New Era of Genetic Testing in Colon Cancer I am going to talk about why family history is an important risk factor for understanding your risk of developing colon cancer. Carol Burke, MD, FACG, FASGE, AGAF, FACP Sanford R Weiss Center for Hereditary Colorectal Neoplasia Department of Gastroenterology and Hepatology Cleveland Clinic, Cleveland, Ohio September 19, 2015

Overview Recognize clues suggestive of a genetic colorectal cancer syndrome Understand the genetic testing process for hereditary colorectal cancer syndromes

Pathways to CRC Sporadic CRC FAP MAP Lynch Syndrome CIN-MSS CIMP MSI MSI MLH1 promotor methylation BRAF mutation FAP MAP Adenoma Sessile Serrated Polyp Colorectal cancer is a molecularly heterogenous disease. It was once believed that only adenomas were precursors to CRC. Over the last decade it has been determined that serrated lesions of the colon are the etiology of up to 20% of the colorectal cancers. The genetic basis of colorectal cancer involves multiple pathways including chromosomal instability, microsatellite instability and CpG Island methylation. While the majority of sporadic cancers develop through the adenoma carcinoma sequence and are typified by CIN and microsatellite stability, serrated cancers and their precursors are associated with activating mutations in the BRAF oncogene, CIMP and can be either MSS or MSI. Lynch Syndrome

Chromosomal Instability Pino MS, et al. NEJM 2010;339;1277

CpG Island Methylation (CIMP) Gene Expression Gene Silencing Turns off MLH1

Multi Target Stool DNA Testing vs FIT Assay: Methylation of BMP3 and NDRG4, KRAS mutations , B-actin and a fecal immunochemical test P < 0.001 P = 0.002 % P < 0.001 Imperiale T, 2014;370:1287

Microsatellite Instability Repeated nucleotide sequences called “microsatellites” DNA fidelity maintained by Mismatch Repair Proteins (MMR) MLH1 PMS2 MSH2 MSH6 Boland CR, Gastroenterology 2010;138:2073

Mismatch Repair Protein Function Nucleotide mismatch Normal MMR Defective MMR Etiology: MLH1 promoter methylation Germline MMR mutation- Lynch Syndrome T C A A G C T G T C G A C A G A T G T C T A C Microsatellite Instability (MSI) Microsatellite Stable (MSS)

Tumor MSI Testing NR21 BAT25 Mono27 Normal Tissue Tumor Tissue MSI-H: > 2/5 (30%) consensus MSI sequences

Immunohistochemistry MLH1 MSH2 Immunohistochemistry

Multi Society Task Force Universal Testing of CRC for dMMR Giardiello FM , Am J Gastro 2014;109:1159

Universal Tumor Testing for LS 1066 unselected tumors assessed for MSI/MMR 19.5% had MSI 11% (21 patients) diagnosed with LS Phenotype: 43% diagnosed > 50 years 22% did not Amsterdam II or revised Bethesda guidelines Germline Testing Results In 21 Proband’s Relatives Relationship Tested Positive Negative First degree 54 25 29 Second degree 22 10 12 > Third degree 41 17 24 Total 117 52 65 Hampel H et al. NEJM 2005;352;18

Typical Genetic Counseling Appointment Collect personal and family history Perform risk assessment (including breast cancer risk models) Educate about genetic syndromes, management options, genetic testing process Informed consent and coordination of genetic testing Psychosocial support and counseling

Who Should Have Genetic Counseling for Lynch Syndrome? Abnormal MSI/IHC testing (Unless MLH1 methylation is proven) Colon or endometrial cancer < 50 > 2 Lynch Syndrome cancers in individual > 2 relatives with LS cancer, 1 < 50 > 3 relatives with LS cancer at any age

Who Should Have Genetic Counseling for other Colon Cancer Syndromes? > 10 colon adenomas Peutz-Jeghers Polyp Juvenile/Inflammatory Polyps Colon cancer < age 50 Close relative diagnosed < age 50 or >2 close relatives with colon cancer

Hereditary Colon Cancer Syndromes Gene(s) Features Lynch MLH1, MSH2, MSH6, PMS2, EPCAM Colon, endometrial, ovarian, gastric, urinary tract, small bowel cancers, brain tumors, sebaceous neoplasms Li Fraumeni TP53 Childhood cancers, sarcoma, leukemia, brain tumors, breast cancer, colon cancer Familial Adenomatous Polyposis (FAP) APC Adenomas, colon cancer, thyroid cancer, osteomas and soft tissue tumors, desmoid tumors MYH-Associated Polyposis (MAP) MUTYH* Adenomas, colon cancer, thyroid cancer Peutz-Jeghers STK11 Mucocutaneous melanin spots, hamartomas, breast, GI, pancreatic, and rare gyn cancers Cowden PTEN Hamartomas, derm lesions, macrocephaly, breast, thyroid, and endometrial cancers Juvenile Polyposis Syndrome BMPR1A, SMAD4 Hamartomas, colon cancer, some with SMAD4 have HHT

Traditional Cancer Risk Assessment and Genetic Testing HNPCC/Lynch syndrome! colon ca 50 d. uterine ca 61 d. stroke 80 47 57 60 59 62 32 30 24 27 35 d. colon ca

Traditional Genetic Testing Utilizes Sanger sequencing and large rearrangement analysis Testing often limited to 1-2 syndromes based on assessment of personal/family history Only testing for high-risk, well known syndrome Variant of uncertain significance rate is low Results take 2-3 weeks

Next Generation Sequencing Whole genome, or several genes can be analyzed at once Allows for testing many genes relatively inexpensively Used for panel genetic testing Cancer specific vs Pan cancer

Benefits Limitations Multi-Gene Panels Increased mutation positive rate Identification of conditions of low clinical suspicion Cost effective Lower turn-around-time then reflex testing Limitations Not all tests are equal Various levels of gene coverage Increased VUS rate Moderate-risk genes Longer turn-around-time than single gene

Insurance Coverage & Cost Most labs offer insurance pre-authorization Many labs billing with same CPT codes as BRCA, Lynch testing Costs range from $1500-$4400

What Are Testing Options? 10 panels (5-28 genes) 10 panels (7-29 genes) Build your own 7 panels (7-29 genes) 1 panel (25 genes) 2 panels (20-52 genes)

Example from Invitae

Genes and Associated Cancer Risks Myriad Genetics Lab myRisk Gene Panel Genes and Associated Cancer Risks Genes associated with rare cancer types and very rare cancer genetic syndromes are not currently included in myRisk (i.e., Hereditary Paraganglioma Syndrome, Von Hippel Lindau Syndrome, Neurofibromatosis). Genes for which there is a theoretical role in inherited cancer risk, i.e. due to the postulated biological function of the protein in a DNA repair pathway, were not included in the absence of meaningful clinical data.     Therefore, 20 out of the 25 genes on the Myriad myRisk Hereditary Cancer Panel have existing society guidelines to direct management changes associated with the increase cancer risk. The remaining 5 genes have at least one well designed study providing evidence for at least a 2 fold increase in cancer over the general population risk. This table illustrates the 25 genes on Myriad myRisk as well as their associated cancers.

High Risk Genes Breast Cancer BRCA1/BRCA2 (HBOC) CDH1 (Hereditary Diffuse Gastric Cancer) PTEN (Cowden) STK11 (Peutz-Jeghers) TP53 (Li-Fraumeni) Colon Cancer APC (Familial Adenomatous Polyposis) BMPR1A/SMAD4 (Juvenile Polyposis) MLH1/MSH2/MSH6/PMS2/EPCAM (Lynch Syndrome) MYH (MYH-Associated Polyposis)

High Risk Genes Significant risk of developing certain types of cancer Considerable research and professional society guidelines for screening and surgery Family members can be tested for the same mutation

Moderate Risk Genes ATM (Ataxia Telangiectasia) Breast, pancreatic, colon CDKN2A (Familial Atypical Mole Malignant Melanoma) Pancreatic, melanoma CHEK2 (Li-Fraumeni Like Syndrome) Breast, prostate, colon PALB2 (Fanconi Anemia) Breast, pancreatic

Moderate Risk Genes 2- to 4-fold risk over the general population risk. Cancer risk is not as elevated as a mutation in a high risk gene. Limited research and no professional society screening or surgical recommendations Family history is often better for risk stratification Unclear if it is beneficial to test other family members for these mutations

Potential Results from myRisk Positive High risk gene Moderate risk gene New moderate risk gene Negative Variant of uncertain significance

Incidental Finding Case 2 Caucasian/N. American AJ Irish 90 85 dx female ca 65 d. 83 dx br ca 60s no info n 55 d. young accident 61 test ca 64 65 45 42 dx br ca 34

Lynch Syndrome PMS2 NCCN 2.2014

Incidental Finding Case 3 d. 54 Panc ca Hungary Sicily 2 89 CRC 89 81 88 br ca 45 d. 64 d. 40 Panc ca 47 50 55 d.55 b/l br ca 44 BRCA - 61 51 26 29 p

Hereditary Diffuse Gastric Cancer CDH1 mutations 39% breast cancer risk by age 80 Diffuse gastric cancer 67% for men and 83% for women by age 80 Average onset 38 years (range of 14-69 years) Options for intense screening or prophylactic gastrectomy/mastectomy

Variants of Uncertain Significance: Our results 235 VUS in 171 patients (41.4%) majority in moderate risk genes (62.2%) VUS rate lower in those of European ancestry than African, Asian, and Middle Eastern (p=0.001) VUS more common in non-European background (54/99) than European background (71/196, p=0.0018).

Important Insurance Updates Companies requiring pre-test genetic counseling: CareSource CCF Employee Health Plan Cigna Medical Mutual of Ohio

Conclusions Variety of “Genetic” tests to determine cause of hereditary colon cancer syndromes Currently, test the tumor first in patients with CRC Germline testing for polyposis or when tumor not available Genetic testing in transformation: NGS lowering costs and driving panel based testing Not all panel tests are created equal Caution: panel testing “easy” to order but complicated to interpret Get a lot of information we might not understand Find unanticipated mutations that highly impact patient care