Translational Molecular Genetics

Translational Molecular Genetics

Harold Frucht, M.D.Division of Digestive and Liver Diseases

Columbia UniversityApril 14, 2005

Sporadic Cases (~70%)

Hamartomatous Polyposis Syndromes

(<1%)

Familial Adenomatous

Polyposis (<1%)

Hereditary Non-Polyposis Colorectal

Cancer (3-5%)

Cases with Familial Risk (~25%)

Disease-Associated Mutations Alter Protein Function

Gene Mutations

Somatic Mutation - Sporadic Cancer

Germ Line Mutation - Inherited Syndrome

Mechanisms of Cancer Gene Action

M (mitosis)M (mitosis)GG11

(cell growth)(cell growth)

S (synthesis)S (synthesis)

GG00 (resting) (resting)

Oncogenes:Oncogenes: promote cell growthpromote cell growth

Suppressor genes:Suppressor genes:inhibit cell cycle; promote apoptosis inhibit cell cycle; promote apoptosis

GG22

Mismatch repairMismatch repairgenes:genes: correct correctreplication errorsreplication errors

Modifier genes:Modifier genes:influence cell functioninfluence cell function

Calvert & Frucht, Ann Int Med, 2002;137:603-613

Somatic Mutation (Sporadic Disease)

2 normal copiesof the genein every cell

One copymutatedin cell(1st hit is acquired)

Second copymutatedin cell(2nd hit is also acquired)


Germline Mutation (Inherited Disease)

One copymutatedin every cell(1st hit is inherited)

Second copymutatedin cell(2nd hit is acquired)


Chung, DC. Gastroenterology 2000; 119: 854-865

CIMP = CpG Island Methylator Phenotype

• epigenetic phenomenom• hypermethylation of the promoter region of

the hMLH1 gene• responsible for MSI in 15% of sporadic

colon cancers

Polymorphism

APC GeneI1307K Mutation

Germline Mutation of Codon 1307

T-A Transversion(Leucine - Isoleucine Substitution)Causes hypermutability in adjacent sequences resulting in somatic alterations which predispose to colon cancer

Incidence in Ashkenzai Jews 6.1 %Lifetime risk of colon cancer in people with mutation 18-30 %

28%

10%

6%

0%

0% 10% 20% 30%

% with mutation

Non-Jewishpopulation

All Ashkenazi Jews

UnselectedAshkenazi Jews w/

CRC

Ashkenazi Jews w/CRC and familyhistory of CRC


Inherited Syndromes Predisposing to Colon Cancer

GeneLifetime

Risk of CRC

Familial Adenomatous Polyposis apc ~100%

Hereditary Non-Polyposis Colon Cancer MMR >80%

Peutz-Jeghers Syndrome STK11 2-13%

Juvenile Polyposis SMAD4 ~<50%

Cowden Syndrome PTEN small

Correlations between the APC Genotype and the Clinical Phenotype

NEJM 2003; 349:1750-1760

Clinical Criteria for Hereditary Non-Polyposis Colorectal Cancer

Amsterdam criteria At least three relatives with colon cancer and all of the following:•One should be the first-degree relative of the other two•Two successive generations should be affected•At least one colon cancer should be diagnosed before the age of 50•FAP should be excluded

Modified Amsterdam criteria

As for the Amsterdam criteria except that the cnacers need to be an HNPCC-associated cancer (colon, endometrium, small bowel, ureter, renal pelvis) instead of specifically colon cancer.

Bethesda criteria Families meeting the Amsterdam criteriaIndividuals with 2 HNPCC-associated cancers, including synchronous or

metachronous cancersIndividuals with colon cancer and a first-degree relative with an HNPCC-

associated cancer and/or colonic adenoma; 1 cancer diagnosed at age < 45 years and the adenoma diagnosed at age < 40 years

Individuals with colon or endometrial cancer diagnosed at < 45 yearsIndividuals with right-sided colon cancer having an undifferentiated pattern

(solid/cribiform) or signet cell histopathology diagnosed at <45 yearsIndividuals with adenomas diagnosed at < 40 years


Autosomal Dominant Inheritance

• Each child has 50% chance of inheriting the mutation

• No “skipped generations”

• Equally transmitted by men and women

Normal

Affected

HNPCC: Direct Mutation Testing

hMLH1hMSH2

hMSH3 hPMS1hMSH6 hPMS2

HEREDITARY COLON CANCER - Germline Mutation

SPORADIC COLON CANCER - Somatic Mutation

FAMILIAL COLON CANCER - Germline Mutation Causing Hypermutability and Subsequent Somatic Mutation

Germline Mutations - Inherited Disease

APC - Familial Polyposis ColiMMR - HNPCC (Lynch Syndrome)MYH- Familial Polyposis Coli

Somatic Mutations - Sporadic Disease

Oncogenes: myc, ras, srcTumor Suppressors: p53, DCC, APC, MCCMismatch Repair Genes: MSH2, MSH3, MSH6,

MLH1, PMS1, PMS2

“Genetic Polymorphisms” - Familial Disease

APC - Familial Colon Cancer

APC Gene

Germline Mutation - Familial Polyposis ColiSomatic Mutation - Sporadic Colon Cancer

I1307K Germline Mutation - Familial Colon Cancer

GENETIC COUNSELING

GENETIC TESTING

Failure to Diagnose Hereditary Colorectal Cancer and Its Medicolegal Implications

A Hereditary Nonpolyposis Colorectal Cancer Case

Henry T. Lynch, M.D.,* Jane Paulson, J.D.,† Matthew Severin, J.D., Ph.D.,* Jane Lynch, B.S.N.,* Patrick Lynch, J.D., M.D.‡

From the *Department of Preventive Medicine, Creighton University School of Medicine, Omaha, Nebraska, †Paulson & Baisch, Portland, Oregon, and ‡Department of GI Oncology/Digestive Diseases, MD Anderson Cancer Center, Houston, Texas

Diseases of the Colon & Rectum 1999: Jan 42(1); 31-35

The Use and Interpretation of Commercial APC Gene Testing for Familial Adenomatous PolyposisFrancis M. Giardiello, M.D., Jill D. Brensinger, M.S., Gloria M. Petersen, Ph.D., Michael C. Luce, Ph.D., Linda M. Hylind, B.S., R.N., Judith A. Bacon, B.S., Susan V. Booker, B.A., Rodger D. Parker, Ph.D., and Stanley R. Hamilton, M.D.

From the Departments of Medicine (F.M.G., J.D.B., L.M.H., J.A.B., S.V.B.) and Pathology (S.R.H.) and the Oncology Center (F.M.G., G.M.P., S.R.H.), John Hopkins University School of Medicine, Baltimore; the Departments of Epidemiology (G.M.P.) and Health Policy and Management (R.D.P.), John Hopkins University School of Hygiene and Public Health, Baltimore; and the Department of Molecular Biology, LabCorp, Research Triangle Park, N.C. (M.C.L.). Address reprint requests to Dr. Giardiello at Blalock 935, Johns Hopkins Hospital, 600 N. Wofe St., Baltimore, MD 21287-4461.

NEJM 1997; 336:823-27

Background The use of commercially available tests for genes linked to familial cancer has aroused concern about the impact of these tests on patients. Familial adenomatous polyposis is an autosomal dominant disease caused by a germ-line mutation of the adenomatous polyposis coli (APC ) gene that causes colorectal cancer if prophylactic colectomy is not performed. We evaluated the clinical use of commercial APC gene testing.

Methods We assessed indications for APC gene testing, whether informed consent was obtained and genetic counseling was offered

before testing, and the interpretation of the results through telephone interviews with physicians and genetic counselors in a nationwide sample of 177 patients from 125 families who underwent testing during 1995.

Results Of the 177 patients tested, 83.0 percent had clinical features of familial adenomatous polyposis or were at risk for the disease — both valid indications for being tested. The appropriate strategy for presymptomatic testing was used in 79.4 percent (50 of 63 patients). Only 18.6 percent (33 of 177) received genetic counseling before the test, and only 16.9 percent (28 of 166) provided written informed consent. In 31.6 percent of the cases the physicians misinterpreted the test results. Among the patients with unconventional indications

for testing, the rate of positive results was only 2.3 percent (1 of 44).

Conclusions Patients who underwent genetic tests for familial adenomatous polyposis often received inadequate counseling and would have been given incorrectly interpreted results. Physicians should be prepared to offer genetic counseling if they order genetic tests.

Microsatellite Instability (MSI)

• 10% - 15% of sporadic tumors have MSI• 95% of HNPCC tumors have MSI• Routine MSI assays soon available

Normal MSI tumor

Electrophoresis gel

Genetic Testing Lab Methods

DNADNA

mRNAmRNA

ProteinProtein

GelGel

NormalNormal MutatedMutatedLinkage Analysis:Linkage Analysis: Probability of Inheritance. Probability of Inheritance.MSI Assays:MSI Assays: Highly predictive for MMR mutation. Highly predictive for MMR mutation.Gene Sequencing:Gene Sequencing: Approaches 100%. Approaches 100%.DGGE:DGGE: Highly sensitive (>90%).SSCP:SSCP: Detects 60%-95% of mutations.

Protein Truncation:Protein Truncation: Point of mutation dependent. Point of mutation dependent. Immunohistochemistry:Immunohistochemistry: Antibody dependent. Antibody dependent.


History Suggestive of Inherited Colon Cancer

Probable HNPCCProbable FAP

APC genetic test of anaffected individual

Genetic test of anaffected individual

negative positive positive negative

annual endoscopy for all family

members

APC gene testing of family members

HNPCC genetic testing of family

members

continued high risk colon cancer screening of the

individual and all family members

no adenomas

adenomasannual endoscopy

prophylactic colectomy

continued survellance for rectal adenomas and extra-colonic tumors.

consider chemoprevention.

positive

annual endoscopy

no adenomas

negative

Colon Cancer screening as

recommended for the general population

positivenegative

negativepositivefor colon

cancer

colectomy

Calvert & Frucht, Ann Int Med, 2002:137;603-613

FAMILY JW-3911-17-94

Legend: = male; = female; = deceased; = probandSolid figures = cancer; BR = breast cancer; CO = colon cancer; LU = lung cancer;EN = endometrial cancer; number refers to age diagnosis

60heart

disease

88 32 52

70’sBR, 70

70’sBR, 70

48

heartdisease

85 60’s

LU, 60

9072

CO, 72

84

uBR, 54

50EN, 42

53EN, 45

29 27CO, 26

ascending,Dukes, C

25


FAMILY JW-379-7-94

Legend: = male; = female; = deceased; = probandSolid figures = cancer; CO = colon cancer; EN = Endometrial cancer; BL = Bladder cancer; BO = Bone cancer; RE = Rectal cancer; number refers to age diagnosis

70 77CO, 59EN, 76

75 90

68RE, 68

73 65CO, 39CO, 64

73BO, 73

4746CO, 37

27 21

67CO, 39BL, 66

12

65EN, 65

60’s, 70’s

49


Incidence of Pancreatic Cancer by Number of Affected First Degree Relatives

Klein AP, et al., Cancer Research 2004; 64: 2634-2638

Number of FDRs with Pancreatic Cancer

Incidence (per 100,000) in the U.S. Population

General U.S. (reference) 9

1 41

2 58

3 or more 288

• 10% of patients with pancreatic cancer have a familial aggregation or an inherited predisposition

END OF PRESENTATION

Clinical Features of Inherited Cancer Syndromes

Feature FAP HNPCC

Age of onset Early Early

No. of adenomas > 100 < 10

Adenoma distribution Total Mainly right side

Cancer distribution Random Mainly right side

Other cancers Periampullary Endometrial, other

Lynch HT et al, Clinical Risk Factors for Colorectal Cancer

Cancer Family Syndromes

Colon (63 %)Endometrium (8/28 %)

Gastric (6 %)Biliopancreatic (4 %)Genitourinary (2 %)

Ovary (1/3 %)Breast (2/6 %)Sarcomas (2 %)Skin (2 %)Small Bowel (1 %)Lung (1 %)Other (2 %)

GENETIC TESTING FOR FAP

1. Linkage Analysis2. In vitro truncated protein testing (transcription -

translation method)3. Mutation Testing

GENETIC TESTING FOR HNPCC

1. Linkage Analysis2. Truncated Protein Testing3. Mutation Testing

4. Microsatellite Instability Testing

Clincial Cancer Screening Recommendations*

Colon Cancer Screening RecommendationsRISK SCREENING

MODALITYAGE AT WHICH

TO BEGINFREQUENCY

Average FOBTSigmoidoscopyFOBT and SigmoidoscopyDCBEColonoscopy

5050505050

annuallyevery 5 yearsevery 5 yearsevery 5-10 yearsevery 10 years

First-degree relative with colon cancer or adenomatous polyp at age 60 years

Same as for average risk individuals

40 same as for average risk individuals

Two or more first-degree relatives with colon cancer or adenomatous polyp at age < 60 years

Colonoscopy preferred 40 or 10 years younger than the earlier diagnosis

every 3-5 years

FAP Sigmoidoscopy 10-12 years annually

HNPCC Colonoscopy 20-25 years or 10 years younger than the earliest colon

cancer diagnosis

every 1-2 years

Extracolonic Cancer Screening RecommendationsFAP:Duodenal cancer EGD 20-25 years every 1-3 years

HNPCC:Endometrial and

ovarian cancer

Gastric cancer

Pelvic examTrans vaginal ultrasound

EGD

25-35 years

30-35 years

every 1-2 years

every 1-2 yeas

*DCBE = double contrast barium enema; EGD = esophagogastroduodenoscopy; FOBT = fecal occult blood test.

Columbia Colon Cancer Prevention Program (C3P2)

History and PhysicalRisk Assessment

Screening GuidelinesGenetic Counseling and Testing

Chemoprevention

Documents

Translational Molecular Genetics