1. Genetic Testing for Cancer: Diagnostic Medicine & Cancer Susceptibility Gail H. Vance, M.D. Professor, Medical & Molecular Genetics Indiana University School of Medicine

2. Objectives  Differential genetic testing for somatic mutations from genetic testing for heritable mutations.  Use breast cancer as an example of disease utilizing both.  Demonstrate that both forms of testing provide information regarding risk and treatment

3. What is Cancer?

4. Cancer is a heterogeneous disease that will claim more than 560,000 lives in our country this year.

5. 2009 Estimated US Cancer Deaths* ONS=Other nervous system. Source: American Cancer Society, 2009. Men 292,540 Women 269,800  26% Lung and bronchus  15%Breast  9%Colon and rectum  6%Pancreas  5%Ovary  3%Leukemia  3%Non-Hodgkin lymphoma  3%Uterine corpus  2%Liver  2%Brain/ONS  22% All other sites Lung and bronchus30% Prostate9% Colon and rectum9% Pancreas6% Leukemia4% Esophagus4% Liver and intrahepatic4% bile duct Non-Hodgkin 3% Lymphoma Urinary bladder3% Kidney3% All other sites 24%

6. Cancer is not a single disease.  Rather, any cancer may involve: Multiple tissue lineages. Aberrant expression of genes with a variety of cellular functions. Variation in the number of deregulated genes

8. Cancer is a genetic disease.  All cancers involve genetic changes in somatic cells, the germ line, or both.

9. What is Genetic Testing  The analysis of human DNA in any of its forms or related products (chromosomes, RNA, proteins)

10. Cancer Genes  Most gene mutations in cancer occur in somatic cells and are acquired (multifactorial etiology).  However, some mutations do occur in the germline and may be inherited and passed on to future generations.

13.  Individuals genetically predisposed to cancer account for 5-10% of the cancer population.  1,000,000 x 0.1=100,000 CANCER BURDEN

15. Features suggesting an inherited predisposition to cancer:  Two or more close relatives affected.  Early age of onset.  Cancers of a specific type occurring together (breast and ovary).  Multiple or bilateral cancers occurring in one person.

17. Breast Cancer  Breast cancer is a common disorder  Lifetime risk is 1 in 8.  180,000 new case each year

19. Breast Cancer  Two major susceptibility genes, BRCA1 and BRCA2 have been identified.  Mutations in these genes account for 3-5% of all breast cancers.

22. BRCA1 Gene on chromosome 17 Autosomal dominant transmission Protein has role in genomic stability >1300 different mutations reported

23. Gene on chromosome 13 Autosomal dominant transmission Protein has role in genomic stability >1300 different mutations reported BRCA2

25. BRCA1 and BRCA2-Linked Mutations in the Ashkenazi Jewish Population

28. Cancer Detection/Screening  Increased surveillance Monthly self-breast exams Semiannual clinical breast exams Semiannual radiography (mammogram/MRI) Annual transvaginal ultrasound, CA125

29. Cancer Prevention  Chemoprevention Tamoxifen Aromatase-inhibitors Oral contraceptives

30. Cancer Prevention  Prophylactic surgery Prophylactic mastectomy reduces risk by 90% Prophylactic salpingo-oophorectomy reduces risk by 96%

31. d. 53 BR CA 87 BR CA dx 80 d. 65 BR CA dx 48 d. 43 panc Ca 55 melanoma dx 49 d. 80d. 90 8075 62 57 51 222 59 BR CA dx 58 85d. 8075 d. 75d. 80 BRCA2 mutation Paternal lineage

33. Genetic Testing for Somatic Mutations in Breast Cancer  HER2 amplification  Oncotype Dx  MammaPrint

34. How do we assess risk in BC patients?  Classical pathological criteria  Lymph node  Age/Tumor size/Tumor grade  ER/PR/HER2  Oncotype Dx  MammaPrint

35. HER2 in Breast Cancer  Human Epidermal Growth Factor Receptor 2, HER2 (ERBB2) Amplified in ~20% breast cancers Prognostic marker Predictive for several systemic therapies  Relative resistance to endocrine therapies  Resistance or sensitivity to various types of chemotherapy  Response to agents that target HER2 (Herceptin ® )

36. Methods for HER2 Detection (IHC and FISH) +2+30+1 AmplifiedNormal Red – HER2; Green – Control Probe Amplified: HER2/CEP17 > 2.2 or average HER2 > 6 Courtesy of D. Persons

37. Assessing HER2 with IHC 1+2+3+0 Herceptin Treatment Indicated for 3+ and some 2+

38. Oncotype Dx  Molecular test performed on formalin-fixed breast tumors.  Used to assess recurrence risk and predicts benefit from hormonal/chemotherapy.  Utility for ER+, LN negative tumors.

39. Oncotype DX ® 21-Gene Recurrence Score ® (RS) Assay PROLIFERATION Ki-67 STK15 Survivin Cyclin B1 MYBL2 ESTROGEN ER PR Bcl2 SCUBE2 INVASION Stromelysin 3 Cathepsin L2 HER2 GRB7 HER2 BAG1GSTM1 REFERENCE Beta-actin GAPDH RPLPO GUS TFRC CD68 16 Cancer and 5 Reference Genes From 3 Studies Paik et al. N Engl J Med. 2004;351: 2817-2826

40. Oncotype DX ® 21-Gene Recurrence Score ® (RS) Assay Calculation of the Recurrence Score Result CategoryRS (0-100) Low riskRS <18 Int riskRS ≥18 and <31 High riskRS ≥31 Paik et al. N Engl J Med. 2004;351: 2817-2826 RS = Coefficient x Expression Level + 0.47 x HER2 Group Score - 0.34 x ER Group Score + 1.04 x Proliferation Group Score + 0.10 x Invasion Group Score + 0.05 x CD68 - 0.08 x GSTM1 - 0.07 x BAG1 Coefficient x Expression Level + 0.47 x HER2 Group Score - 0.34 x ER Group Score + 1.04 x Proliferation Group Score + 0.10 x Invasion Group Score + 0.05 x CD68 - 0.08 x GSTM1 - 0.07 x BAG1 Coefficient x Expression Level RS= +0.47 x HER2 Group Score -0.34 x ER Group Score +1.04 x Proliferation Group Score +0.10 x Invasion Group Score +0.05 x CD 68 -0.08 x GSTM1 -0.07 X BAG1

41. Paik et al. N Engl J Med. 2004;351:2817-2826 The Recurrence Score ® Result Stratifies Patients by their 10-Year Distant Recurrence-Free Survival

43. MammaPrint  DNA microarray-based in vitro diagnostic test used to assess risk of cancer recurrence.  70 gene signature of critical molecular pathways associated with breast cancer metastasis.  Estimates high or low risk of recurrence.  Fresh frozen tumor specimens.

44. Summary  Genetic testing performed for both heritable and somatic gene mutations.  Heritable (germline) mutations may be predictive and performed in healthy women from blood.  Heritable mutations also predict risk for future disease in multiple organs.  Somatic mutation testing performed on the tumor to better assess risk and inform therapy.