The Human Genome 23 Pairs of Chromosomes About 30,000 Genes

Sporadic ~75% Familial (gene(s)) not known 15-20% ~20-25% HNPCC ~2-3% FAP (<1%) MYH (<1%)

Practical Colorectal Cancer Genetics 100 new colorectal cancer patients 1 with FAP 2 with HNPCC 17 with FH of polyps or cancer 80 with no FH of polyps or cancer

Exons 1-14Exon 15 AAPC Severe Desmoids Profuse Colorectal Polyposis 1309 Adenomatous Polyposis Coli Gene (APC) Wild Type Truncated Mutant Proteins

19 yo with Colon Cancer

D17S250 NTNT NT – +LOH 123 Tumor Testing for Microsatellite Instability

The Canadian Contribution - Family C

Hereditary Nonpolyposis Colorectal Cancer Germline mutations in mismatch repair genes MSH2, MLH1, PMS1, PMS2, MSH6

5’ 3’ hMSH2 hMLH1 Promoter Exon Mutation

5’ 3’ hMSH2 hMLH1 Promoter Exon

DNA Mismatch Repair & MSI Colorectal Cancer Cancer A Cancer B MLH1MSH2 Courtesy: Aaron Pollett

3 Colon dx ~ 50 Bowel dx 55Colon dx 55 Colon dx ? pancreas, prostate, liver Endo. dx 31 Rectal dx 34 Cecum dx 51 Transverse colon dx 52 Duodenum dx 53 d. 64 d. 80 d MSH2

MSH2 Immunohistochemistry Duodenal AdenomaDuodenal Cancer Courtesy: Aaron Pollett

Kids with Colorectal Cancer

ATCG ACCACC ACCACC ACCACC ACCACC GCCGCC GCCGCC III - 1 III - 2 IV - 1 IV - 2 IV - 3

Inherited Colorectal Cancer - It’s ‘Easy’ Familial Adenomatous Polyposis (FAP) - APC Gene Hereditary Nonpolyposis Colorectal Cancer (HNPCC) - mismatch repair genes

What have we learned from Hereditary Colorectal Cancer Syndromes? Germline MutationSomatic Mutation Familial Adenomatous APC gene APC gene Polyposis (FAP) Hereditary Nonpolyposis Mismatch Repair Mismatch Repair Colorectal Cancer (HNPCC) Genes Genes Sporadic Colorectal Cancer…….. APC gene Mismatch Repair Genes

5’ 3’ hMLH1 Promoter Exon Hypermethylation Hypermethylation of MLH1 Promoter as an Epigenetic Cause of MLH1 Inactivation in SPORADIC COLORECTAL CANCER

MSS vs MSI-H colorectal cancer Survival by Stage Gryfe et al, NEJM 342:69-77;2000 Hazard Ratio 0.45 ( ) P<0.001

Adjuvant 5FU and Colon Cancer Survival N Engl J Med 345(15) , 2001

Chemotherapy and Mismatch Repair Deficiency Meyers et al; 61:5193, 2001 HCT 116 HCT chromosome 3

National Cancer Institute of Canada 292 North Central Cancer Treatment Group Protocol Protocol Gastrointestinal Intergroup, NCI 143 Fondation Francaise de Cancerologie Digestive 35 Total 570 No. Cases MULTISITE COLLABORATION

Colorectal Cancer Genetics & 5-FU Ribic CM NEJM 2003 MSS MSI Hazard Ratio 0.69 ( ) p= ( ) p=0.10

What about….? CPT11 Oxaliplatin Avastin Erbitux ? Palliative Rx NEJM 2000 Aduvant Rx NEJM 2004 Palliative Rx NEJM 2004

Genetic Basis of Colorectal Cancers with Microsatellite Instability 100 CRC 15 MSI-H 85 MSS (1 FAP) 2 HNPCC 1 MLH1 germline mutation 1 MSH2 germline mutation 13 Sporadic 13 MLH1 promoter methylation

Attenuated Polyposis

Family N - Autosomal Recessive Colorectal Cancer? multiple polyps

DNA Base Excision Repair Slupska et al, J Bacteriol. 178, 3885, 1996

Exon 7 (codon 165) …GGGCTACTATT… Exon 7 (codon 165) …GGGCTGCTATT… tyrosine cysteine Exon 13 (codon 382) …ctcaGGTCTGC… Exon 13 (codon 382)… ctcaGATCTGC… glycine aspartic acid Functionally Important MYH Mutations

MYH Associated Polyposis (MAP) Autosomal Recessive Colorectal Cancer multiple polyps Y165C G382D Y165CG382D

How did it happen?

Pancreatic cancer: Causes Environmental factors Smoking Alcohol Coffee Diet Chemicals Host factors Past medical history Pancreatitis Genetic predisposition Familial cancer syndromes Familial Aggregation of cases

Somatic Mutations in Pancreatic Cancer Gene or RegionFrequency of Alteration (% of tumors) K-ras>90 p16>95 p DPC455 Chromosome 19q/AKT Chromosome 6q/MYB10 Chromosome 20q/AIB110 BRCA27 – 10 LKB1/STK114 MKK44 TGF-β R-I or R-II<5 RB1<5 Kern S. Molecular genetic alterations in ductal pancreatic adenocarcinomas. Med Clin North Am 2000(84):

Familial Cancer Syndromes PANCREATIC CANCER Familial Melanoma (p16) Familial Breast- ovarian cancer syndrome (BRCA1, BRCA2) HNPCC (hMSH2, hMLH1 & other mismatch repair genes) Peutz-Jeghers, Li-Fraumeni etc. (less common)

Melanoma + Pancreas Cancer

Breast/Ovarian Cancer + Pancreas Cancer

Lifetime Risk of Pancreas Cancer General Population ~ % BRCA2, PJS, HNPCC, p16, Familial Pancreatitis ~ 5-10 % Familial Pancreas Cancer ~ 20-30%

High Risk Pancreas Cancer Screening Program Who? BRCA2, p16, Familial Pancreas Cancer, Peutz- Jeghers, FAP, Hereditary NonPolyposis Colorectal Cancer, Familial Pancreatitis What? Yearly MRI, Ultrasound, Blood collection/banking

What Joe General Surgeon should know BRCA1/2, MMR genes, APC, MYH, RET, p16, VHL Keep your eyes and ears open for new ones! Microsatellite instability/18q LOH in colorectal cancer Somatic genetics of Wilms, neuroblastoma Molecular based therapies - eg Herceptin, Gleevec Non-cancer genetic syndromes

Sporadic ~75% Familial (gene(s)) not known 15-20% ~20-25% HNPCC ~2-3% FAP (<1%) MYH (<1%) Is it ALL genetic? If it is, how do we figure it out?

Allelic architecture and mapping strategy Magnitude of effect Frequency in population Family-based linkage studies Association studies in populations Unlikely to exist Slide thanks to D. Altshuler

Predisposing mutationDisease Common complex genetic diseases Normal cell Cancer high penetrance BRCA1, BRCA2, others APC, MMR genes, others Mutant Variant A Cancer low penetrance

Predisposing mutationDisease Common complex genetic diseases Normal cell Cancer high penetrance BRCA1, BRCA2, others APC, MMR genes, others Mutant Variant A Cancer low penetrance SNP Example:Subject 1: GCGCTTAG A TTCCAG GCGCTTAG A TTCCAG Subject 2: GCGCTTAG G TTCCAG GCGCTTAG A TTCCAG

Predisposing mutationDisease Common complex genetic diseases Normal cell Cancer high penetrance BRCA1, BRCA2, others APC, MMR genes, others Mutant Variant A Mutant Variant B Cancer low penetrance Cancer low penetrance

Predisposing mutationDisease Common complex genetic diseases Normal cellCancer high penetrance BRCA1, BRCA2, others APC, MMR genes, others Mutant Variant A Mutant Variant B Cancer low penetrance + Gene:gene interactions

Predisposing mutationDisease Common complex genetic diseases Normal cell Cancer high penetrance BRCA1, BRCA2, others APC, MMR genes, others Mutant Variant A Mutant Variant B Cancer low penetrance + Gene:environment interactions

Genome Wide Association Studies Definition Study of genetic variation across the genome, designed to identify genetic associations with observable traits (eg. blood pressure), or the presence or absence of a disease (eg. colorectal cancer)

Genome Wide Association Studies of Common Multigenic Diseases Risk Variants in an individual Asthma Diabetes Arthritis Autism Colon Cancer

Risch, Nature 2000 How many subjects do you need for a powerful GWAS?

THE ARCTIC PROJECT Assessment of Risk of Colo-Rectal Tumors in Canada GWS Stage 1 Design: 1200 Cases (Ontario) 1200 Controls (Ontario) 1.4 billion genetic tests Brent Zanke, Steve Gallinger, Celia Greenwood, Michele Cotterchio, Tom Hudson

Progress to Date Genotyping (1200 CRC and 1200 controls)  1536 SNPs from candidate genes   10K coding non synonymous SNPs.   100K Affymetrix gene chip SNP array.   500K Affymetrix gene chip SNP array.  (In Analysis) Validation

250 ng Genomic DNA RE Digestion Adaptor Ligation Xba Fragmentation and Labeling PCR: One Primer Amplification Complexity Reduction AA BB AB Hyb & Wash Affymetrix Centurion 100K+500K SNP chips

Multi-Stage Analysis of ~100,000 SNPs Stage 1: Ontario 1257 cases/1336 controls 99,632 SNPs Stage 2: Seattle + Newfoundland 1139 cases/1055 controls Stage 3: Scotland 975 cases/1002 controls Stage 4: Scotland 1910 cases/1985 controls Validation: EPIC & France 2199 cases/2401 controls 1142 SNPs 76 SNPs 9 SNPs 2 SNPs 1 SNP confirmed

Outcome of Two Best SNPs OR95%CIp-value rs Stages E-08 French/EPIC E-04 All cohorts E-11 rs Stages E-05 French/EPIC All cohorts

rs (8q24 locus)

8q24 locus AffymetrixIllumina