UPDATE OF LYNCH SYNDROME(LS):GENETICS, NATURAL HISTORY, HETEROGENEITY, SCREENING AND MANAGEMENT HENRY T. LYNCH, MD JANE F. LYNCH, BSN Creighton University School of Medicine Omaha, Nebraska

Colorectal Cancer Worldwide estimates for colorectal cancer during 2008*: Incidence – 1,233,711 Mortality – 608,644 Worldwide estimates for familial/hereditary CRC during 2008*: Lynch syndrome 3-5% of all CRC 37,011-61,686 FAP <1% of all CRC <12,337 Familial 20% of all CRC 246,742 *GLOBOCAN. The International Agency for Research on Cancer web site. URL: http:///www.iarc.fr/

Search for LS Among CRC Affecteds* Among 500 CRC patients, 18 (3.6%) had LS. Of these 18:  18 (100%) had MSI-H CRCs;  17 (94%) were correctly predicted by IHC;  only 8 (44%) were dx < 50 years;  only 13 (72%) met the revised Bethesda guidelines;  1/35 cases of CRC show LS. *Hampel et al. J Clin Oncol 26:5783-5788, 2008.

Screening for LS Recommendation*: All incident CRC and EC cases should be molecularly screened for LS. MSI highly sensitive (89.3%). IHC equally sensitive (91.2%), is inexpensive, is more readily available, and predicts the nonworking gene. IHC is preferred method to screen for LS. *Hampel et al. J Clin Oncol 26:5783-5788, 2008.

Screening for Amsterdam Criteria LS* a) Screening of all CRC patients meeting Amsterdam Criteria (AC) would fail to detect half of all cases; b) Screening those aged £ 50 would detect only half of all cases; c) Screening of all patients using Bethesda Guidelines for MSI would fail to detect at least 1/3 of all cases. *Boland & Shike. Gastroenterology 138:2197.e1- 2197.e7, 2010.

Magnitude of the Problem Question: Why are these figures of such significant public health impact? Answer: Each hereditary cancer comes from a family that could benefit immensely from genetic counseling. DNA testing, surveillance, and highly-targeted management are the key!

Cost-effectiveness of DNA Testing Estimate the cost-effectiveness of genetic testing strategies to identify LS among newly dx CRC patients using MSI and IHC.* Conclusion: Preliminary tests seem cost-effective from the U.S. health care system perspective. Detects nearly twice as many cases of LS as targeting younger patients. MMR testing is not cost effective. *Genet Med 12:93-104, 2010.

Cardinal Features of Lynch Syndrome • Family pedigree shows autosomal dominant inheritance pattern for syndrome cancers. • Earlier average age of CRC onset than in the general population: - Lynch syndrome: 45 years; - general population: 63 years. • Accelerated carcinogenesis, i.e., shorter time for a tiny adenoma to develop into a carcinoma: - Lynch syndrome: 2-3 years; - general population: 8-10 years. High risk of additional CRCs: 25-30% of patients who have surgery for a LS-associated CRC will have a second primary CRC within 10 years, if surgery was < a subtotal colectomy.

Increased risk for certain extracolonic malignancies Endometrial Ovary Stomach Small bowel Pancreas Liver and biliary tree Muir-Torre cutaneous features Brain, (glioblastoma) Possible Prostate cancer and others.

N Engl J Med 354: 261-269, 2006.

Cardinal Features of Lynch Syndrome • Differentiating pathology features of LS CRCs: - more often poorly differentiated; - excess of mucoid and signet-cell features; - Crohn’s-like reaction; - significant excess of infiltrating lymphocytes within the tumor. • Increased survival from CRC. • Sine qua non for diagnosis is identification of germline mutation in MMR gene (most commonly MLH1, MSH2, MSH6) segregating in the family.

Targeted CRC Screening Screening is melded to LS’s natural history: Proximal location  colonoscopy Early age of onset  beginning at age 25 Accelerated carcinogenesis  every 1-2 yrs < age 40, then annually Pattern of extra-clonic cancers  targeted screening

Could this be hereditary Colon Cancer

Familial Hereditary Sporadic AC-1 without MMR (Familial CRC of syndrome “X”) Lynch Syndrome TACSTD1 (EPCAM) Sporadic FAP; AFAP Mixed Polyposis Syndrome Ashkenazi I1307K CHEK2 (HBCC) MUTYH (MAP) TGFBR1 PJS FJP CD BRRS = as yet undiscovered hereditary cancer variants Hamartomatous Polyposis Syndromes

FAP, Survival Increase, Managed Polyposis Registry* Regular systematic surveillance needed to ensure appropriate prophylactic colectomy before CRC develops. Registry programs ↑ survival and age of onset of CRC, ↓ incidence of CRC in FAP. *Mallinson et al. Gut 59:1378-1382, 2010.

FAP, Survival Increase, Managed Polyposis Registry* Results:  Survival ↑ from 57.8 yrs to 70.4 yrs (P<0.001) by screening;  from 58.1 yrs to 69.6 yrs (P=0.007) following establishment of polyposis registry;  CRC incidence ↓ from 43.5% to 3.8% by  from 28.7% to 14.0% following establishment of polyposis registry. *Mallinson et al. Gut 59:1378-1382, 2010.

FAP, Survival Increase, Managed Polyposis Registry* Conclusion: A regular systematic large bowel screening program, managed by a polyposis registry, significantly improves the prognosis of FAP. *Mallinson et al. Gut 59:1378-1382, 2010.

Attenuated FAP Later onset (CRC ~age 50) Few colonic adenomas Not associated with CHRPE UGI lesions Associated with mutations at extreme 5’, 3' ends of APC gene, & exon 9A 14

Molecular Diagnosis of LS: Toward a Consensus If tumor is MSI-positive, IHC is then done to direct mutational testing to a specific MMR gene, which MSI alone cannot do.* If tumor is MSS, must weigh low probability of an informative IHC test and cost of performing it.** *Engel et al. Int J Cancer 118:115-122, 2006. **Lynch et al. J Natl Cancer Inst 99:261-263, 2007.

BRAF V600E mutation and LS BRAF V600E mutation can sort this out since when detected it excludes LS and contributes to improved cost- effectiveness of genetic testing for LS. *Clin Gastroenterol Hepatol 6:206-214, 2008.

Frequency of MMR Mutations* ~60% of Amsterdam+ LS families with clinically defined phenotype carry point mutations or large genomic deletions in the transcription of either MLH1 or MSH2 genes. Conversely, the pathogenic change inactivating the MMR system is not known or not fully understood in the remaining ~40%. *Lagerstedt-Robinson et al. J Natl Cancer Inst 99:291-199, 2007.

Frequency of MMR Mutations* A portion of this ~40% lacking MMR mutations is caused by a mutation mechanism in the gene known as EPCAM. Others have been classified as familial colorectal cancer Type “X”.** *Kovacs et al. Hum Mutat 30:197-203, 2009. **Lindor et al. JAMA 293:1979-1985, 2005.

Epithelial Cell Adhesion Molecule (EPCAM) Gene and Its Lynch Syndrome Connection Impacts Diagnosis Genetic Counseling Phenotype site specific CRC Pathogenesis Pharmacogenetics

History of Family R* Ascertained by us in 1970 and followed continuously. 700 blood line relatives 327 individuals age ≥ 18, ≥ 25% pedigree risk Phenotype strikingly similar to LS but integral extracolonic cancers absent (site-specific CRCs) *Lynch et al. Cancer 56:934-938, 1985. Lynch et al. Cancer 56:939-951, 1985.

First patient identified with EPCAM mutation CRC affecteds EPCAM results

Exons 8 and 9 and polyadenylation sequence 5’ EPCAM deletion Exons 8 and 9 and polyadenylation sequence Polyadenylation Sequence Transcriptional read through Hypermethylation of the MSH2 promoter Ligtenberg MJ, Nature Genetics 2009.

Why LS with Site-Specific CRC? Deletion in EPCAM results in hypermethylation and incomplete silencing of MSH2. EPCAM mutation carriers may have phenotypic features that differ from carriers of MSH2 mutations – namely, an almost exclusive expression of site-specific CRC, thereby lacking extracolonic cancers.

American and Dutch families have the same deletion in the EPCAM gene MSH2 Deletion c.859-1462_*1999del (4.9 kb, starting in intron 7 and including exons 8 & 9) Lightenberg, Nature Genetics 2009.

American and Dutch EPCAM mutations originate from a common ancestor Family R and the Dutch families share a 6.1 MB region surrounding the same EPCAM deletion indicating a common ancestor. Based on the size of the shared region it is estimated the deletion occurred 10 generations ago. Dutch Families Chromosome 2 Family R Chromosome 2 Deletion and Region inherited from common ancestor

Location and Age at diagnosis Cancer diagnoses in 12 Dutch founder EPCAM mutation patients reported from the Netherlands Subject (Ref) Location and Age at diagnosis Family A:III:3 (1) Cecum (56) Family A:III:4 (1) Ileum (56) Family A:IV:1 (1) Cecum (47) Family A:IV:3 (1) Cecum (52) Family A:V:1 (1) Rectum (18) Family B:III:1 (1) Rectum (45) Family B:III:4 (1) Transverse colon (50) Family C (1) Ileocecum (53) Family D (1) Cecum (40) Patient A (2) Colon (44), Duodenum (54) Patient B (2) Colon (32) Patient C (2) Colon (43,60,70) (1) Lightenberg, Nature Genetics 2009. (2) Neissen, Genes, Chromo and Cancer 2010.

Conclusions for EPCAM Conclusions: 1) Cancer control compliance in Family R profound; 2) 40% of AC-I cases lack MMR mutations – how many may qualify as EPCAM? 3) Likely EPCAM phenotype site-specific CRC; 4) What can we learn from molecular features of EPCAM for pharmacologic benefit? 5) 1/35 CRC affecteds likely LS (Hampel et al.*). *J Clin Oncol 26:5783-5788, 2008.