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 FAP1 with FAP 2 with HNPCC2 with HNPCC 17 with FH 17 with FH of polyps of polyps or canceror cancer

80 with no 80 with no FH of polyps FH of polyps or canceror cancer

Exons 1-14 Exon 15

AAPC AAPC

Severe DesmoidsProfuse Colorectal Polyposis

1309

Adenomatous Polyposis Coli Gene (APC)

Wild Type

Truncated Mutant Proteins

19 yo with Colon Cancer

D17S250

N T N T N T

– + LOH

1 2 3

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’

5’

3’

3’

hMSH2

hMLH1

Promoter

Exon

Mutation

5’

5’

3’

3’

hMSH2

hMLH1

Promoter

Exon

DNA Mismatch Repair & MSI Colorectal Cancer

CancerA

CancerB

MLH1 MSH2

Courtesy: Aaron Pollett

3

Colon dx~50

Bowel dx 55 Colon dx 55 Colon dx ?

Colon dx ?

pancreas, prostate, liver

Endo. dx 31Rectal dx 34Cecum dx 51Transverse colon dx 52Duodenum dx 53

d. 64

d. 80

d. 55

d. 55

28

52

84

MSH2 MSH2

MSH2 Immunohistochemistry

Duodenal Adenoma Duodenal Cancer

Courtesy: Aaron Pollett

Kids with Colorectal Cancer

d.12Duodenal ca dx 11

MetsCAL spots

36

0.06250

34

0.06250

9CRC dx 8

+ 3 dysplastic polypsAux. freckling

6CAF spots

Plexiform neurofibroma (tongue)

d.70Gastric dx 60s d. childbirth

30 d.18-19 d.war

d.62d. Lung ca

55

d.90CRC dx 60s

73

7

Both sides: Afganistan

ATCG ATCG

ATCG

ACC

ATCGATCG

ACC

ACC

ACC

GCC

GCC

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 Mutation Somatic MutationFamilial 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 Ratio0.45 (0.30-0.68)P<0.001

Adjuvant 5FU and Colon Cancer Survival

N Engl J Med 345(15) 1091-1097, 2001

Chemotherapy and Mismatch Repair Deficiency

Meyers et al; 61:5193, 2001

HCT 116

HCT 116 + chromosome 3

National Cancer Institute of Canada 292

North Central Cancer Treatment Group Protocol 784852 66

Protocol 874651 34 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 (0.50-0.94) p=0.02

2.17 (0.84-5.55) 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 HNPCC1 MLH1 germline mutation

1 MSH2 germline mutation

13 Sporadic 13 MLH1 promoter methylation

Attenuated Polyposis

Family N - Autosomal Recessive Colorectal Cancer?

multiple polyps multiple polyps 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 multiple polyps multiple polyps

Y165C G382D

Y165C G382D

Y165C G382D

Y165C G382D

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 Region Frequency of Alteration (% of tumors)

K-ras >90

p16 >95

p53 50 - 75

DPC4 55

Chromosome 19q/AKT2 10 - 20

Chromosome 6q/MYB 10

Chromosome 20q/AIB1 10

BRCA2 7 – 10

LKB1/STK11 4

MKK4 4

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): 691-695.

Familial Cancer Syndromes

PANCREATICCANCER

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 ~ 0.2-0.5 %

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

Mag

nitu

de o

f ef

fect

Frequency in population

Family-based linkage studies

Association studies in populations

Unlikely to exist

Slide thanks to D. Altshuler

http://pgaedu.net/GtD_Presentations/snp_palmer.ppt

Predisposing mutation Disease

Common complex genetic diseases

Normal cell Cancerhigh penetrance

BRCA1, BRCA2, othersAPC, MMR genes, others

MutantVariant A Cancer

low penetrance

Predisposing mutation Disease

Common complex genetic diseases

Normal cell Cancerhigh penetrance

BRCA1, BRCA2, othersAPC, MMR genes, others

MutantVariant A Cancer

low penetrance

SNP Example: Subject 1: GCGCTTAG A TTCCAGGCGCTTAG A TTCCAG

Subject 2: GCGCTTAG G TTCCAGGCGCTTAG A TTCCAG

Predisposing mutation Disease

Common complex genetic diseases

Normal cell Cancerhigh penetrance

BRCA1, BRCA2, othersAPC, MMR genes, others

MutantVariant A

MutantVariant B

Cancerlow penetrance

Cancerlow penetrance

Predisposing mutation Disease

Common complex genetic diseases

Normal cell Cancerhigh penetrance

BRCA1, BRCA2, othersAPC, MMR genes, others

MutantVariant A

MutantVariant B

Cancerlow penetrance

+

Gene:gene interactions

Predisposing mutation Disease

Common complex genetic diseases

Normal cell Cancerhigh penetrance

BRCA1, BRCA2, othersAPC, MMR genes, others

MutantVariant A

MutantVariant B

Cancerlow 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?

http://pgaedu.net/GtD_Presentations/snp_palmer.ppt

THE ARCTIC PROJECTAssessment 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, CeliaGreenwood, 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 XbaXba

Fragmentationand 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: Ontario1257 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 & France2199 cases/2401 controls

1142 SNPs

76 SNPs

9 SNPs

2 SNPs

1 SNP confirmed

Outcome of Two Best SNPs

OR 95%CI p-value

rs10505477      

Stages 1-4 1.18 1.12-1.25 1.41E-08

French/EPIC 1.16 1.07-1.26 5.05E-04

All cohorts 1-7 1.17 1.12-1.23 3.16E-11

       

rs719725      

Stages 1-4 1.14 1.07-1.20 1.32E-05

French/EPIC 0.98 0.90-1.06 0.61

All cohorts 1-7 1.08 1.01-1.15 0.023

rs10505477 (8q24 locus)

8q24 locusAffymetrix Illumina