1. 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

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

3. 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

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

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

6. 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

7. 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

8. 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)

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

10. Immunohistochemistry
MLH1
MSH2
Immunohistochemistry

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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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)

23. Example from Invitae

24. 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.

25. 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)

26. 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

27. 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

28. 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

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

30. 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

31. Lynch Syndrome PMS2
NCCN

32. 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

33. 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 years)
Options for intense screening or prophylactic gastrectomy/mastectomy

34. 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).

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

36. 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