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

  2. Disease-Associated Mutations Alter Protein Function

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

  4. Mechanisms of Cancer Gene Action Oncogenes: promote cell growth G1 (cell growth) M (mitosis) G2 S (synthesis) G0 (resting) Mismatch repair genes: correct replication errors Modifier genes: influence cell function Suppressor genes: inhibit cell cycle; promote apoptosis Calvert & Frucht, Ann Int Med, 2002;137:603-613

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

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

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

  8. 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

  9. Polymorphism

  10. 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 %

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

  12. Inherited Syndromes Predisposing to Colon Cancer

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

  14. Clinical Criteria for Hereditary Non-Polyposis Colorectal Cancer Calvert & Frucht, Ann Int Med, 2002;137:603-613

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

  16. HNPCC: Direct Mutation Testing

  17. Germline Mutations - Inherited DiseaseAPC - Familial Polyposis ColiMMR - 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

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


  20. 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

  21. 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. Background The use of commercially available tests for geneslinked to familial cancer has aroused concern about the impactof these tests on patients. Familial adenomatous polyposis isan autosomal dominant disease caused by a germ-line mutationof the adenomatous polyposis coli (APC ) gene that causes colorectalcancer if prophylactic colectomy is not performed. We evaluatedthe clinical use of commercial APC gene testing. Methods We assessed indications for APC gene testing, whetherinformed consent was obtained and genetic counseling was offeredbefore testing, and the interpretation of the results throughtelephone interviews with physicians and genetic counselorsin a nationwide sample of 177 patients from 125 families whounderwent testing during 1995. Results Of the 177 patients tested, 83.0 percent had clinicalfeatures of familial adenomatous polyposis or were at risk forthe disease — both valid indications for being tested.The appropriate strategy for presymptomatic testing was usedin 79.4 percent (50 of 63 patients). Only 18.6 percent (33 of177) received genetic counseling before the test, and only 16.9percent (28 of 166) provided written informed consent. In 31.6percent of the cases the physicians misinterpreted the testresults. Among the patients with unconventional indicationsfor testing, the rate of positive results was only 2.3 percent(1 of 44). Conclusions Patients who underwent genetic tests for familialadenomatous polyposis often received inadequate counseling andwould have been given incorrectly interpreted results. Physiciansshould be prepared to offer genetic counseling if they ordergenetic tests. NEJM 1997; 336:823-27

  22. 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

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

  24. History Suggestive of Inherited Colon Cancer Probable FAP Probable HNPCC APC genetic test of an affected individual Genetic test of an affected individual negative positive positive negative APC gene testing of family members Colon Cancer screening as recommended for the general population annual endoscopy for all family members HNPCC genetic testing of family members continued high risk colon cancer screening of the individual and all family members positive negative no adenomas annual endoscopy positive negative negative positive for colon cancer no adenomas adenomas annual endoscopy prophylactic colectomy colectomy continued survellance for rectal adenomas and extra-colonic tumors. consider chemoprevention. Calvert & Frucht, Ann Int Med, 2002:137;603-613

  25. FAMILY JW-3911-17-94 60 heart disease 88 32 52 84 uBR, 54 72 CO, 72 70’s BR, 70 70’s BR, 70 48 heart disease 85 60’s LU, 60 90 53 EN, 45 50 EN, 42 27 CO, 26 ascending, Dukes, C 25 29 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 Calvert & Frucht, Ann Int Med, 2002:137;603-613

  26. FAMILY JW-379-7-94 70 77 CO, 59 EN, 76 75 90 73 BO, 73 65 EN, 65 68 RE, 68 67 CO, 39 BL, 66 73 65 CO, 39 CO, 64 60’s, 70’s 46 CO, 37 49 47 27 21 12 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 Calvert & Frucht, Ann Int Med, 2002:137;603-613

  27. Incidence of Pancreatic Cancer by Number of Affected First Degree Relatives • 10% of patients with pancreatic cancer have a familial aggregation or an inherited predisposition Klein AP, et al., Cancer Research 2004; 64: 2634-2638


  29. Clinical Features of Inherited Cancer Syndromes Lynch HT et al, Clinical Risk Factors for Colorectal Cancer

  30. 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 %)

  31. GENETIC TESTING FOR FAP • Linkage Analysis • In vitro truncated protein testing (transcription - translation method) • Mutation Testing

  32. GENETIC TESTING FOR HNPCC • Linkage Analysis • Truncated Protein Testing • Mutation Testing • Microsatellite Instability Testing

  33. Clincial Cancer Screening Recommendations*

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