Am. J. Pathol. July 2005. 2 Molecular Pathology in Clinical Practice Fundamental in almost every aspect of healthcare delivery: diagnosis prognosis therapeutic choice therapeutic outcome monitoring prediction of disease risk beginning-of-life choices patient and specimen ID molecular epidemiology 1953 Major Breakthroughs in DNA Technology 1962 Wilkins, Max Perutz (X-ray analysis of hemoglobin), Crick, Steinbeck, Watson, John Kendrew (myoglobin and the structure of proteins) Rosalind Franklin X-Ray difraction image of DNA Beaumont DNA Symposium 2006 Modified from Dan Farkas, Ph.D. 3 Major Breakthroughs in DNA Technology 1958 DNA Polymerases: Kornberg & others 1966 Elucidation of the Genetic Code: Nirenberg, Khorana, Jacob, Monod 1970 Reverse Transcriptase: Baltimore, Temin & Mizutani 1970 Restriction Endonucleases: Nathans, Smith, Wilcox, & others 1975 Southern Blotting: Southern 1977 DNA Sequencing: Sanger, Maxam & Gilbert Early Molecular Diagnostics The 1980s Transition from “Revolution” in Molecular Biology to Clinical Applications 4 Manual DNA/RNA Extraction, Gel Electrophoresis and Southern Blots •Highly labor intensive •Very long turnaround time (7-10 d) •Extremely poor reimbursement •Replaced by new techniques Beaumont DNA Symposium 2006 Modified from Dan Farkas, PhD Major Breakthroughs in the 1980s 1985 Restriction fragment length polymorphism analysis 1985 Invention of PCR 1986 Development of fluorescence in situ hybridization (FISH) 1988 Discovery of thermostable DNA polymerase: PCR automation 5 Polymerase Chain Reaction Described in 1985 by Kary Mullis Nobel Prize in 1993 Amplifies DNA up to approximately 35 kb Multiple cycles of - DNA denaturation - Primer annealing - Polymerase extension PCR Amplification Process Template DNA Denaturation and Primer Annealing 5’ 5’ Extension of Primers 6 PCR Changed Everything PCR Moved Molecular Diagnostics Forward Exponentially: Menu (Hematology, Oncology, Genetics, Infectious Diseases, Paternity Testing, Forensics, etc.) Reimbursement (new codes/new reimbursement/better economic model for labs) Progress towards automation PCR “wannabes” (competition = good) Major Breakthroughs in the 1990s and Beyond 1992 1993 1996 2001 2003, Conception of real time PCR Discovery of structurestructure-specific endonucleases for cleavage assays First applications of DNA microarrays First draft versions of the human genome sequence April 14 Completion of the human genome sequencing announced in a Joint Proclamation of the International Human Genome Sequencing Consortium. “We, the Heads of Government of the United States of America, the United Kingdom, Japan, France, Germany, and China, are proud to announce that scientists from our six countries have completed the essential sequence of three billion base pairs of DNA of the human genome, the molecular instruction book of human life.” life.” 7 History of Genome Project – the summit - 2001 HGP consortium publishes its working draft in Nature (15 Feb) Celera publishes its draft in Science (16 Feb). Complete sequence of the entire set of human chromosomes 8 “Omics” The Next Generation Beyond the GENOME TRANSCRIPTOME PROTEOME METABOLOME KINOME PHARMACOGENOME NUTRIGENOME 9 REAL TIME QUANTITATIVE PCR Arguably THE big development in molecular diagnostics in the last five years Quantitative Combines Amplification with Detection Fast Automated Kits don’t necessarily require molecular MTs Small footprint 10 DNA Microarrays Shift from the static standard genomics to functional genomics Gene expression profiling: transcriptomics (which of the 30,000 genes are expressed and at what levels) Endpoint of transcriptomics = Proteomics – to monitor gene translation – currently only 1% of proteins have 3Dstructure determined Microarray Applications Monitoring of gene expression: global gene expression to determine transcriptional profile = transcriptome as the major determinant of cell phenotype and function Gene expression can yield important pathogenesis and prognostic information Key advance: cluster analysis 11 Chronic Lymphocytic Leukemia AmpliChip CYP450 Array Array consists of: •2D6 gene: 27 alleles (7 are duplications) •2C19 gene: 2 alleles Beaumont DNA Symposium 2006 Courtesy of Dan Farkas, PhD 12 William Beaumont Hospital Royal Oak Campus Beaumont Reference Laboratory 13 William Beaumont Hospital Royal Oak, Michigan Department of Clinical Pathology Molecular Pathology Services • INHERITED DISORDERS • GENETIC RISK FACTORS • MOLECULAR DETECTION OF INFECTIOUS AGENTS • MOLECULAR HEMATOLOGY • PHARMACOGENOMICS Molecular Center of Excellence (MCOE) Award 2004 • FIRST MCOE IN MICHIGAN • 30 MCOE LABORATORIES IN US (SECOND MCOE LAB IN MICHIGAN – 2006) Inherited Disorders Inherited thrombophilia – Factor V. Leiden – Prothrombin mutation – MTHFR genotyping (2 mutation detection) Hereditary hemochromatosis (2 mutation detection) Cystic Fibrosis: ACOG panel of mutations, extended panel (40 + 4) 14 Inherited Disorders, cont’d Ashkenazi Jewish Panel – – – – – – – – – Bloom Syndrome Canavan disease Cystic fibrosis Familial dysautonomia Fanconi anemia Gaucher disease Mucolipidosis type IV NiemannNiemann-Pick disease TayTay-Sachs disease Genetic Risk Factors for Cardiovascular Disease APO E genotype ACE genotype Angiotensinogen genotype Angiotensin II receptor type I genotype Glycoprotein Pl A1/A2 genotype MTHFR for Hyperhomocysteinemia genotyping (2 mutation detection) 15 Infectious Diseases Chlamydia trachomatis Neisseria Gonorrhoeae M. tuberculosis Cytomegalovirus Herpes Simplex virus Human papilloma virus Hepatitis B virus – quantitation Hepatitis C virus: qualitative (ultrasensitive) quantitative genotype VZV HIV: quantitative standard quantitative ultrasensitive genotype Enterovirus Infectious Diseases Respiratory Virus Panel Influenza A Influenza B Parainfluenza Types 1 – 3 Adenovirus Respiratory Syncytial Virus Types A and B Rhinovirus Metapneumovirus 16 Molecular Hematology Tests BCR major breakpoint (p210) - qualitative BCR minor breakpoint (p190) - qualitative BCR-ABL quantitation FLT 3 gene mutations (ITD and D835) JAK2 gene mutation - qualitative JAK2 gene mutation – quantitative CLL gene expression analysis and IgVH mutational status Molecular Pathology Laboratory Test Volumes 70000 60000 50000 40000 30000 20000 10000 07 06 20 05 20 20 03 04 20 02 20 01 20 00 20 99 20 98 19 97 19 96 19 95 19 94 19 93 19 19 19 92 0 17 Molecular Pathology Laboratory Test Volumes Year # tests performed 1992 307 % increase from previous year 1993 550 79% 1994 1747 218% 1995 3604 106% 1996 4472 24% 1997 9376 110% 1998 10821 15.4% 1999 13292 22.8% 2000 20,953 57.6% 2001 23,392 11.6% 2002 28,531 22% 2003 38,792 36% 2004 43,461 12% 2005 47,450 9% 2006 53,552 12.9% 2007 64,346 20.2% Molecular Pathology Laboratory Test BCR gene rearrangement B/T cell gene rearrangement BclBcl-2 gene rearrangement DNA Bank Fragile X Syndrome HerHer-2/neu gene amplification HCV qualitative Chlamydia trachomatis PCR LCR PCR Year Offered 1991 1992 1992 1992 1992 1993 1994 1994 1996 2001 18 Molecular Pathology Laboratory Test Factor V Leiden Neisseria gonorrhea LCR Year Offered 1995 1996 PCR HCV quantitative HLA α class II typing Hereditary Hemochromatosis MTHFR mutation (homocysteine) Cytomegalovirus Herpes Simplex virus HIV quantitation (standard and ultrasensitive) Prothrombin mutation Angiotensin converting enzyme (ACE) 2001 1996 1996 1997 1998 1998 1998 1998 1998 1999 Molecular Pathology Laboratory Test Glycoprotein Pl A1/A2 HCV genotyping Human papillomavirus Angiotensinogen mutation Angiotensin receptor type 1 CYP2C9 genotype (Coumadin hyperhyperresponsiveness) HBV quantitation Apo E genotype HIV genotyping Herpes Simplex Virus Cystic Fibrosis Year Offered 2000 2001 2001 2002 2002 2002 2002 2002 2002 2003 2003 19 Molecular Pathology Laboratory Test Year Offered Varicella Zoster Virus 2004 FLT 3 gene mutations 2004 BCR qualitative 2004 Enterovirus Ashkenazi Jewish Panel JAK2 mutation - qualitative 2005 2005 2006 BCR quantitative 2007 JAK2 mutation – quantitative 2007 Molecular Pathology Laboratory Test Year Offered Chronic lymphocytic leukemia (CLL) gene expression and IgVH mutational status 2007 Respiratory Virus Panel (Influenza A and B, Parainfluenza types 1 -3, Adenovirus, Rhinovirus, Respiratory Syncytial Virus Type A and B, Metapneumovirus) Metapneumovirus) 2008 20 Current and Near Future Molecular Breakthroughs Respiratory Viral Panel Circulating tumor cell detection Specific prostate cancer molecular markers Pharmacogenomics Nutrigenomics Microarray applications Epigenetics Respiratory Viral Panel Luminex xTAGTM RVP: FDA approved January 3, 2008 First multiplexed molecular test FDA-cleared for respiratory infections First test cleared for detection of human metapneumovirus First test cleared for Flu A subtyping First test cleared for adenovirus Detects >85% of respiratory virus infections 21 RVP Targets Viral Family Respiratory Syncytial Virus (RSV) Influenza A Viral Subtype A B NonNon-specific Influenza A H1 H3 Influenza B Parainfluenza 1 Parainfluenza 2 Parainfluenza 3 Metapneumovirus Rhinovirus Adenovirus Sample Collection NA Amplification Bead Hybridization Luminex xMAP Bead Analysis Target Specific Primer Extension xTAG RVP Data Analysis 22 RVP Testing Sites North Shore - LIJ Health Systems Laboratories, Long Island, NY William Beaumont Hospital, Royal Oak, MI St. Joseph’s Healthcare, McMaster University, Hamilton, ON, Canada Mount Sinai Hospital, Toronto, ON, Canada Respiratory Virus Statistics 7th leading cause of death in the US. More than 60,000 deaths annually 80% of respiratory infections are viral Viral respiratory infections responsible for 75-80% of all doctor visits 11 million adults diagnosed annually with lower respiratory viral infections and 208,000 hospitalized 23 Respiratory Virus Statistics (cont’d) 5.3 million children diagnosed with lower respiratory viral infections (2.8 million <5 years old) and 430,000 hospitalized (90% under the age of 5) Annual cost to society $10 billion Approx 60% of viral infections are misdiagnosed or diagnosed late and inaccurately treated with antibiotics RVP Viruses - Statistics Influenza • can affect up to 20% of US population each year • hospitalizations >200,000 annually • deaths: 36,000 annually RSV A and B • most common cause of bronchiolitis and pneumonia in infants and children • deaths: 17,000 annually Metapneumovirus • recently discovered virus causing fluflu-like symptoms in all age groups • second most frequent cause of lower respiratory infections in children 24 RVP Viruses - Statistics (cont’d) Adenovirus • generally causing mild infections Rhinovirus • CDC recently reported multiple deaths (NY, OR, TX, WA) • common cold • 66 million in US annually Parainfluenza 1,2,3 • lower respiratory infections in adults and children • cause 4040-50% of croup cases • cause 1010-15% of bronchiolitis and bronchitis Advantages of Multiplex RVP Testing Collection: noninvasive NP swab One patient sample- 12 virus detection High sensitivity and specificity (important for reference lab with long distance shipping and decline in infectivity) Automated Rapid TAT 6-8 hours vs traditional testing for this number of viruses, requiring multiple individual diagnostic tests and up to several days to provide a complete diagnosis 25 Sensitivity and Specificity of xTAG RVP Virus Influenza A Flu A Subtype H1 Flu A Subtype H3 Influenza B RSV A RSV B Parainfluenza 1 Parainfluenza 2 Parainfluenza 3 Rhinovirus Adenovirus Metapneumovirus Sensitivity 96.4% 100% 91.7% 91.5% 100% 100% 100% 100% 84.2% 100% 78.3% 96% Specificity 95.9% 100% 98.7% 96.7% 98.4% 97.4% 99.8% 99.8% 99.6% 91.3% 100% 98.8% Source: Luminex package insert for xTAG RVP kit reagents. Figures are based on testing of 544 prospectively prospectively collected specimens at four North American clinical laboratories during the 20052005-6 flu season in a trial that compared xTAG with DFA and/or culture. Advantages of Multiplex RVP Testing (cont’d) Allows starting appropriate treatment quickly Promote appropriate antiviral use Avoid inappropriate use of antibiotics Cohorting: guide accurate isolation procedures Reduce unnecessary diagnostic studies Surveillance: identify types of virus circulating in the community; assist MDs and public health officials in preventing outbreaks 26 Cost and Reimbursement Cost to perform: $80-90 CPT code 87798: Infectious agent detection by nucleic acid; amplified probe technique, each organism Reimbursement: • hospital-based clinical labs vs reference labs • $49.04 (Medicare Michigan) x multiple Limitations Cost: development, validation, equipment Personnel training Need for automation of NA extraction Not rapid enough for ER patients (Rapid immunoassays and DFA have shorter TAT, but low sensitivity) A positive molecular test may be analytically true, but of unclear clinical significance (detect viable and non-viable viruses, viruses shed for daysweeks after recovery from clinical illness, or after live attenuated vaccine) 27 Circulating Tumor Cell Detection FDA approved tests CellSearchTM (Veridex) Metastatic breast cancer - January 2004 Metastatic colorectal cancer - November 2007 Metastatic prostate cancer - February 2008 CellSearch detection Detection and quantitation of circulating tumor cells (CTC) of epithelial origin that detach from solid tumor and enter the blood stream Principle: identification of CTCs CD45-, EpCAM + cytokeratins + in whole blood Presence and number of CTCs correlate with progression-free survival (PFS) and overall survival (OS) in all 3 types of cancer 28 Clinical Trial Metastatic Breast Cancer (MBC) Multi-institutional - 20 centers 177 MBC patients 145 healthy individuals 200 patients with benign diseases Clinical outcomes monitored up to 50 months Predictive Value: OS of MBC Patients with <5 or >5 CTC at Baseline (N=177) 100% CTC / 7.5mL at Baseline <5 CTC >5 CTC %Probability of Survival 90% 80% 70% Logrank p < 0.0001 60% N (%) 89 (50%) 88 (50%) Median OS in Months (95% C.I.) 21.9 (20.1 to 28.6) 10.9 ( 7.0 to 15.2) Cox Hazards Ratio = 2.4 chi-square = 19.54 (p-value < 0.0001) 21.9 Months 50% 10.9 Months 40% 30% 20% 10% 0% 0 5 10 15 20 25 30 35 Time from Baseline (Months) 40 45 50 29 %Probability of Progression Free Survival Predictive Value: PFS of MBC Patients with <5 or ≥5 CTC at Baseline (N=177) 100% 90% CTC / 7.5mL at Baseline <5 CTC >5 CTC 80% N (%) 89 (50%) 88 (50%) Median PFS in Months (95% C.I.) 7.0 (5.6 to 8.9) 2.7 (2.1 to 4.4) 70% 60% 50% 40% 7.0 Months Cox Hazards Ratio = 1.9 chi-square = 14.44 (p-value = 0.0001) 2.7 Months 30% 20% Logrank p = 0.0001 10% 0% 0 5 10 15 20 25 30 35 Time from Baseline (Months) 40 45 50 Clinical Trial Metastatic Colorectal Cancer (MCRC) Multi-institutional 413 MCRC patients 158 healthy individuals 55 patients with benign diseases Clinical outcomes monitored up to 36 months 30 Predictive Value: OS of MCRC Patients with <3 or >3 CTC at Baseline (N=413) CTC / 7.5mL at Baseline N (%) <3 CTC 305 (74%) 108 (26%) >3 CTC 100% 90% %Probability of Survival 80% Median OS in Months (95% C.I.) 18.5 (15.5 to 21.2) 9.4 ( 7.5 to 11.6) Cox Hazard Ratio = 2.5 chi-square = 31.48 (p-value < 0.0001) 70% 60% 18.5 Months 50% 9.4 Months 40% Logrank p < 0.0001 30% 20% 10% 0% 0 2 6 4 8 10 12 14 16 18 20 22 24 Time from Baseline Blood Draw (Months) 26 28 30 %Probability of Progression Free Survival Predictive Value: PFS of MCRC Patients with <3 or ≥3 CTC at Baseline (N=413) 100% CTC / 7.5mL at Baseline N (%) <3 CTC 305 (74%) 108 (26%) >3 CTC 90% 80% Median PFS in Months (95% C.I.) 7.9 (7.0 to 8.6) 4.5 (3.7 to 6.3) Cox Hazard Ratio = 1.6 chi-square = 12.19 (p-value = 0.0002) 70% 60% 7.9 Months 50% 4.5 Months 40% Logrank p = 0.0002 30% 20% 10% 0% 0 2 4 6 8 10 12 14 16 18 20 22 24 Time from Baseline Blood Draw (Months) 26 28 30 31 Predictive Value: OS of MPC Patients with <5 or >5 CTC at Baseline (N=219) CTC / 7.5mL at Baseline N (%) <5 CTC 94 (43%) 125 (57%) >5 CTC 100% %Probability of Survival 90% Median OS in Months (95% C.I.) 21.7 (21.3 to ------) 11.5 ( 9.3 to 13.7) 80% 70% Logrank p < 0.0001 60% 21.7 Months 50% 11.5 Months 40% 30% 20% Cox Hazard Ratio = 3.3 chi-square = 34.48 (p-value < 0.0001) 10% 0% 0 2 4 6 8 10 12 14 16 18 20 22 24 Time from Baseline Blood Draw (Months) 26 28 30 A Reduction in CTC Below 5 After the Initiation of Therapy Predicts Longer OS whereas an Increase in CTC Count to 5 or above Predicts a Shorter OS in MPC Patients Group Description 1 <5 CTC at All Draws 2 >5 CTC at BL & <5 CTC at Last Draw 3 <5 CTC at Early Draw & >5 CTC at Last Draw 4 >5 CTC at All Draws N (%) 88 (38%) 45 (20%) 26 (11%) 71 (31%) Median OS in Months (95% C.I.) >26 (21.4 to ------) 21.3 (18.4 to ------) 9.3 ( 8.2 to 11.3) 6.8 ( 5.8 to 10.3) 100% %Probability of Survival 90% 80% 70% 60% 1 50% 40% 30% 20% 10% Curve Comparison 1 vs. 2 1 vs. 3 1 vs. 4 2 vs. 3 2 vs. 4 3 vs. 4 Logrank p-Value* 0.1528 <0.0001 <0.0001 <0.0001 <0.0001 0.5013 *p-values not adjusted for multiple hypothesis tests 0% 0 2 4 6 4 2 3 8 10 12 14 16 18 20 22 24 Time from Baseline Blood Draw (Months) 26 28 30 32 MBC, MCRC & MPC Overall Survival Comparison 100% 90% 80% 70% 60% 50% 40% 1 30% 20% 10% 0% 0 5 10 15 20 25 30 35 40 45 50 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 4 0% 0 5 10 15 20 25 30 35 40 45 50 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 MBC MCRC MPC <cut-off at all time points 22.6 18.6 >26 ≥cut-off at all time points 4.1 3.9 6.8 ≥cut-off at baseline and <cut-off at final draw 19.8 11.7 21.3 <cut-off at early draw and ≥cut-off at final draw 10.6 7.1 9.3 2 5 10 15 20 25 30 35 40 45 50 3 5 10 15 20 25 30 35 40 45 50 Molecular Pathology Staff, 2008 33 34