DNA Mismatch Repair Genes MLH1 Research Articles

Classification of MLH1 Missense VUS Using Protein Structure-Based Deep Learning-Ramachandran Plot-Molecular Dynamics Simulations Method.

Pathogenic variation in DNA mismatch repair (MMR) gene MLH1 is associated with Lynch syndrome (LS), an autosomal dominant hereditary cancer. Of the 3798 MLH1 germline variants collected in the ClinVar database, 38.7% (1469) were missense variants, of which 81.6% (1199) were classified as Variants of Uncertain Significance (VUS) due to the lack of functional evidence. Further determination of the impact of VUS on MLH1 function is important for the VUS carriers to take preventive action. We recently developed a protein structure-based method named "Deep Learning-Ramachandran Plot-Molecular Dynamics Simulation (DL-RP-MDS)" to evaluate the deleteriousness of MLH1 missense VUS. The method extracts protein structural information by using the Ramachandran plot-molecular dynamics simulation (RP-MDS) method, then combines the variation data with an unsupervised learning model composed of auto-encoder and neural network classifier to identify the variants causing significant change in protein structure. In this report, we applied the method to classify 447 MLH1 missense VUS. We predicted 126/447 (28.2%) MLH1 missense VUS were deleterious. Our study demonstrates that DL-RP-MDS is able to classify the missense VUS based solely on their impact on protein structure.

Tuberous sclerosis complex: a complex case.

Tuberous sclerosis complex (TSC) is an inheritable disorder characterized by the formation of benign yet disorganized tumors in multiple organ systems. Germline mutations in the TSC1 (hamartin) or more frequently TSC2 (tuberin) genes are causative for TSC. The malignant manifestations of TSC, pulmonary lymphangioleiomyomatosis (LAM) and renal angiomyolipoma (AML), may also occur as independent sporadic perivascular epithelial cell tumor (PEComa) characterized by somatic TSC2 mutations. Thus, discerning TSC from the copresentation of sporadic LAM and sporadic AML may be obscured in TSC patients lacking additional features. In this report, we present a case study on a single patient initially reported to have sporadic LAM and a mucinous duodenal adenocarcinoma deficient in DNA mismatch repair proteins. Moreover, the patient had a history of Wilms’ tumor, which was reclassified as AML following the LAM diagnosis. Therefore, we investigated the origins and relatedness of these tumors. Using germline whole-genome sequencing, we identified a premature truncation in one of the patient's TSC2 alleles. Using immunohistochemistry, loss of tuberin expression was observed in AML and LAM tissue. However, no evidence of a somatic loss of heterozygosity or DNA methylation epimutations was observed at the TSC2 locus, suggesting alternate mechanisms may contribute to loss of the tumor suppressor protein. In the mucinous duodenal adenocarcinoma, no causative mutations were found in the DNA mismatch repair genes MLH1, MSH2, MSH6, or PMS2. Rather, clonal deconvolution analyses were used to identify mutations contributing to pathogenesis. This report highlights both the utility of using multiple sequencing techniques and the complexity of interpreting the data in a clinical context.

Induction of mismatch repair deficiency, compromised DNA damage signaling and compound hypermutagenesis by a dietary mutagen in a cell-based model for Lynch syndrome.

The prevalent cancer predisposition Lynch syndrome (LS, OMIM #120435) is caused by an inherited heterozygous defect in any of the four core DNA mismatch repair (MMR) genes MSH2, MSH6, MLH1 or PMS2. MMR repairs errors by the replicative DNA polymerases in all proliferating tissues. Its deficiency, following somatic loss of the wild-type copy, results in a spontaneous mutator phenotype that underlies the rapid development of, predominantly, colorectal cancer (CRC) in LS. Here, we have addressed the hypothesis that aberrant responses of intestinal stem cells to diet-derived mutagens may be causally involved in the restricted cancer tropism of LS. To test this we have generated a panel of isogenic mouse embryonic stem (mES) cells with heterozygous or homozygous disruption of multiple MMR genes and investigated their responses to the common dietary mutagen and carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Our data reveal that PhIP can inactivate the wild-type allele of heterozygous mES cells via the induction of either loss of heterozygosity (LOH) or intragenic mutations. Moreover, while protective DNA damage signaling (DDS) is compromised, PhIP induces more mutations in Msh2, Mlh1, Msh6 or Pms2-deficient mES cells than in wild-type cells. Combined with their spontaneous mutator phenotypes, this results in a compound hypermutator phenotype. Together, these results indicate that dietary mutagens may promote CRC development in LS at multiple levels, providing a rationale for dietary modifications in the management of LS.

Classification of MSH6 Variants of Uncertain Significance Using Functional Assays.

Lynch syndrome (LS) is one of the most common hereditary cancer predisposition syndromes worldwide. Individuals with LS have a high risk of developing colorectal or endometrial cancer, as well as several other cancers. LS is caused by autosomal dominant pathogenic variants in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, PMS2 or MSH6, and typically include truncating variants, such as frameshift, nonsense or splicing variants. However, a significant number of missense, intronic, or silent variants, or small in-frame insertions/deletions, are detected during genetic screening of the MMR genes. The clinical effects of these variants are often more difficult to predict, and a large fraction of these variants are classified as variants of uncertain significance (VUS). It is pivotal for the clinical management of LS patients to have a clear genetic diagnosis, since patients benefit widely from screening, preventive and personal therapeutic measures. Moreover, in families where a pathogenic variant is identified, testing can be offered to family members, where non-carriers can be spared frequent surveillance, while carriers can be included in cancer surveillance programs. It is therefore important to reclassify VUSs, and, in this regard, functional assays can provide insight into the effect of a variant on the protein or mRNA level. Here, we briefly describe the disorders that are related to MMR deficiency, as well as the structure and function of MSH6. Moreover, we review the functional assays that are used to examine VUS identified in MSH6 and discuss the results obtained in relation to the ACMG/AMP PS3/BS3 criterion. We also provide a compiled list of the MSH6 variants examined by these assays. Finally, we provide a future perspective on high-throughput functional analyses with specific emphasis on the MMR genes.

Pilomatricomas and café au lait macules as herald signs of constitutional mismatch repair deficiency (CMMRD) syndrome—A case report

Constitutional mismatch repair deficiency (CMMRD) syndrome results from bi-allelic mutations in DNA mismatch repair genes-MLH1, MSH2, MSH6, or PMS2. We present two siblings with CMMRD having p.Arg802Ter (c.2404C>T) homozygous mutations in PMS2 exon 14 with typical cutaneous features. This case report highlights the role of the dermatologist in early diagnosis of this condition.

Ectopic Expression of Hematopoietic SHIP1 in Human Colorectal Cancer.

Colorectal cancer (CRC) is a heterogeneous disease that results from the accumulation of mutations in colonic mucosa cells. A subclass of CRC is characterized by microsatellite instability, which is thought to occur mainly through inactivation of the DNA mismatch repair genes MLH1 and MSH2. The inositol 5-phosphatase SHIP1 is expressed predominantly in hematopoietic cells. In this study, the expression of SHIP1 in carcinomas and its putative correlation with clinicopathologic parameters, expression of DNA repair genes and microsatellite instability was investigated. By analyzing a multi-tumor tissue microarray, expression of SHIP1 was detected in 48 out of 72 cancer entities analyzed. The expression of SHIP1 protein of 145 kDa was confirmed by Western blot analysis in 7 out of 14 carcinoma cell lines. Analysis of a large colorectal cancer tissue microarray with 1009 specimens revealed SHIP1 expression in 62% of the samples analyzed. SHIP1 expression was inversely correlated with lymph node metastasis, vascular invasion and tumor grade, and it was positively associated with left-sided tumor localization. Interestingly, a strong relationship between the expression of SHIP1 and nuclear and membranous beta-catenin and the DNA repair genes MLH1 and MSH2 was observed.

Molecular pathology of Lynch syndrome

Lynch syndrome (LS) is characterised by predisposition to colorectal, endometrial, and other cancers and is caused by inherited pathogenic variants affecting the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2. It is probably the most common predisposition to cancer, having an estimated prevalence of between 1/100 and 1/180. Resources such as the International Society for Gastrointestinal Hereditary Cancer's MMR gene variant database, the Prospective Lynch Syndrome Database (PLSD), and the Colon Cancer Family Register (CCFR), as well as pathological and immunological studies, are enabling advances in the understanding of LS. These include defined criteria by which to interpret gene variants, the function of MMR in the normal control of apoptosis, definition of the risks of the various cancers, and the mechanisms and pathways by which the colorectal and endometrial tumours develop, including the critical role of the immune system. Colorectal cancers in LS can develop along three pathways, including flat intramucosal lesions, which depend on the underlying affected MMR gene. This gives insights into the limitations of colonoscopic surveillance and highlights the need for other forms of anti-cancer prophylaxis in LS. Finally, it shows that the processes of autoimmunisation and immunoediting fundamentally constrain the development of tumours in LS and explain the efficacy of immune checkpoint blockade therapy in MMR-deficient tumours. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The most common RNF43 mutant G659Vfs*41 is fully functional in inhibiting Wnt signaling and unlikely to play a role in tumorigenesis

RNF43 is an E3 ligase that inhibits Wnt signaling by ubiquitinating Wnt receptors for degradation. It is mutated in various cancer types with the most recurrent mutation being the frameshift G659Vfs*41 with frequencies of ~5–8% in colon, stomach and endometrial cancers. This mutation, a deletion of G in a 7-G repeat, has been assumed to encode an inactive enzyme that would lead to increased Wnt signaling and drive tumorigenesis, yet no functional characterization has been reported. We analyzed the distribution of G659Vfs*41 and its association with other cancer gene mutations, and found that the mutation occurred nearly exclusively in tumors with low expression of the DNA mismatch repair gene MLH1. Mutant RNF43-G659Vfs*41 was no different from wild type RNF43 in expression, stability, localization, R-spondin binding, and inhibition of Wnt signaling. No dominant negative activity of the mutant was observed. Colon tumors with RNF43-G659Vfs*41 had low Wnt/β-catenin signaling and were frequently mutated in BRAF. A colon cancer cell line with RNF43-G659Vfs*41 and BRAF-V600E mutations was sensitive to activation of Wnt/β-catenin signaling. These findings suggest that the frequent occurrence of RNF43-G659Vfs*41 may result from error-prone replication of the 7-G repeat in MLH1-deficient tumors and that the mutation itself does not inactivate enzyme.

1651 Colon, Urothelial and Sebaceous Malignancy All in One: A Rare Presentation of Muir-Torre Syndrome

INTRODUCTION: Muir-Torre syndrome (MTS) is a rare autosomal-dominant condition and phenotypic variant of Lynch syndrome caused by mutations in DNA mismatch repair genes MSH2, MSH6, and MLH1. Clinical diagnostic criteria include the presence of at least one sebaceous gland neoplasm and one visceral malignancy, often colonic or genitourinary. Compared to Lynch syndrome, the prevalence of MTS is likely underreported. We present a unique case of a patient with incidental findings of multiple visceral malignancies associated with skin cancer. CASE DESCRIPTION/METHODS: A 57-year-old Caucasian man presented for surveillance colonoscopy. His family history was significant for father with colon cancer at age 50, lung, and renal cancer. The patient was diagnosed with sebaceous adenocarcinoma, invasive squamous cell, and basal cell carcinomas requiring excisional surgery at age 46. Screening colonoscopy at that time was normal, with repeat after 7 years noting diminutive sigmoid tubular adenomas that were resected. Current colonoscopy revealed 14 colon polyps, multiple with high grade dysplasia, and an invasive sigmoid adenocarcinoma. Staging CT A/P was negative for metastatic disease. CEA was normal. The patient opted for left hemicolectomy instead of total proctocolectomy. Tumor pathology revealed moderately differentiated adenocarcinoma with negative margins (pT4N0M0). Colon cancer genetic testing showed lack of expression of MSH2/MSH6 and germline MSH2 mutation, confirming the diagnosis of MTS. A month later he developed hematuria and acute kidney injury that culminated in a new diagnosis of invasive high-grade urothelial carcinoma. Neoadjuvant chemotherapy was started with plan for cystectomy. DISCUSSION: MTS is a rare condition that warrants early recognition, regular follow-up and observation for new malignancies. Our case highlights the importance of awareness and the need for early, universal screening in high-risk individuals. MTS patients should undergo screening colonoscopy at ages 20–25 with surveillance every year thereafter to offer mortality benefit. If colon cancer is diagnosed, total rather than partial colectomy is preferred due to a high metachronous cancer rate of 25%. Patients and relatives should be counseled on increased risk of future malignancies. Reconstruction of family history may allow for the identification and selection of patients in whom to proceed with clinical, molecular, and immunohistochemical analysis. Clinicians must therefore be cognizant of this disease and its enduring effects.

Yield of Lynch Syndrome Surveillance for Patients With Pathogenic Variants in DNA Mismatch Repair Genes

Patients with Lynch syndrome are offered the same colorectal cancer (CRC) surveillance programs (colonoscopy every 2 years), regardless of the pathogenic DNA mismatch repair gene variant the patient carries. We aimed to assess the yield of surveillance for patients with these variants in MLH1, MSH2, MSH6, and PMS2. We analyzed data on colonoscopy surveillance, including histopathology analysis, from all patients diagnosed with Lynch syndrome (n = 264) at a single center. We compared the development of (advanced) adenomas and CRC among patients with pathogenic variants in the DNA mismatch repair genes MLH1 (n = 55), MSH2 (n = 44), MSH6 (n = 143), or PMS2 (n = 22) over 1836 years of follow-up (median follow-up of 6 years per patient). At first colonoscopy, CRC was found in 8 patients. During 916 follow-up colonoscopies, CRC was found in 9 patients. No CRC was found in patients with variants in MSH6 or PMS2 over the entire follow-up period. There were no significant differences in the number of colonoscopies with adenomas or advanced adenomas among the groups. The median time of adenoma development was 3 years (IQR, 2-6 years). There were no significant differences in time to development of adenoma. However, patients with variants in MSH6 had a significant longer time to development of advanced neoplasia (advanced adenoma or CRC) than patients in the other groups. Six carriers died during follow up (5 from cancer, of which 3 from pancreatic cancer). No CRC was found during follow-up of patients with Lynch syndrome carrying pathogenic variants in MSH6; advanced neoplasia developed over shorter follow-up time periods in patients with pathogenic variants in MLH1 or MSH2. The colonoscopy interval for patients with pathogenic variants in MSH6 might be increased to 3 years from the regular 2-year interval.

Mutational landscape of T-cell lymphoma in mice lacking the DNA mismatch repair gene Mlh1: no synergism with ionizing radiation.

Biallelic germline mutations in the DNA mismatch repair gene MLH1 lead to constitutional mismatch repair-deficiency syndrome and an increased risk for childhood hematopoietic malignancies, including lymphoma and leukemia. To examine how Mlh1 dysfunction promotes lymphoma as well as the influence of ionizing radiation (IR) exposure, we used an Mlh1-/- mouse model and whole-exome sequencing to assess genomic alterations in 23 T-cell lymphomas, including 8 spontaneous and 15 IR-associated lymphomas. Exposure to IR accelerated T-cell lymphoma induction in the Mlh1-/- mice, and whole-exome sequencing revealed that IR exposure neither increased the number of mutations nor altered the mutation spectrum of the lymphomas. Frequent mutations were evident in genes encoding transcription factors (e.g. Ikzf1, Trp53, Bcl11b), epigenetic regulators (e.g. Suv420h1, Ep300, Kmt2d), transporters (e.g. Rangap1, Kcnj16), extracellular matrix (e.g. Megf6, Lrig1), cell motility (e.g. Argef19, Dnah17), protein kinase cascade (e.g. Ptpro, Marcks) and in genes involved in NOTCH (e.g. Notch1), and PI3K/AKT (e.g. Pten, Akt2) signaling pathways in both spontaneous and IR-associated lymphomas. Frameshift mutations in mononucleotide repeat sequences within the genes Trp53, Ep300, Kmt2d, Notch1, Pten and Marcks were newly identified in the lymphomas. The lymphomas also exhibited a few chromosomal abnormalities. The results establish a landscape of genomic alterations in spontaneous and IR-associated lymphomas that occur in the context of mismatch repair dysfunction and suggest potential targets for cancer treatment.

Cancer Risks for PMS2-Associated Lynch Syndrome.

Lynch syndrome due to pathogenic variants in the DNA mismatch repair genes MLH1, MSH2, and MSH6 is predominantly associated with colorectal and endometrial cancer, although extracolonic cancers have been described within the Lynch tumor spectrum. However, the age-specific cumulative risk (penetrance) of these cancers is still poorly defined for PMS2-associated Lynch syndrome. Using a large data set from a worldwide collaboration, our aim was to determine accurate penetrance measures of cancers for carriers of heterozygous pathogenic PMS2 variants. A modified segregation analysis was conducted that incorporated both genotyped and nongenotyped relatives, with conditioning for ascertainment to estimates corrected for bias. Hazard ratios (HRs) and corresponding 95% CIs were estimated for each cancer site for mutation carriers compared with the general population, followed by estimation of penetrance. In total, 284 families consisting of 4,878 first- and second-degree family members were included in the analysis. PMS2 mutation carriers were at increased risk for colorectal cancer (cumulative risk to age 80 years of 13% [95% CI, 7.9% to 22%] for males and 12% [95% CI, 6.7% to 21%] for females) and endometrial cancer (13% [95% CI, 7.0%-24%]), compared with the general population (6.6%, 4.7%, and 2.4%, respectively). There was no clear evidence of an increased risk of ovarian, gastric, hepatobiliary, bladder, renal, brain, breast, prostate, or small bowel cancer. Heterozygous PMS2 mutation carriers were at small increased risk for colorectal and endometrial cancer but not for any other Lynch syndrome-associated cancer. This finding justifies that PMS2-specific screening protocols could be restricted to colonoscopies. The role of risk-reducing hysterectomy and bilateral salpingo-oophorectomy for PMS2 mutation carriers needs further discussion.

Abstract 5369: Tumorigenesis in Lynch syndrome: Somatic mutation profiles compared to sporadic counterparts

Abstract Lynch syndrome (LS) is the most prevalent cancer predisposition syndrome in which germline mutation in one of four DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 causes significantly increased lifetime risks of colorectal, endometrial, ovarian, and other cancers. Whether or not breast cancer is part of LS tumor spectrum is currently debated. Moreover, the reasons for organ selectivity in germline mutation carriers are unknown. We have reported that breast carcinoma from LS individuals resembles common breast carcinoma in many respects, but differs with respect to genomic instability (Lotsari et al. 2012, Cancer Res.). To extend these preliminary observations, we are now carrying out an in-depth investigation into the spectra of somatic mutations in LS and sporadic breast carcinomas. All available breast carcinomas among 300 LS families from the nation-wide Hereditary Colon Cancer Registry of Finland have been identified and the tumors investigated for MMR status, DNA methylator phenotype, mechanisms of two-allele inactivation for MMR genes, and somatic mutation status. For the latter, we use massive parallel sequencing as described in our recent publication (Porkka et al. 2017, Oncotarget). To define the unique vs. shared molecular features of LS breast tumorigenesis, profiles of somatic alterations in LS breast carcinomas are compared to those of established LS spectrum carcinomas (colorectal and ovarian) as well as sporadic breast carcinomas with sequence information available from large electronic data-sets. Of 14 LS breast carcinoma samples, 9 (64 %) were MMR-deficient and 5 (36 %) MMR-proficient. This contrasts with LS colorectal and ovarian carcinomas, all of which were MMR-deficient (p=0.0012). MMR-deficient LS breast carcinomas harbored an average of 783 non-synonymous mutations and MMR-proficient tumors 555 mutations (statistically non-significant). In contrast to colorectal and ovarian carcinomas, in which loss of heterozygosity (LOH) or somatic MMR gene mutation provided a second hit consistent with two-hit inactivation in 89 % (25/28) of the tumors, a detectable second hit (mainly LOH) was present in only 43 % (6/14) of breast carcinomas (p=0.0025). Genes harboring high-frequency mutations in at least one-third of tumors were regarded candidates for driver genes. There were 133 such genes in LS colorectal carcinomas, 10 in ovarian carcinomas, and 18 in breast carcinomas. Genes involved in epigenetic regulation were significantly enriched in all three tumor types. In addition, a significant enrichment of NOTCH signaling associated genes characterized LS breast carcinomas. This research is likely to shed light to the mechanisms of organ-specific cancer susceptibility in germline carriers of MMR gene mutations. Our results are expected to guide the surveillance and other clinical management of LS individuals with breast carcinoma. Citation Format: Noora Porkka, Alisa Olkinuora, Satu Mäki-Nevala, Samuli Eldfors, Päivi Peltomäki. Tumorigenesis in Lynch syndrome: Somatic mutation profiles compared to sporadic counterparts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5369.

PO-005 Prevention strategies for intestinal cancer development in lynch syndrome

IntroductionLynch syndrome (LS) is an autosomal dominant hereditary cancer predisposition syndrome which is mostly caused by defects in DNA mismatch repair (MMR) genes MLH1 and MSH2. LS patients have an increased lifetime risk of developing a range of neoplasms including a non-polyposis form of colorectal cancer (HNPCC) which typically manifests at a young age. In this project, we focus on distinct factors that may influence the pathogenesis of CRC in LS.Material and methodsThe mouse models that have been used in the past to study LS do not correspond well to the human pathology. Therefore, our research group has recently developed an inducible mouse model that closely mimics the clinical situation in MSH2-deficient LS patients.Wojciechowicz et al., 2014 After administration of tamoxifen, Lgr5-CreERT2;Msh2flox/- (Msh2-Lynch) mice have been shown to carry about 5% MSH2-deficient intestinal crypts that remained present over time. Since the cells of these crypts lack functional MMR, they have a high mutational burden and may develop into intestinal tumours.Results and discussionsExposure of Msh2-Lynch mice to the methylating agent temozolomide (TMZ) led to expansion of the MSH2-deficient cell compartment and accelerated tumour development.Wojciechowicz et al., 2014 All intestinal tumours were shown to be MSH2 deficient and especially the tumours from TMZ treated mice showed a high mutational burden. Strikingly, a change in the housing conditions (conventional housing to SPF) almost fully ablated intestinal tumour development in our mouse model. We hypothesise that a change in the microbiome underlies this phenotype. Therefore, we are currently dissecting the role of the microbiome, the immune system and inflammation in intestinal tumour development in Msh2-Lynch mice.ConclusionThe strong effect of housing conditions on tumour development in Msh2-Lynch mice may help us to divulge distinct factors that modulate cancer risk in LS. The results of this study may ultimately contribute to a reduction of tumour incidence among LS patients and improve treatment options.

Haplotype analysis suggest that the MLH1 c.2059C\u2009>\u2009T mutation is a Swedish founder mutation

Lynch syndrome (LS) predisposes to a spectrum of cancers and increases the lifetime risk of developing colorectal- or endometrial cancer to over 50%. Lynch syndrome is dominantly inherited and is caused by defects in DNA mismatch-repair genes MLH1, MSH2, MSH6 or PMS2, with the vast majority detected in MLH1 and MSH2. Recurrent LS-associated variants observed in apparently unrelated individuals, have either arisen de novo in different families due to mutation hotspots, or are inherited from a founder (a common ancestor) that lived several generations back. There are variants that recur in some populations while also acting as founders in other ethnic groups. Testing for founder mutations can facilitate molecular diagnosis of Lynch Syndrome more efficiently and more cost effective than screening for all possible mutations. Here we report a study of the missense mutation MLH1 c.2059C > T (p.Arg687Trp), a potential founder mutation identified in eight Swedish families and one Finnish family with Swedish ancestors. Haplotype analysis confirmed that the Finnish and Swedish families shared a haplotype of between 0.9 and 2.8 Mb. While MLH1 c.2059C > T exists worldwide, the Swedish haplotype was not found among mutation carriers from Germany or France, which indicates a common founder in the Swedish population. The geographic distribution of MLH1 c.2059C > T in Sweden suggests a single, ancient mutational event in the northern part of Sweden.

Genetic anticipation in Swedish Lynch syndrome families.

Among hereditary colorectal cancer predisposing syndromes, Lynch syndrome (LS) caused by mutations in DNA mismatch repair genes MLH1, MSH2, MSH6 or PMS2 is the most common. Patients with LS have an increased risk of early onset colon and endometrial cancer, but also other tumors that generally have an earlier onset compared to the general population. However, age at first primary cancer varies within families and genetic anticipation, i.e. decreasing age at onset in successive generations, has been suggested in LS. Anticipation is a well-known phenomenon in e.g neurodegenerative diseases and several reports have studied anticipation in heritable cancer. The purpose of this study is to determine whether anticipation can be shown in a nationwide cohort of Swedish LS families referred to the regional departments of clinical genetics in Lund, Stockholm, Linköping, Uppsala and Umeå between the years 1990–2013. We analyzed a homogenous group of mutation carriers, utilizing information from both affected and non-affected family members. In total, 239 families with a mismatch repair gene mutation (96 MLH1 families, 90 MSH2 families including one family with an EPCAM–MSH2 deletion, 39 MSH6 families, 12 PMS2 families, and 2 MLH1+PMS2 families) comprising 1028 at-risk carriers were identified among the Swedish LS families, of which 1003 mutation carriers had available follow-up information and could be included in the study. Using a normal random effects model (NREM) we estimate a 2.1 year decrease in age of diagnosis per generation. An alternative analysis using a mixed-effects Cox proportional hazards model (COX-R) estimates a hazard ratio of exp(0.171), or about 1.19, for age of diagnosis between consecutive generations. LS-associated gene-specific anticipation effects are evident for MSH2 (2.6 years/generation for NREM and hazard ratio of 1.33 for COX-R) and PMS2 (7.3 years/generation and hazard ratio of 1.86). The estimated anticipation effects for MLH1 and MSH6 are smaller.

Universal screening for Lynch syndrome in endometrial cancers: frequency of germline mutations and identification of patients with Lynch-like syndrome

Lynch syndrome (LS) is an inherited clinical syndrome characterized by a high risk of colorectal, endometrial (lifetime risk of up to 60%), ovarian, and urinary tract cancers. The diagnosis is confirmed by identification of germline mutations in the DNA mismatch repair genes MLH1, PMS2, MSH2, MSH6, or EPCAM. In 2015, our institution implemented universal screening of endometrial cancer (EC) hysterectomy specimens by mismatch repair immunohistochemistry (IHC) with reflex MLH1 promoter hypermethylation analysis for tumors with loss of MLH1/PMS2 expression. Patients with tumors negative for MLH1 methylation and those with a loss of the heterodimer pair MSH2 and MSH6, or isolated loss of either PMS2 or MSH6 were referred to the Familial Cancer Program for genetic counseling and consideration of germline testing. Between May 2015 to Dec 2016, 233 EC patients were screened by IHC for LS with a median age of 63 years. Sixty tumors (27%) had abnormal IHC staining results. Fifty-one (22%) harbored heterodimeric loss of MLH1 and PMS2, 49 of which showed MLH1 promoter methylation (1 failure, 1 negative). One showed loss of MLH1/PMS2 and MSH6, 2 showed loss of MSH2/MSH6, and 6 had isolated loss of MSH6 only. Ten patients underwent genetic counseling, and germline testing was performed in 8; LS was confirmed in 5 patients (2.1%). In addition, 3 patients with negative germline testing and presumed Lynch-like syndrome were identified and offered additional somatic testing. Universal screening for LS in EC patients has yielded positive results for identification of patients at risk for this inherited syndrome.

Association between promoter methylation of MLH1 and MSH2 and reactive oxygen species in oligozoospermic men-A pilot study.

MLH1 and MSH2 are important genes for DNA mismatch repair and crossing over during meiosis and are implicated in male infertility. Therefore, the methylation patterns of the DNA mismatch repair genes MLH1 and MSH2 in oligozoospermic males were investigated. Ten oligozoospermic patients and 29 normozoospermic donors were analysed. Methylation profiles of the MLH1 and MSH2 promotors were analysed. In addition, sperm motility and seminal reactive oxygen species (ROS) were recorded. Receiver operating characteristic (ROC) analysis was conducted to determine the accuracy of the DNA methylation status of MLH1 and MSH2 to distinguish between oligozoospermic and normozoospermic men. In oligozoospermic men, MLH1 was significantly (p=.0013) more methylated compared to normozoospermic men. Additionally, there was a significant positive association (r=.384; p=.0159) between seminal ROS levels and MLH1 methylation. Contrary, no association between MSH2 methylation and oligozoospermia was found. ROC curve analysis for methylation status of MLH1 was significant (p=.0275) with an area under the curve of 61.1%, a sensitivity of 22.2% and a specificity of 100.0%. This pilot study indicates oligozoospermic patients have more methylation of MLH1 than normozoospermic patients. Whether hypermethylation of the MLH1 promoter plays a role in repairing relevant mismatches of sperm DNA strands in idiopathic oligozoospermia warrants further investigation.

Abstract 3806: Integrative omics analysis in gastric cancer reveals SLC7A5 functional related to DNA mismatch repair system as a potential therapeutic biomarker in GC

Abstract Background: Metastasis is a major cause leading to high mortality rate and poor prognosis in Gastric Cancer (GC) patients. Our previous studies showed that SLC7A5 is an amino acid transporter gene meditated by a pivotal post-transcriptional tumor suppressor mir-126 and associated with tumor metastasis in GC. Purpose: (a) to further determine the SLC7A5 functional mechanisms in GC by involving more bio-omics data such as DNA methylation, copy number variation (CNV) and mRNA expression; b) to study the association of SLC7A5 bio-omics data with the clinicopathologic features, prognosis and subtypes in GC patients. Methods: We intensively performed the genome-wide association study for bio-omics data of GC patients available from The Cancer Genome Atlas (TCGA, n=421) and Gene Expression Omnibus published studies (GEO, GSE13911, n=69, GSE15459, n=209). We did Kaplan-Meier survival analysis and univariate Cox regression model analyses in SLC7A5 mRNA expression from curated database (http://www.kmplot.com) with 882 GC patients for overall survival and 646 GC patients for progression free survival analysis. Results: CNV of SLC7A5 is positively correlated with mRNA expression (p=3.18e-07), which suggests that SLC7A5 is potentially a target driver gene for GC. Further integrative analysis identified two important methylation probes in promotor region (TSS200 position) negatively correlated with mRNA expression of SLC7A5 (p=1.03e-3). In addition, the aberrant high methylation and expression of SLC7A5, which were significantly higher than the corresponding nonmalignant tissues, were identified in a subset of gastric cancer patients. Interestingly, SLC7A5 showed statistically significant correlation with the mismatch repair-deficient phenotype in sporadic microsatellite instability gastric cancer (p=2.12e-5). Data mining from two independent datasets of GEO validated SLC7A5 is positively correlated with DNA mismatch repair genes MLH1, BRCA1 and BRCA2. And further Gene Ontology (GO) pathway analysis suggested that SLC7A5 and DNA mismatch repair genes (MMR) together shared the same metabolic processes. Importantly, the overall survival from 882 GC patients and progression free survival analysis from 646 GC patients showed SLC7A5 high expression is associated with poor prognosis tumorigenesis, which demonstrated that SLC7A5 is a potential independent biomarker for GC. Conclusion: We found SLC7A5 has positive interaction with DNA mismatch genes through database mining, which result in microsatellite instability phenotype in GC patients. We also found SLC7A5 over expression is associated with poor prognosis and potentially an pivotal therapeutic target in GC. Note: This abstract was not presented at the meeting. Citation Format: Yunyun Zhou, Xuehua Chen, Zhenggang Zhu, Junqing Wang. Integrative omics analysis in gastric cancer reveals SLC7A5 functional related to DNA mismatch repair system as a potential therapeutic biomarker in GC [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 3806. doi:10.1158/1538-7445.AM2017-3806

Abstract 4870: Mutational landscape and timing of resistant clone emergence in 104 Chinese pediatric patients with relapsed acute lymphoblastic leukemia

Abstract Relapsed pediatric acute lympoblastic leukemia (ALL), occurring in 15-20% of cases, is one of the leading causes of cancer-related death in children, conferring a dismal survival rate of less than 40% primarily due to drug resistance. We performed whole-genome sequencing (WGS) and RNA-seq of matched diagnosis and relapse tumors as well as WGS of remission DNA from 104 pediatric ALL patients treated at Shanghai Children’s Medical Center (n=89), Institute of Hematology & Blood Disease Hospital in Tianjin (n=14) and the Second Hospital of Anhui Medical University (n=1) between 2003 and 2014. The median age at diagnosis was 6.29 (range, 0.23-16.05) years. Relapse occurred during chemotherapy in 85 patients and after completion of therapy in 19 patients. Somatic mutation rate in ALL increased significantly from an average of 0.23 per Mb at diagnosis to 0.65 per Mb at relapse. We identified 34 potential driver genes at diagnosis and 45 at relapse based on significance of mutation recurrence and pathogenicity. The four most significant relapse-specific driver genes harbor known point mutations on purine metabolism genes NT5C2 (n=13) and PRPS1/PRPS2 (n=7), novel alterations affecting glucocorticoids receptor NR3C1 (n=13) and folylpolyglutamate synthetase FPGS (n=8). Functional characterization showed that all NR3C1 mutations resulted in significant loss of transcription activator function and subcellular mislocalization. Ectopic expression of mutant NR3C1 in ALL cell lines consistently led to prednisone resistance. Five driver genes (TP53, CREBBP, SMARCA4, WHSC1 and NIPBL) showed the highest increase (2-5 fold) of mutation frequency from diagnosis to relapse with mutations collectively present in 28% of relapsed ALL. Truncation mutations in DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 occurred exclusively at relapse in four ALL with the highest mutation burden. Targeted ultra-deep sequencing at 5,000X of 268 somatic alterations was performed on 214 serial bone-marrow samples collected during ALL therapy for 17 cases. The resulting mutant allele fraction (MAF) values showed high concordance with minimum residual disease (MRD) measured by flow cytometry, indicating suitability for quantifying leukemia burden. Temporal change of MAF in the serial samples also allows us to track clonal identity and emergence of drug-resistant clones during and after therapy. For example, one case had deep sequencing performed on 11 somatic lesions from 10 bone barrow samples collected during the 15 months between diagnosis and relapse. We found that a minor clone present in 0.5-1% of ALL blasts at diagnosis emerged five months prior to relapse, from which two new subclones arose harboring NT5C2 mutations L406>SSF and D407>DRD. Preliminary data generated from serial bone marrow samples provides the first insight into the use of somatic alterations for monitoring leukemia progress during relapse. Citation Format: Benshang Li, Yongjin Li, Shuhong Shen, Xiaofan Zhu, Xiaotu Ma, Ningling Wang, Yanling Liu, Yu Liu, Hui Zhang, Ting-Nien Lin, Michael Rusch, Michael Edmonson, John Easton, Yingchi Zhang, Jingliao Zhang, Cheng Cheng, Jingyan Tang, James R. Downing, Ching-Hon Pui, Jun J. Yang, Jinghui Zhang. Mutational landscape and timing of resistant clone emergence in 104 Chinese pediatric patients with relapsed acute lymphoblastic leukemia [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 4870. doi:10.1158/1538-7445.AM2017-4870