1. Translational Molecular Genetics Harold Frucht, M.D. Division of Digestive and Liver Diseases Columbia University April 14, 2005

3. Disease-Associated Mutations Alter Protein Function

4. Gene Mutations Somatic Mutation - Sporadic Cancer Germ Line Mutation - Inherited Syndrome

5. Mechanisms of Cancer Gene Action M (mitosis) G 1 (cell growth) S (synthesis) G 0 (resting) Oncogenes: promote cell growth Suppressor genes: inhibit cell cycle; promote apoptosis G2G2G2G2 Mismatch repair genes: correct replication errors Modifier genes: influence cell function Calvert & Frucht, Ann Int Med, 2002;137:603-613

6. Somatic Mutation (Sporadic Disease) 2 normal copies of the gene in every cell One copy mutated in cell (1st hit is acquired) Second copy mutated in cell (2nd hit is also acquired) Calvert & Frucht, Ann Int Med, 2002;137:603-613

7. Germline Mutation (Inherited Disease) One copy mutated in every cell (1st hit is inherited) Second copy mutated in cell (2nd hit is acquired) Calvert & Frucht, Ann Int Med, 2002;137:603-613

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

9. 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

10. Polymorphism

11. APC Gene I1307K 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 Jews6.1 % Lifetime risk of colon cancer in people with mutation18-30 %

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

13. Inherited Syndromes Predisposing to Colon Cancer Gene Lifetime Risk of CRC Familial Adenomatous Polyposisapc~100% Hereditary Non-Polyposis Colon CancerMMR>80% Peutz-Jeghers SyndromeSTK112-13% Juvenile PolyposisSMAD4~<50% Cowden SyndromePTENsmall

14. Correlations between the APC Genotype and the Clinical Phenotype NEJM 2003; 349:1750-1760

15. Clinical Criteria for Hereditary Non-Polyposis Colorectal Cancer Amsterdam criteriaAt 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 criteriaFamilies meeting the Amsterdam criteria Individuals with 2 HNPCC-associated cancers, including synchronous or metachronous cancers Individuals 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 years Individuals with right-sided colon cancer having an undifferentiated pattern (solid/cribiform) or signet cell histopathology diagnosed at <45 years Individuals with adenomas diagnosed at < 40 years Calvert & Frucht, Ann Int Med, 2002;137:603-613

16. Autosomal Dominant Inheritance Each child has 50% chance of inheriting the mutation No “skipped generations” Equally transmitted by men and women Normal Affected

17. HNPCC: Direct Mutation Testing hMLH1 hMSH2 hMSH3hPMS1 hMSH6hPMS2

18. HEREDITARY COLON CANCER -Germline Mutation SPORADIC COLON CANCER -Somatic Mutation FAMILIAL COLON CANCER -Germline Mutation Causing Hypermutability and Subsequent Somatic Mutation

19. Germline Mutations - Inherited Disease APC - Familial Polyposis Coli MMR - HNPCC (Lynch Syndrome) MYH- Familial Polyposis Coli Somatic Mutations - Sporadic Disease Oncogenes: myc, ras, src Tumor Suppressors: p53, DCC, APC, MCC Mismatch Repair Genes: MSH2, MSH3, MSH6, MLH1, PMS1, PMS2 “Genetic Polymorphisms” - Familial Disease APC - Familial Colon Cancer

20. APC Gene Germline Mutation - Familial Polyposis Coli Somatic Mutation - Sporadic Colon Cancer I1307K Germline Mutation - Familial Colon Cancer

21. GENETIC COUNSELING GENETIC TESTING

22. 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

23. The Use and Interpretation of Commercial APC Gene Testing for Familial Adenomatous Polyposis Francis 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.

24. Microsatellite Instability (MSI) 10% - 15% of sporadic tumors have MSI 95% of HNPCC tumors have MSI Routine MSI assays soon available NormalMSI tumor Electrophoresis gel

25. Genetic Testing Lab Methods DNA mRNA Protein Gel Normal Mutated Linkage Analysis: Probability of Inheritance. MSI Assays: Highly predictive for MMR mutation. Gene Sequencing: Approaches 100%. DGGE: DGGE: Highly sensitive (>90%). SSCP: SSCP: Detects 60%-95% of mutations. Protein Truncation: Point of mutation dependent. Immunohistochemistry: Antibody dependent. Calvert & Frucht, Ann Int Med, 2002;137:603-613

26. History Suggestive of Inherited Colon Cancer Probable HNPCCProbable FAP APC genetic test of an affected individual Genetic test of an affected individual negativepositive 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 adenomas annual 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 positive negative positive for colon cancer colectomy Calvert & Frucht, Ann Int Med, 2002:137;603-613

27. FAMILY JW-39 11-17-94 Legend: = male;= female;= deceased;= proband Solid figures = cancer; BR = breast cancer; CO = colon cancer; LU = lung cancer; EN = endometrial cancer; number refers to age diagnosis 60 heart disease 88 3252 70’s BR, 70 70’s BR, 70 48 heart disease 8560’s LU, 60 90 72 CO, 72 84 uBR, 54 50 EN, 42 53 EN, 45 29 27 CO, 26 ascending, Dukes, C 25 Calvert & Frucht, Ann Int Med, 2002:137;603-613

28. FAMILY JW-37 9-7-94 Legend: = male;= female;= deceased;= proband Solid figures = cancer; CO = colon cancer; EN = Endometrial cancer; BL = Bladder cancer; BO = Bone cancer; RE = Rectal cancer; number refers to age diagnosis 7077 CO, 59 EN, 76 7590 68 RE, 68 7365 CO, 39 CO, 64 73 BO, 73 47 46 CO, 37 2721 67 CO, 39 BL, 66 12 65 EN, 65 60’s, 70’s 49 Calvert & Frucht, Ann Int Med, 2002:137;603-613

29. 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)9 141 258 3 or more288 10% of patients with pancreatic cancer have a familial aggregation or an inherited predisposition

31. END OF PRESENTATION

32. Clinical Features of Inherited Cancer Syndromes FeatureFAPHNPCC Age of onsetEarly No. of adenomas> 100< 10 Adenoma distributionTotalMainly right side Cancer distributionRandomMainly right side Other cancersPeriampullaryEndometrial, other Lynch HT et al, Clinical Risk Factors for Colorectal Cancer

33. 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 %)

34. GENETIC TESTING FOR FAP 1.Linkage Analysis 2.In vitro truncated protein testing (transcription - translation method) 3.Mutation Testing

35. GENETIC TESTING FOR HNPCC 1.Linkage Analysis 2.Truncated Protein Testing 3.Mutation Testing 4.Microsatellite Instability Testing

36. Clincial Cancer Screening Recommendations* Colon Cancer Screening Recommendations RISKSCREENING MODALITY AGE AT WHICH TO BEGIN FREQUENCY AverageFOBT Sigmoidoscopy FOBT and Sigmoidoscopy DCBE Colonoscopy 50 annually every 5 years every 5-10 years every 10 years First-degree relative with colon cancer or adenomatous polyp at age  60 years Same as for average risk individuals 40same as for average risk individuals Two or more first-degree relatives with colon cancer or adenomatous polyp at age < 60 years Colonoscopy preferred40 or 10 years younger than the earlier diagnosis every 3-5 years FAPSigmoidoscopy10-12 yearsannually HNPCCColonoscopy20-25 years or 10 years younger than the earliest colon cancer diagnosis every 1-2 years Extracolonic Cancer Screening Recommendations FAP: Duodenal cancerEGD20-25 yearsevery 1-3 years HNPCC: Endometrial and ovarian cancer Gastric cancer Pelvic exam Trans 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.

37. Columbia Colon Cancer Prevention Program (C3P2) History and Physical Risk Assessment Screening Guidelines Genetic Counseling and Testing Chemoprevention