EPCAM Deletions Research Articles

Biallelic EPCAM deletions induce tissue-specific DNA repair deficiency and cancer predisposition

We report a case of Mismatch Repair Deficiency (MMRD) caused by germline homozygous EPCAM deletion leading to tissue-specific loss of MSH2. Through the use of patient-derived cells and organoid technologies, we performed stepwise in vitro differentiation of colonic and brain organoids from reprogrammed EPCAMdel iPSC derived from patient fibroblasts. Differentiation of iPSC to epithelial-colonic organoids exhibited continuous increased EPCAM expression and hypermethylation of the MSH2 promoter. This was associated with loss of MSH2 expression, increased mutational burden, MMRD signatures and MS-indel accumulation, the hallmarks of MMRD. In contrast, maturation into brain organoids and examination of blood and fibroblasts failed to show similar processes, preserving MMR proficiency. The combined use of iPSC, organoid technologies and functional genomics analyses highlights the potential of cutting-edge cellular and molecular analysis techniques to define processes controlling tumorigenesis and uncovers a new paradigm of tissue-specific MMRD, which affects the clinical management of these patients.

The roles EpCAM plays to enhance the malignancy of gastric cancer.

Gastric cancer (GC) remains a global challenge due to its high morbidity and mortality rates especially in Asia as well as poor response to treatment. As a member of the adhesion protein family and transmembrane glycoprotein, EpCAM expressed excessively in cancer cells including GC cells. The database assay showed that EpCAM is excessively expressed and easily mutated in cancers, especially in early stage of GC. To explore the roles EpCAM plays in oncogenesis and progression of GC, the expression of EpCAM was deleted in GC cells with CRISPR/Cas9 method, and then the changes of cell proliferation, apoptosis, motility and motility associated microstructures in EpCAM-deleted GC cells (EpCAM-/-SGC7901) were detected to evaluate the rules EpCAM played. The results showed that EpCAM deletion caused cell proliferation, motility and the development of motility-relevant microstructures inhibited significantly, apoptotic trend and contact inhibition enhanced in EpCAM-deleted GC cells. The results of western blot suggested that EpCAM modulates the expression of epithelial/endothelial mesenchymal transition (EMT) correlated genes. All results as above indicated that EpCAM plays important roles to enhance the oncogenesis, malignancy and progression as a GC enhancer. Combining our results and published data together, the interaction of EpCAM with other proteins was also discussed and concluded in the discussion. Our results support that EpCAM can be considered as a novel target for the diagnosis and therapy of GC in future.

Lynch syndrome: influence of additional susceptibility variants on cancer risk.

Some patients with Lynch syndrome (LS) have extreme phenotypes, i.e. cancer before the recommended screening age, or cancer for which there are no screening guidelines. We made the hypothesis that additional germline variants in cancer susceptibility genes (CSG) could explain some of these phenotypes. We compared the prevalence of additional CSG variants in LS patients with a cancer diagnosis before age 30 (early-onset, EO group) and after 40 (usual-onset, UO group). While there was no overall difference, we did find an excess of pathogenic variants and variants of unknown significance in EO cases when only gastrointestinal CSG were considered (OR 2.25; 95% CI: 1.01-5.06, p value = 0.04). Four EO cases stood out: two with POLE/POLD1 variants in the key exonuclease domain, one with a BMPR1A duplication and one with an EPCAM deletion. Additional germline variants should be considered in future screening recommendations, as they might influence cancer risk.

The Somatic Mutational Landscape of Mismatch Repair Deficient Prostate Cancer.

Prostate cancers with mismatch repair deficiency (MMR-d) have aggressive clinical and histological features, and they are potentially responsive to immunotherapy. However, its rarity prevents the analysis of the underlying biology. Here, we collected the genomic data of 2664 primary prostate tumors and 1409 metastatic prostate tumors from the GENIE and TCGA databases. A total of 69 (2.59%) primary and 60 (4.26%) metastatic MMR-d tumors were identified among these tumors. Single nucleotide variant (SNV) frequencies of 34 candidate genes (including KMT2D (46.4%), ZFHX3 (33.3%), JAK1 (31.9%), and RNF43 (27.5%)) and 16 candidate genes (including KMT2D (33.3%) and JAK1 (28.3%)) were higher in MMR-d primary tumors and MMR-d metastatic tumors, respectively. The tumor mutation burden (TMB) was higher in primary MMR-d tumors. Homozygous deletions of EPCAM and EPAS1 were enriched in MMR-d primary tumors, while EPCAM deletions were enriched in metastatic MMR-d tumors. For genomic rearrangement events, TMPRSS2-ETS fusions were less frequent in primary MMR-d tumors. Our study indicates MMR-d prostate cancers have unique genomic features. These may play an important role in providing therapeutic targets for the treatment of this subset of prostate cancer patients.

Extracolonic tumours in a pedigree with EPCAM-related Lynch Syndrome

Lynch Syndrome is characterized by phenotypic and genotypic heterogeneity. Despite scarce evidence, individuals with an EPCAM deletion appear to have a comparable risk of colorectal cancer (CRC) as MSH2 mutation carriers, but a lower risk of extracolonic cancer (such as endometrial cancer) unless the deletion extends close to the promoter of MSH2.A genotype-phenotype correlation is yet to be established for EPCAM alterations. In this report, we describe a family with EPCAM deletion characterized by a particularly aggressive phenotype and extracolonic cancer.We present a family with 5 members carrying an EPCAM deletion encompassing exons 8 and 9. Three female family members presented CRC at the ages of 32, 44 and 60 (mucinous moderately and well-differentiated adenocarcinoma); in two of them metachronous colon cancers and advanced adenomas were diagnosed in the intensive surveillance program.Two female patients (42 and 63 years-old) presented with gastric cancer (GC). Two patients presented with small bowel cancer at 51 and 60 years-old - the first one presented a metachronous jejunal cancer at 68 years. Only one family member was submitted to hemithyroidectomy due a right-lobe Hürthle cell carcinoma at 56 years-old.This report illustrates the existence of intrafamilial clinical heterogeneity among carriers of this EPCAM alteration, and hence the difficulty in predicting phenotype for EPCAM-associated Lynch syndrome.

Lynch Syndrome-Associated Endometrial Cancer With Combined EPCAM-MSH2 Deletion: A Case Report.

BackgroundLynch syndrome (LS), an autosomal dominant disorder, is characterized by germline pathogenic variants in DNA mismatch repair (MMR) genes like MSH2. EPCAM deletions cause a minority (3%) of LS cases. However, there are only a few reports of LS-associated endometrial cancer (LS-EC) induced by the inactivation of the MSH2 gene due to EPCAM deletions.Case PresentationWe present the case of a 45-years old woman diagnosed with endometrial cancer (EC). Definitive surgery revealed meso-differentiated endometrioid adenocarcinoma, stage IA without lymph-vascular space invasion. Four months later, she received radiation therapy (125I radioactive seeds implantation), and platinum-containing regimen combined chemotherapy because of vaginal stump metastasis of EC. After five years, we performed immunohistochemistry (IHC) on pelvic mass because of presacral metastatic lymph node. IHC showed the absence of MSH2 and MSH6 protein expression in the pelvic mass tissue. Peripheral blood was used for genetic testing based on her cancer diagnosis and family history of cancer in close relatives. Genetic testing revealed deletions of exon 8 and 9 in EPCAM and deletions of exon 1 and 8 in MSH2; thus, we diagnosed the presence of LS. The patient underwent interstitial brachytherapy (BT) of the presacral metastatic lymph node.ConclusionThis case highlights that patients with LS-EC who are carriers of combined EPCAM-MSH2 deletion might experience better oncologic outcomes even with early recurrence.

Interval between the First Cancer and the Genetic Diagnosis in Lynch Syndrome Probands.

Objective Little is known about the time from developing a first cancer to confirming the presence of a mismatch repair (MMR) gene mutation for Lynch syndrome (LS) probands. Methods This was a retrospective single center study. LS probands, who have an MMR gene mutation that was confirmed first in a pedigree and thereafter developed at least one cancer, were included in this study. Results There were 21 LS probands who had developed at least one cancer; 6 with MLH1 mutations, 9 with MSH2 mutations, 4 with MSH6 mutations, and 2 with EPCAM deletions. The median ages at the first cancer and the genetic diagnosis were 47 (34-71) and 62 (38-84) years old, respectively. The mean interval between the first cancer and the genetic diagnosis was 11.0 (0-25) years, and 20 years or longer interval was required for the 5 probands. Six (28.6%) probands were older than 70 years, and 3 (14.3%) were in their 80s when they were diagnosed to have LS. The genetic diagnosis was confirmed at the first, second, third, and fourth cancer or later in 5, 5, 6, and 5 probands, respectively. Of the 16 cancers examined, 2 (12.5%) were microsatellite stable (MSS), both of whom had germline MSH6 mutations. All 17 LS probands who developed colorectal cancer met the revised Bethesda guidelines at the genetic diagnosis, but only 7 of 11 (63.6%) met them at the first cancer. Twelve out of 21 (57.1%) met the revised Amsterdam criteria. Conclusion It took 11 years for the LS probands from the first cancer to the diagnostic confirmation by genetic tests. A quarter of the probands were in their 70s or 80s at genetic diagnosis.

Mismatch Repair Deficiency and Microsatellite Instability in Triple-Negative Breast Cancer: A Retrospective Study of 440 Patients.

PurposeTo investigate the status of mismatch repair (MMR) and microsatellite instability (MSI) in triple-negative breast cancer (TNBC) and to examine correlations between MMR/MSI status and clinicopathological parameters.MethodsWe retrospectively collected tissue samples from 440 patients with TNBC and constructed tissue microarrays. Protein expression of MLH1, MSH2, MSH6, and PMS2 was detected by immunohistochemistry (IHC). We also analyzed 195 patient samples using MSI polymerase chain reaction (PCR) testing. Correlations between MSI status and clinicopathological parameters and prognosis were analyzed.ResultsThe median age of the cohort was 49 years (range: 24–90 years) with a median follow-up period of 68 months (range: 1–170 months). All samples were positive for MLH1, MSH2, MSH6, and PMS2, except for one sample identified as MMR-deficient (dMMR) by IHC, with loss of MSH2 and intact MSH6 expression. MSI PCR revealed no case with high-frequency MSI (MSI-H), whereas 14 (7.2%) and 181 (92.8%) samples demonstrated low-frequency and absence of MSI events, respectively. The dMMR sample harbored low-frequency instability, as revealed by MSI PCR, and a possible EPCAM deletion in the tumor, as observed from next-generation sequencing. No correlations were detected between MMR or MSI status and clinicopathological parameters, programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) expression, or survival.ConclusionsThe incidence of dMMR/MSI-H is extremely low in TNBC, and rare discordant MSI PCR/MMR IHC results may be encountered. Moreover, MMR/MSI status may be of limited prognostic value. Further studies are warranted to explore other predictive immunotherapy biomarkers for TNBC.

Investigation on the hereditary basis of colorectal cancers in an African population with frequent early onset cases

BackgroundApproximately 25% of colorectal cancer patients in sub-Saharan Africa are younger than 40 years, and hereditary factors may contribute. We investigated the frequency and patterns of inherited colorectal cancer among black Zimbabweans.MethodsA population-based cross-sectional study of ninety individuals with a new diagnosis of colorectal cancer was carried out in Harare, Zimbabwe between November 2012 and December 2015. Phenotypic data was obtained using interviewer administered questionnaires, and reviewing clinical and pathology data. Cases were screened for mismatch repair deficiency by immunohistochemistry and/or microsatellite instability testing, and for MLH1, MSH2 and EPCAM deletions using multiplex ligation-dependent probe amplification. Next generation sequencing using a 16-gene panel was performed for cases with phenotypic features consistent with familial colorectal cancer. Variants were assessed for pathogenicity using the mean allele frequency, phenotypic features and searching online databases.ResultsThree Lynch syndrome cases were identified: MSH2 c.2634G>A pathogenic mutation, c.(1896+1_1897–1)_(*193_?)del , and one fulfilling the Amsterdam criteria, with MLH1 and PMS2 deficiency, but no identifiable pathogenic mutation. Two other cases had a strong family history of cancers, but the exact syndrome was not identified. The prevalence of Lynch syndrome was 3·3% (95% CI 0·7–9·4), and that of familial colorectal cancer was 5·6% (95% CI, 1·8–12·5).ConclusionsIdentifying cases of inherited colorectal cancer in sub-Saharan Africa is feasible, and our findings can inform screening guidelines appropriate to this setting.

1406P - Microsatellite instability testing and lynch syndrome screening for colorectal cancer patients through tumour sequencing

BackgroundUniversal tumor screening for Lynch Syndrome (LS), which involves up to 6 sequential tests, is recommended by NCCN guidelines for all patients with colorectal cancer (CRC) at diagnosis. Microsatellite instability (MSI) testing is the first step in the genetic diagnosis for LS. Additionally, MSI status has been approved by FDA to select patients for immunotherapy treatments. Here we evaluate the performance of a single tumor sequencing test using Illumina TruSight™ Oncology 500 (TSO500) for MSI status determination and LS screening. MethodsA total of 233 CRC subjects were screened through a commercial MSI-PCR assay run on tumor-normal DNA. Tumor DNA from 63 selected subjects was sequenced with TSO500. The MSI score was calculated using 130 homopolymer microsatellite loci targeted by the TSO500 panel. Subsequently, BRAF p.V600E status and potential mutations in MMR genes or EPCAM were analyzed from TSO500 results. For LS screening, a method with three filtering criteria was used: 1) MSI-high (MSI-H) status, 2) without BRAF p.V600E mutations, and 3) at least 1 MMR gene variant or EPCAM deletion inferred as germline small variant mutation by TSO500 germline classifier or copy number change. Finally, matched normal samples were sequenced with TSO500 to confirm any germline mutations linked to LS. ResultsUsing MSI-PCR, 45 of the 233 (19.3%) subjects were identified as MSI-H and 188 (80.7%) as microsatellite stable (MSS). TSO500 achieved an overall percent agreement (OPA) of 100.0% (95% CI: 94.3% - 100.0%) with MSI-PCR for the 63 subjects analyzed by both methods. Eight subjects were identified as LS positive through tumor sequencing by TSO500. Matched normal sequencing confirmed all 8 positive cases of identified potential LS mutations as germline. Overall, TSO500 tumor sequencing achieved an OPA of 100.0% (95% CI: 94.3% - 100.0%) with matched normal sequencing. ConclusionsCollectively, our results demonstrated that MSI status can be accurately determined with tumor sequencing. Moreover, LS screening by TSO500 with secondary filtering algorithms can be used as a single upfront test to identify BRAF p.V600E status and potential pathogenic germline mutations linked to LS. Legal entity responsible for the studyIllumina. FundingIllumina. DisclosureL. Liu: Shareholder / Stockholder / Stock options, Full / Part-time employment: Ilumina. C. Garbutt: Full / Part-time employment: Illumina. M. Golkaram: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. S. Kaplan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. L. Costa: Research grant / Funding (institution), PIC/IC/82821/2007: Faculdade de Medicina da Universidade de Lisboa. A. So: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. S. Zhang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. T. Pawlowski: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. All other authors have declared no conflicts of interest.

Toward a better definition of EPCAM deletions in Lynch Syndrome: Report of new variants in Italy and the associated molecular phenotype.

BackgroundInherited epimutations of Mismatch Repair (MMR) genes are responsible for Lynch Syndrome (LS) in a small, but well defined, subset of patients. Methylation of the MSH2 promoter consequent to the deletion of the upstream EPCAM gene is found in about 1%–3% of the LS patients and represents a classical secondary, constitutional and tissue‐specific epimutation. Several different EPCAM deletions have been reported worldwide, for the most part representing private variants caused by an Alu‐mediated recombination.Methods712 patients with suspected LS were tested for MMR mutation in our Institute. EPCAM deletions were detected by multiplex ligation‐dependent probe amplification (MLPA) and then defined by Long‐Range polymerase chain reaction (PCR)/Sanger sequencing. A comprehensive molecular characterization of colorectal cancer (CRC) tissues was carried out by immunohistochemistry of MMR proteins, Microsatellite Instability (MSI) assay, methylation specific MLPA and transcript analyses. In addition, somatic deletions and/or variants were investigated by MLPA and next generation sequencing (NGS).ResultsAn EPCAM deletion was found in five unrelated probands in Italy: variants c.556‐490_*8438del and c.858+1193_*5826del are novel; c.859‐1430_*2033del and c.859‐670_*530del were previously reported. All probands were affected by CRC at young age; tumors showed MSI and abnormal MSH2/MSH6 proteins expression. MSH2 promoter methylation, as well as aberrant in‐frame or out‐of‐frame EPCAM/MSH2 fusion transcripts, were detected in CRCs and normal mucosae.ConclusionAn EPCAM deletion was the causative variant in about 2% of our institutional series of 224 LS patients, consistent with previously estimated frequencies. Early age and multiple CRCs was the main clinical feature of this subset of patients.

Polyp burden in Lynch syndrome patients ascertained via multigene panel testing.

596 Background: Lynch syndrome (LS), also known as hereditary non-polyposis colorectal cancer (HNPCC), is a hereditary colorectal cancer syndrome thought to present with few or no polyps. It has been suggested that the majority of LS patients with colon polyps will develop 1-9 cumulative adenomatous polyps in their lifetime, while approximately 10% will develop ≥10. Our aim was to describe polyp burden in a cohort of LS patients who were ascertained via multi-gene cancer panel testing. Methods: We retrospectively reviewed the personal history for all individuals with a pathogenic or likely pathogenic variant (collectively, PV) in MLH1, MSH2 (including 3’ EPCAM deletions), MSH6, or PMS2. Individuals with more than one PV in a LS-associated or other gene were excluded. All polyp data was obtained from provided test requisition forms and/or pathology reports. Results: A total of 131 individuals reported to have colon polyps were molecularly confirmed as having LS. Of these individuals, 55% (72/131) reported a history of colorectal cancer. Overall, polyp burden was reported as follows: 83% (109/131) with 1-10 polyps, 13% (17/131) with 10-19 polyps, and 4% (5/131) with 20-50 polyps. Of the 5 individuals reporting 20-50 polyps, the average was 31.2 polyps (range 21-50). Adenomatous pathology was reported for one or more polyps in 69% (91/131) of individuals. When limited to those with adenomatous polyps, 85% (77/91) reported 1-10 polyps, 11% (10/91) reported 10-19 polyps, and 4% (4/91) reported 20-50 polyps. When breaking down by presence and absence of colorectal cancer, 18% (13/72) and 15% (9/59), respectively, reported a polyp burden ≥10. Overall, 17% (22/131) of all LS patients reported ≥10 polyps. Conclusions: Similar to previously published data, the majority of LS patients with colon polyps report a polyp burden of < 10. However, our data suggest that the proportion of LS patients with a polyp burden of ≥10 may be higher than previously reported (17% vs. 10%) stressing the importance of including the LS-associated genes for these patients. While LS has historically been referred to as a “non-polyposis” syndrome, a subset of patients actually present with an attenuated polyposis-like phenotype.

Elucidating the molecular basis of MSH2-deficient tumors by combined germline and somatic analysis.

In a proportion of patients presenting mismatch repair (MMR)-deficient tumors, no germline MMR mutations are identified, the so-called Lynch-like syndrome (LLS). Recently, MMR-deficient tumors have been associated with germline mutations in POLE and MUTYH or double somatic MMR events. Our aim was to elucidate the molecular basis of MSH2-deficient LS-suspected cases using a comprehensive analysis of colorectal cancer (CRC)-associated genes at germline and somatic level. Fifty-eight probands harboring MSH2-deficient tumors were included. Germline mutational analysis of MSH2 (including EPCAM deletions) and MSH6 was performed. Pathogenicity of MSH2 variants was assessed by RNA analysis and multifactorial likelihood calculations. MSH2 cDNA and methylation of MSH2 and MSH6 promoters were studied. Matched blood and tumor DNA were analyzed using a customized next generation sequencing panel. Thirty-five individuals were carriers of pathogenic or probably pathogenic variants in MSH2 and EPCAM. Five patients harbored 4 different MSH2 variants of unknown significance (VUS) and one had 2 novel MSH6 promoter VUS. Pathogenicity assessment allowed the reclassification of the 4 MSH2 VUS and 6 probably pathogenic variants as pathogenic mutations, enabling a total of 40 LS diagnostics. Predicted pathogenic germline variants in BUB1, SETD2, FAN1 and MUTYH were identified in 5 cases. Three patients had double somatic hits in MSH2 or MSH6, and another 2 had somatic alterations in other MMR genes and/or proofreading polymerases. In conclusion, our comprehensive strategy combining germline and somatic mutational status of CRC-associated genes by means of a subexome panel allows the elucidation of up to 86% of MSH2-deficient suspected LS tumors.

Abstract 4277: Clinicopathological significance of endometrial cancer with MSH2 deficiency

Abstract Background: Endometrial cancer accounts for the second percentage of Lynch syndrome related tumors following colorectal cancer. Inactivation of MSH2 was frequently observed in endometrial cancer with microsatellite instability (MSI) or mismatch repair complex deficiency (dMMR). With respect to MSH2 deficiency (dMSH2), not like MLH1 deficiency, most of dMSH2 were caused by germline mutations in the MSH2 gene or germline EpCAM deletions. Meanwhile, heritable germline epimutations in MSH2 have been reported in a few Lynch syndrome families that lacked germline mutations in the MSH2 gene. We previously provided evidence for frequent MSH2 hypermethylation in Lynch syndrome colorectal tumors with dMSH2 and MSH2 methylation may serve as the “second hit” at the wild-type allele. Materials and Methods: In this study, we analyzed MSH2 promoter methylation status, as well as MLH1 methylation status, and expression status of the mismatch repair proteins (MLH1, MSH2, PMS2, and MSH6) by immunohistochemistry in a cohort of 138 endometrial cancer tissues surgically resected at Okayama University Hospital. DNA was extracted from formalin-fixed, paraffin-embedded tissue, analyzed MSI status by four mononucleotide markers and both MLH1 and MSH2 promoter methylation status by a fluorescent quantitative bisulfite PCR assay. Results: Endometrial cancers displaying MSI or dMMR were observed in 40 (29.0%) or 41 cases (29.7%), respectively. Endometrial cancers with MSH2 deficiency were observed in eight (5.8%) of 138 tumors (19.5% of dMMR). MSH2 promoter methylation was present in 7 cases (5.1% in 138 tumors), and significantly correlated with dMSH2 (P=0.0041, Fisher's exact probability test). Then, we also examined the family history of first-degree relatives retrospectively. In this cohort, although patients with MMR deficiency were significantly associated with family history of Lynch syndrome related tumor (P<0.001), patients with this family history of Lynch syndrome related tumor are more frequently observed in patients with dMSH2 (P=0.0347). Interestingly, patients with MSH2 promoter methylation were strongly associated with this family history of Lynch syndrome related tumor (P=0.002), though patients with MLH1 methylation were not (P=0.0878). Conclusions: In this study, we demonstrated that MSH2 methylation significantly correlated with dMSH2 and may have strong relation with family history of Lynch syndrome related tumor, taking a role as “second hit” to the MSH2 gene. Citation Format: Junko Haraga, Takeshi Nagasaka, Keiichirou Nakamura, Tomoko Haruma, Takeshi Nishida, Akihiro Nyuya, Kazuya Yasui, Hisashi Masuyama, Toshiyoshi Fujiwara, Yuji Hiramatsu. Clinicopathological significance of endometrial cancer with MSH2 deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4277. doi:10.1158/1538-7445.AM2017-4277

Abstract A37: MSI is not an important feature in early onset pancreatic carcinoma

Abstract Background and Aim: There is scant information on pancreatic cancer occurring at young age. About 5 to 10 percent of patients with pancreatic cancer have a family history of pancreatic cancer, which is partially due to defined hereditary syndromes like Lynch syndrome. Recently, we have identified deletions of the 3’ end of the EPCAM gene as a novel cause of this hereditary syndrome. In the cohort of patients with EPCAM deletions a relatively high number of pancreatic cancers and duodenal cancers were observed. We therefore aimed to assess whether development of pancreatic or duodenal cancer at young age might be an indication of EPCAM-associated Lynch syndrome. Methods: We searched the Dutch Pathology Registry (PALGA) to identify all patients who were diagnosed with pancreatic carcinoma (PC), ampulla of Vater carcinoma (VC) or duodenal carcinoma (DC) before the age of 50 in The Netherlands between January 2000 and December 2012. Immunohistochemical analysis was used to analyze mismatch repair (MMR)- and EPCAM-protein expression. MS-status was determined by a panel of 5 mononucleotide repeats in cases with an aberrant MMR protein pattern. Furthermore, multiplex ligation-dependent probe amplification will be performed in case of an absent EPCAM staining or loss of MSH2 and MSH6 protein expression. Results: An aberrant MMR-IHC for PMS2 combined with microsatellite instability (MSI) was identified in 1 of 48 (2%) PC diagnosed before age 50. In 24 VC patients, one tumor (4%) showed MSI with absence of both MLH1 and PMS2 staining. This patient had a history of colon carcinoma at the age of 43 years. All patients showed a normal immunohistochemical staining for MSH2 and MSH6. EPCAM protein expression was present in all cases, except one case with an anaplastic pancreas carcinoma where EPCAM was absent in the vimentine-positive area of the tumor. Eight out of 22 DC tumors (36%) showed a MMR-defective phenotype with MSI, of which 2 with an aberrant MSH2 and MSH6 staining and a positive EPCAM protein expression. One of these two patients had a history of colon carcinoma at the age of 24 and 26 years. Conclusion: This nationwide study cohort suggests that early onset pancreatic cancer and ampulla of Vater cancer are at most rarely associated with (EPCAM-associated) Lynch syndrome, in contrast to early onset duodenal cancer. Citation Format: Monica AJ Van Zanten, Marlies JE Kempers, Marjolijn JL Ligtenberg, Iris D. Nagtegaal. MSI is not an important feature in early onset pancreatic carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A37.

491PD - Lynch-Like Syndrome: Characterization and Comparison with Epcam Deletion Carriers

ABSTRACT Introduction: Colorectal cancers (CRCs) with MSI+ but without detectable germline mutation or hypermethylation in DNA mismatch repair (MMR) genes can be classified as Lynch-like Syndrome (LLS). The underlying mechanism and clinical significances of LLS are largely unknown. Methods: We measured MSI and MMR protein expression in 4,765 consecutive CRC cases. Among these, MSI+ cases were further classified based on clinical parameters, sequencing of MMR genes or polymerase e (POLE) and d (POLD1) and promoter methylation analysis of MLH1 and MSH2. Results: We found that MSI+ and MMR protein deficient CRCs comprised 6.3% (N = 302) of this cohort. Based on germline sequencing of 124 cases, we identified 54 LS with MMR germline mutation (LS-MMR), 15 LS with EPCAM deletions (LS-EPCAM) and 55 LLS patients. Of the 55 LLS patients, six (10.9%) had variants of unknown significance in the genes tested, and one patient had a novel somatic mutation (p.S459P) in POLE. In patients with biallelic deletions of EPCAM, all tumors and their matched normal mucosa showed promoter hypermethylation of MSH2. Conclusion: We found that patients with LLS and LS-EPCAM shared clinical features that differed from LS-MMR patients, including lower frequency of fulfillment of the revised Bethesda guidelines (83.6% and 86.7% vs. 98.1% for LS-MMR) and older mean age at CRC diagnosis (52.6 and 52.7 years vs. 43.9 years for LS-MMR). The similarity between LLS and LS-EPCAM suggests LLS as a subset of familial MSI+ CRC. Disclosure: All authors have declared no conflicts of interest.

Lynch-like syndrome: characterization and comparison with EPCAM deletion carriers.

Colorectal cancers (CRCs) with microsatellite instability-high (MSI+) but without detectable germline mutation or hypermethylation in DNA mismatch repair (MMR) genes can be classified as Lynch-like syndrome (LLS). The underlying mechanism and clinical significances of LLS are largely unknown. We measured MSI and MMR protein expression in 4,765 consecutive CRC cases. Among these, MSI+ cases were further classified based on clinical parameters, germline sequencing of MMR genes or polymerase ε (POLE) and δ (POLD1) and promoter methylation analysis of MLH1 and MSH2. We found that MSI+ and MMR protein-deficient CRCs comprised 6.3% (N = 302) of this cohort. On the basis of germline sequencing of 124 cases, we identified 54 LS with MMR germline mutation (LS-MMR), 15 LS with EPCAM deletions (LS-EPCAM) and 55 LLS patients. Of the 55 LLS patients, six (10.9%) had variants of unknown significance in the genes tested, and one patient had a novel somatic mutation (p.S459P) in POLE. In patients with biallelic deletions of EPCAM, all tumors and their matched normal mucosa showed promoter hypermethylation of MSH2. Finally, we found that patients with LLS and LS-EPCAM shared clinical features that differed from LS-MMR patients, including lower frequency of fulfillment of the revised Bethesda guidelines (83.6 and 86.7% vs. 98.1% for LS-MMR) and older mean age at CRC diagnosis (52.6 and 52.7 years vs. 43.9 years for LS-MMR). We identified somatic mutation in POLE as a rare underlying cause for MMR deficiency in LLS. The similarity between LLS and LS-EPCAM suggests LLS as a subset of familial MSI+ CRC.

Germline deletions in the EPCAM gene as a cause of Lynch syndrome – literature review

Lynch syndrome (clinically referred to as HNPCC – Hereditary Non-Polyposis Colorectal Cancer) is a frequent, autosomal, dominantly-inherited cancer predisposition syndrome caused by various germline alterations that affect DNA mismatch repair genes, mainly MLH1 and MSH2. Patients inheriting this predisposition are susceptible to colorectal, endometrial and other extracolonic tumors. It has recently been shown that germline deletions of the last few exons of the EPCAM gene are involved in the etiology of Lynch syndrome. Such constitutional mutations lead to subsequent epigenetic silencing of a neighbouring gene, here, MSH2, causing Lynch syndrome. Thus, deletions of the last few exons of EPCAM constitute a distinct class of mutations associated with HNPCC. Worldwide, several investigators have reported families with EPCAM 3’end deletions. The risk of colorectal cancer in carriers of EPCAM deletions is comparable to situations when patients are MSH2 mutation carriers, and is associated with high expression levels of EPCAM in colorectal cancer stem cells. A lower risk of endometrial cancer was also reported. Until now the standard diagnostic tests for Lynch syndrome have contained analyses such as immunohistochemistry and tests for microsatellite instability of mismatch repair genes. The identification of EPCAM deletions or larger EPCAM-MSH2 deletions should be included in routine mutation screening, as this has implications for cancer predisposition.

Identification of a founder EPCAM deletion in Spanish Lynch syndrome families

Germline deletions at the 3'-end of EPCAM have been involved in the etiology of Lynch syndrome (LS). The aim of this study was to characterize at the molecular level Spanish families harboring EPCAM deletions. Non-commercial multiplex ligation-dependent probe amplification (MLPA) probes and long-range polymerase chain reaction (PCR) amplification were used to characterize each deletion. Haplotyping was performed by analyzing eight microsatellite markers and five MSH2single nucleotide polymorphisms (SNPs). Methylation of MSH2 was analyzed by methylation specific-MLPA. Tumors diagnosed in seven Spanish families harboring EPCAM deletions were almost exclusively colorectal. Mosaicism in MSH2 methylation was observed in EPCAM deletion carrier samples, being average methylation levels higher in normal colon and colorectal tumors (27.6% and 31.1%), than in lymphocytes and oral mucosa (1.1% and 0.7%). Three families shared the deletion c.858 + 2568_*4596del, with a common haplotype comprising 9.9 Mb. In two families the novel EPCAM deletion c.858 + 2488_*7469del was identified. This study provides knowledge on the clinical and molecular characteristics of mosaic MSH2 epimutations. The identification of an EPCAM founder mutation has useful implications for the molecular diagnosis of LS in Spain.

An Update on the Pathogenesis of Lynch Syndrome: Recently Described Novel Molecular Mechanisms

Lynch syndrome, originally described in 1913 and previously known as hereditary nonpolyposis colorectal carcinoma syndrome, is the most common hereditary cancer syndrome. This syndrome is classically due to germline mutations in the mismatch repair genes MLH1, MSH2, MSH6, or PMS2. The cancer risk for patients with Lynch syndrome is not limited to the colorectum; women with Lynch syndrome are at risk for endometrial cancer, and Lynch patients of both genders are at risk for other cancers as well. There are cases of cancers in families that meet the clinical criteria (Amsterdam and Bethesda criteria) for Lynch syndrome but do not have a mutation in the one of the four classic mismatch repair genes. For some time, there has been speculation that other mutations or mechanisms were responsible for a subset of Lynch syndrome patients; much research has gone into identifying those alternative mutations and mechanisms. Recently, EPCAM deletion, CHEK2 mutations, and germline MLH1 hypermethylation have been identified as alternative mutations that cause Lynch syndrome in mismatch repair-negative patients. This article reviews these novel mechanisms and mutations, their clinical significance, and the pathogenesis of these Lynch causing mutations.