1 LYNCH SYNDROME EPCAM FAMILY WITH PREDOMINANT COLORECTAL CANCER HENRY T. LYNCH, MD; STEPHEN N. THIBODEAU, PHD; CARRIE SNYDER, MSN, RN, APNG; JENNIFER RHEES; BRITTANY THOMAS, MS, CGC; JANE F. LYNCH, BSN; PATRICK LYNCH, JD, MD; JANINE M. DAVIES, MD; C. RICHARD BOLAND, MD; DOUGLAS L. RIEGERT-JOHNSON, MD;

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

3 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: , 2007.

4 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: , **Lindor et al. JAMA 293: , 2005.

5 Frequency of EPCAM EPCAM mutation cosegregated with the LS phenotype in 19% (5/27) of families tested who lacked MLH1/MSH2 mutation. Previously not described in any cancer predisposition syndrome. *Kovacs et al. Hum Mutat 30: , 2009.

6 EPCAM Likely Tip of the Iceberg* Similar alterations of genes that regulate somatic methylation across the genome are likely. *Ligtenberg et al. Nat Genet 41: , Kobelka. Clin Genet 75: , 2009.

7 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.

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

10 Dutch Founder Mutation: EPCAM c _*1999del (4.9 kb, starting in intron 7 and including exons 8 & 9). EPCAM MSH2 Family “R” – Lynch Syndrome type I: Colon specific Lynch Syndrome. Location by MLPA probes. ? ? Confirmed by MLPA End points to be confirmed

11 Sequencing of the Deletion Breakpoints Alignment to Intron 7 of EpCAM Alignment to 5’ region of MSH2 (~5700bp upstream of ATG) BLAT Alignment of Deletion PCR product sequence Alignment of Deletion PCR product sequence to EpCAM and 5’ MSH2 sequences Both Deletion breakpoints are located in AluSx sequences The Deletion breakpoints are the same as reported for family CL177 by Wagner et al.

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13 History of Family R* Ascertained by us in 1970 and followed continuously. 701 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: , Lynch et al. Cancer 56: , 1985.

14 History of Family R Genealogy, cancer pathology verification, in consenting patients with genetic counseling (Lynch syndrome protocol). We constantly advised family members of their inordinately high CRC risk. Compliance was very strong!

15 Cancer History of Family R Colon Cancer Site Specific? At Risk Individuals (n=327) 80 Individuals developed cancer: a) 49 (61%) Colon cancer (14 metachronous) b) 3 (4%) Small bowel cancer (1 EPCAM positive, 1 negative and 1 not tested) c) 1 (3%) Endometrial cancer (EPCAM negative) d) No Ovarian cancer cases

16 History of Family R In mid-1990s when LS MMR germline mutations became known,* testing of Family R consistently showed absence of MMR mutations. *Fishel et al. Cell 75: , Leach et al. Cell 75: , Bronner et al. Nature 368: , Papadopoulos et al. Science 263: , 1994.

17 Methods for EPCAM Analysis performed using a commercially available MLPA kit (PO72 version 6, MRC Holland) on patients’ DNA.

18 First patient identified with EPCAM mutation CRC affecteds

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20 Family Information Service (FIS)* Educational session welcoming all family members. Enables free-wheeling discussion with family support, where areas of communication are open and inhibition decreased. Highly effective (we have done >100 FISs over past 40 years). *Lynch et al. Cancer 91: , 2001.

21 Genetic Counseling: EPCAM 46 have been tested: 16 positive, 30 negative; 44 have been disclosed with genetic counseling 14 at FIS, 30 through telephone counseling; 14 additional samples submitted with results pending; All results were well-received with the exception of one individual wanting to confirm the test result through an independent sample which has been completed, confirmed and disclosed.

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24 Origin of EPCAM mutation Family R EPCAM mutation most probably identical by descent (“founder mutation”) from Dutch ancestor. –MLPA shows Family R and Dutch mutation span same regions –Family R has Dutch ancestry (following slide) –Breakpoint mapping and haplotype analysis underway for confirmation

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26 Founder Event – Needs More Research Our initial EPCAM findings in Family R suggestive of Ligtenberg-Netherlands Founder findings. Problems – differences in size of the deletions; Need more sequencing of the breakpoints defining the ends of the deletions.

27 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: , 2008.