DNA Mismatch Repair Genes MLH1 Research Articles

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

Loss of MSH2 and MSH6 due to heterozygous germline defects in MSH3 and MSH6.

Lynch Syndrome (LS) is the most common dominantly inherited colorectal cancer (CRC) predisposition and is caused by a heterozygous germline defect in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2. High microsatellite instability (MSI-H) and loss of MMR protein expression in tumours reflecting a defective MMR are indicators for LS, as well as a positive family history of early onset CRC. MSH2 and MSH6 form a major functional heterodimer, and MSH3 is an alternative binding partner for MSH2. So far, the role of germline MSH3 variants remains unclear, as to our knowledge heterozygous truncating variants are not regarded causative for LS, but were detected in patients with CRC, and recently biallelic MSH3 defects have been identified in two patients with adenomatous polyposis. By gene screening we investigated the role of MSH3 in 11 LS patients with truncating MSH6 germline variants and an unexplained MSH2 protein loss in their corresponding MSI-H tumours. We report the first two LS patients harbouring heterozygous germline variants c.1035del and c.2732T>G in MSH3 coincidentally with truncating variants in MSH6. In the patient with truncating germline variants in MSH3 and MSH6, two additional somatic second hits in both genes abrogate all binding partners for the MSH2 protein which might subsequently be degraded. The clinical relevance of MSH3 germline variants is currently under re-evaluation, and heterozygous MSH3 defects alone do not seem to induce a LS phenotype, but might aggravate the MSH6 phenotype in affected family members.

Platinum-Based Chemotherapy Induces Methylation Changes in Blood DNA Associated with Overall Survival in Patients with Ovarian Cancer.

Purpose: DNA damage repair can lead to epigenetic changes. DNA mismatch repair proteins bind to platinum DNA adducts and at sites of DNA damage can recruit the DNA methylating enzyme DNMT1, resulting in aberrant methylation. We hypothesised that DNA damage repair during platinum-based chemotherapy may cause aberrant DNA methylation in normal tissues of patients such as blood.Experimental Design: We used Illumina 450k methylation arrays and bisulphite pyrosequencing to investigate methylation at presentation and relapse in blood DNA from patients with ovarian cancer enrolled in the SCOTROC1 trial (n = 247) and in a cohort of ovarian tumor DNA samples collected at first relapse (n = 46). We used an ovarian cancer cell line model to investigate the role of the DNA mismatch repair gene MLH1 in platinum-induced methylation changes.Results: Specific CpG methylation changes in blood at relapse are observed following platinum-based chemotherapy and are associated with patient survival, independent of other clinical factors [hazard ratio, 3.7; 95% confidence interval, 1.8-7.6, P = 2.8 × 10-4]. Similar changes occur in ovarian tumors at relapse, also associated with patient survival (hazard ratio, 2.6; 95% confidence interval, 1.0-6.8, P = 0.048). Using an ovarian cancer cell line model, we demonstrate that functional mismatch repair increases the frequency of platinum-induced methylation.Conclusions: DNA methylation in blood at relapse following chemotherapy, and not at presentation, is informative regarding survival of patients with ovarian cancer. Functional DNA mismatch repair increases the frequency of DNA methylation changes induced by platinum. DNA methylation in blood following chemotherapy could provide a noninvasive means of monitoring patients' epigenetic responses to treatment without requiring a tumor biopsy. Clin Cancer Res; 23(9); 2213-22. ©2016 AACR.

Abstract 127: The genomic landscapes of inflammatory bowel disease-associated colorectal cancers

Abstract Long duration inflammatory bowel disease (IBD) increases the risk for colorectal cancer (CRC). Mutation analysis of limited numbers of genes has suggested that CRCs arising in an IBD background differ from those that do not. We sought to characterize the genetic landscape of these tumors through whole-exome sequencing. Thirty-one IBD patients with CRC were identified, including 15 with ulcerative colitis, 14 with Crohn's disease and 2 with indeterminate colitis. Whole-exome sequencing was performed on micro-dissected tumor and matched non-neoplastic formalin-fixed paraffin-embedded (FFPE) tissues. Somatic alterations were identified by comparing matched specimens. Mutation prevalence in sporadic CRC was obtained from previously published exome-sequencing studies. An average of 11.6 Gb were sequenced per sample. The depth of quality coverage of the targeted region was 29 to 100-fold in the tumors; and 17- to 123-fold in non-tumor samples. Two specimens harbored somatic mutations in the DNA proofreading or mismatch repair genes POLE, MLH1 and MSH6 and exhibited hypermutable phenotype. The remaining cases had a median mutation rate of 1.33 mutations/Mb and an average of 71 alterations per sample. The pattern of base changes in non-hypermutated tumors showed a preponderance of C to T transitions (62%), a majority of which (48% of all mutations) occurred at 5′-CpG-3′ sites. An excess of A to C transversions at AA dinucleotides, particularly in a context of AAG trinucleotides, was also noted. This pattern has been previously observed in esophageal and gastric cancers, both of which are also tied to chronic inflammation. There were no apparent differences in the spectrum of substitutions between tumors arising in UC and CD patients. We identified 28 genes recurrently mutated in three or more non-hypermutated samples. TP53 was the most commonly mutated gene, with incidence prevalence similar to sporadic CRC (63% of cases), but with different mutation spectrum. APC and KRAS were mutated at significantly lower rates than in sporadic CRCs (13% and 20% of cases, respectively). Genes mutated more commonly or uniquely in IBD-CRC, included SOX9 and EP300, encoding proteins in the WNT pathway, NRG1, encoding an ERBB ligand, and IL16, encoding a cytokine. Three cases featured hotspot PIK3CA mutations. Likely oncogenic driver gene mutations in BRAF, CTNNB1, and CREBBP were found in one case each. Mutated genes were enriched for gene ontologies associated with cell communication, cell-cell signaling, and cell adhesion. Our study also revealed recurrent mutations in components of the Rho/Rac GTPase network, suggesting a role for non-canonical WNT signaling in IBD-CRC. CRCs arising in IBD patients demonstrate a unique molecular profile, which provides clues to their etiology. Our study also sets the stage for improved early detection of IBD-associated CRCs based on their unique genetic compositions as well as for the tailoring of therapies in this patient population. Citation Format: Ana I. Robles, Giovanni Traverso, Ming Zhang, Nicholas Roberts, Mohammed A. Khan, Christine Joseph, Gregory Lauwers, Florin Selaru, Maria Popoli, Xiquan Ke, Ralph H. Hruban, Stephen J. Meltzer, Kenneth W. Kinzler, Bert Vogelstein, Curtis C. Harris, Nickolas Papadopoulos. The genomic landscapes of inflammatory bowel disease-associated colorectal cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 127.

Systematic immunohistochemical screening for Lynch syndrome in colorectal cancer: a single centre experience of 486 patients.

Germline mutations in DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause autosomal dominantly inherited Lynch syndrome. Lynch syndrome patients and their families benefit from life-saving intensive cancer surveillance. Approximately one in 30 colorectal cancers arises in the setting of Lynch syndrome. The aim of this study was to assess the detection rate of Lynch syndrome at our institution after introduction of systematic immunohistochemical screening for MMR deficiency in colorectal cancers from 2011 to 2015. Following the recommendations by the Evaluation of Genomic Applications in Practice and Prevention working group all colorectal cancers were immunohistochemically stained for the presence of MMR proteins MLH1, PMS2, MSH2 and MSH6, independent of clinical criteria. In the case of loss of MLH1, the somatic BRAF mutation V600E was assessed with molecular testing and/or immunohistochemistry. Clinical follow-up of potential Lynch syndrome carriers (patients with tumours showing loss of MLH1 expression with absence of BRAFV600E, loss of PMS2, MSH2 or MSH6) was evaluated. Of all patients (n = 486), loss of MMR protein expression was found in 73 (15.0%) tumours. Twenty-eight (6.0%) were classified as potential Lynch syndrome carriers. Of the genetically tested potential Lynch syndrome carriers (10 out of 28 patients), 40% were first diagnosed with Lynch syndrome. Implementation of systematic immunohistochemistry screening for Lynch syndrome showed that 6% of colorectal cancers were potentially Lynch-syndrome related. Tumour board protocols should systematically contain information on MMR status of all colorectal cancers and, in MMR deficient cases, include clear recommendations for genetic counselling for all potential Lynch syndrome patients.

Novel Mutations in MLH1 and MSH2 Genes in Mexican Patients with Lynch Syndrome.

Background. Lynch Syndrome (LS) is characterized by germline mutations in the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2. This syndrome is inherited in an autosomal dominant pattern and is characterized by early onset colorectal cancer (CRC) and extracolonic tumors. The aim of this study was to identify mutations in MMR genes in three Mexican patients with LS. Methods. Immunohistochemical analysis was performed as a prescreening method to identify absent protein expression. PCR, Denaturing High Performance Liquid Chromatography (dHPLC), and Sanger sequencing complemented the analysis. Results. Two samples showed the absence of nuclear staining for MLH1 and one sample showed loss of nuclear staining for MSH2. The mutations found in MLH1 gene were c.2103+1G>C in intron 18 and compound heterozygous mutants c.1852_1854delAAG (p.K618del) and c.1852_1853delinsGC (p.K618A) in exon 16. In the MSH2 gene, we identified mutation c.638dupT (p.L213fs) in exon 3. Conclusions. This is the first report of mutations in MMR genes in Mexican patients with LS and these appear to be novel.

Acute lymphoblastic leukemia and lymphoma in the context of constitutional mismatch repair deficiency syndrome

Constitutional mismatch repair deficiency (CMMRD) syndrome is one of the rare diseases associated with a high risk of cancer. Causative mutations are found in DNA mismatch repair genes PMS2, MSH6, MSH2 or MLH1 that are well known in the context of Lynch syndrome. CMMRD follows an autosomal recessive inheritance trait and is characterized by childhood brain tumors and hematological malignancies as well as gastrointestinal cancer in the second and third decades of life. There is a high risk of multiple cancers, occurring synchronously and metachronously. In general, the prognosis is poor. About one third of CMMRD patients develop hematological malignancies as primary (sometimes the only) malignancy or as secondary neoplasm. T-cell non-Hodgkin lymphomas, mainly of mediastinal origin, are the most frequent hematological malignancies. Besides malignant diseases, non-neoplastic features are frequently observed, e.g. café-au-lait spots sometimes resembling neurofibromatosis type I, hypopigmented skin lesions, numerous adenomatous polyps, multiple pilomatricomas, or impaired immunoglobulin class switch recombination. Within the present review, we summarize previously published CMMRD patients with at least one hematological malignancy, provide an overview of steps necessary to substantiate the diagnosis of CMMRD, and refer to the recent most relevant literature.

Review: Clinical aspects of hereditary DNA Mismatch repair gene mutations.

Inherited mutations of the DNA Mismatch repair genes MLH1, MSH2, MSH6 and PMS2 can result in two hereditary tumor syndromes: the adult-onset autosomal dominant Lynch syndrome, previously referred to as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and the childhood-onset autosomal recessive Constitutional Mismatch Repair Deficiency syndrome. Both conditions are important to recognize clinically as their identification has direct consequences for clinical management and allows targeted preventive actions in mutation carriers. Lynch syndrome is one of the more common adult-onset hereditary tumor syndromes, with thousands of patients reported to date. Its tumor spectrum is well established and includes colorectal cancer, endometrial cancer and a range of other cancer types. However, surveillance for cancers other than colorectal cancer is still of uncertain value. Prophylactic surgery, especially for the uterus and its adnexa is an option in female mutation carriers. Chemoprevention of colorectal cancer with aspirin is actively being investigated in this syndrome and shows promising results. In contrast, the Constitutional Mismatch Repair Deficiency syndrome is rare, features a wide spectrum of childhood onset cancers, many of which are brain tumors with high mortality rates. Future studies are very much needed to improve the care for patients with this severe disorder.

The pathological significance of selected MLH1 missense mutations based on the theoretical in silico predictions.

Germline mutations in the human DNA mismatch repair (MMR) genes MSH2 and MLH1 are associated with the inherited cancer disorder Lynch syndrome (LS). Up to 30% of MLH1 variants found are missense mutations. The functional consequences in regard to pathogenicity of many of these variants are unclear. Missense mutations affect protein structure or function, but may also cause aberrant splicing. In this study, in silico prediction tools (SIFT, PolyPhen-2 and ESEfinder 3.0), literature and an online MMR mutation database review were used to evaluate the clinical significance of 26 MLH1 missense mutations identified in patients from Polish families, suspected of Lynch syndrome. Six MLH1 VUS were classified as pathogenic, nine MLH1 missense mutations are classified as neutral. 11 variants require additional research. The results show that in silico prediction tools can be utilized in an appropriate and efficient manner to determine the pathogenicity of MMR gene variations.

Aspirin, Ibuprofen, and the Risk of Colorectal Cancer in Lynch Syndrome.

Inheritance of a germline mutation in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 causes a high risk of colorectal and other cancers (Lynch Syndrome). Use of aspirin has been shown to be associated with a reduced risk of colorectal cancer for the general population as well as for MMR gene mutation carriers. The aim of this study was to determine whether use of aspirin and ibuprofen in a nontrial setting is associated with the risk of colorectal cancer risk for MMR gene mutation carriers. We included 1858 participants in the Colon Cancer Family Registry who had been found to have a pathogenic germline mutation in a MMR gene (carriers). We used weighted Cox proportional hazards regression to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). All statistical tests were two-sided. A total of 714 carriers (38%) were diagnosed with colorectal cancer at a mean age of 42.4 (standard deviation 10.6) years. A reduced risk of colorectal cancer was associated with aspirin use (for 1 month to 4.9 years: HR = 0.49, 95% CI = 0.27 to 0.90, P = .02; for ≥5 years: HR = 0.25, 95% CI = 0.10 to 0.62, P = .003) and ibuprofen use (for 1 month to 4.9 years: HR = 0.38, 95% CI = 0.18 to 0.79, P = .009; for ≥5 years: HR = 0.26, 95% CI = 0.10 to 0.69, P = .007), compared with less than one month of use. Our results provide additional evidence that, for MMR gene mutation carriers, use of aspirin and ibuprofen might be effective in reducing their high risk of colorectal cancer.

A model-based assessment of the cost–utility of strategies to identify Lynch syndrome in early-onset colorectal cancer patients

BackgroundLynch syndrome is an autosomal dominant cancer predisposition syndrome caused by mutations in the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2. Individuals with Lynch syndrome have an increased risk of colorectal cancer, endometrial cancer, ovarian and other cancers. Lynch syndrome remains underdiagnosed in the UK. Reflex testing for Lynch syndrome in early-onset colorectal cancer patients is proposed as a method to identify more families affected by Lynch syndrome and offer surveillance to reduce cancer risks, although cost-effectiveness is viewed as a barrier to implementation. The objective of this project was to estimate the cost–utility of strategies to identify Lynch syndrome in individuals with early-onset colorectal cancer in the NHS.MethodsA decision analytic model was developed which simulated diagnostic and long-term outcomes over a lifetime horizon for colorectal cancer patients with and without Lynch syndrome and for relatives of those patients. Nine diagnostic strategies were modelled which included microsatellite instability (MSI) testing, immunohistochemistry (IHC), BRAF mutation testing (methylation testing in a scenario analysis), diagnostic mutation testing and Amsterdam II criteria. Biennial colonoscopic surveillance was included for individuals diagnosed with Lynch syndrome and accepting surveillance. Prophylactic hysterectomy with bilateral salpingo-oophorectomy (H-BSO) was similarly included for women diagnosed with Lynch syndrome. Costs from NHS and Personal Social Services perspective and quality-adjusted life years (QALYs) were estimated and discounted at 3.5% per annum.ResultsAll strategies included for the identification of Lynch syndrome were cost-effective versus no testing. The strategy with the greatest net health benefit was MSI followed by BRAF followed by diagnostic genetic testing, costing £5,491 per QALY gained over no testing. The effect of prophylactic H-BSO on health-related quality of life (HRQoL) is uncertain and could outweigh the health benefits of testing, resulting in overall QALY loss.ConclusionsReflex testing for Lynch syndrome in early-onset colorectal cancer patients is predicted to be a cost-effective use of limited financial resources in England and Wales. Research is recommended into the cost-effectiveness of reflex testing for Lynch syndrome in other associated cancers and into the impact of prophylactic H-BSO on HRQoL.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1254-5) contains supplementary material, which is available to authorized users.

Splicing analysis for exonic and intronic mismatch repair gene variants associated with Lynch syndrome confirms high concordance between minigene assays and patient RNA analyses.

A subset of DNA variants causes genetic disease through aberrant splicing. Experimental splicing assays, either RT-PCR analyses of patient RNA or functional splicing reporter minigene assays, are required to evaluate the molecular nature of the splice defect. Here, we present minigene assays performed for 17 variants in the consensus splice site regions, 14 exonic variants outside these regions, and two deep intronic variants, all in the DNA mismatch-repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, associated with Lynch syndrome. We also included two deep intronic variants in APC and PKD2. For one variant (MLH1 c.122A>G), our minigene assay and patient RNA analysis could not confirm the previously reported aberrant splicing. The aim of our study was to further investigate the concordance between minigene splicing assays and patient RNA analyses. For 30 variants results from patient RNA analyses were available, either performed by our laboratory or presented in literature. Some variants were deliberately included in this study because they resulted in multiple aberrant transcripts in patient RNA analysis, or caused a splice effect other than the prevalent exon skip. While both methods were completely concordant in the assessment of splice effects, four variants exhibited major differences in aberrant splice patterns. Based on the present and earlier studies, together showing an almost 100% concordance of minigene assays with patient RNA analyses, we discuss the weight given to minigene splicing assays in the current criteria proposed by InSiGHT for clinical classification of MMR variants.

Defective DNA mismatch repair activity is common in sebaceous neoplasms, and may be an ineffective approach to screen for Lynch syndrome.

A subset of individuals with Lynch syndrome (LS) has a variant called Muir-Torre syndrome (MTS) where patients develop multiple sebaceous neoplasms. Absence of gene expression and microsatellite instability (MSI) have been welldocumented in LS neoplasms. It is unclear whether the presence of these abnormalities in isolated sebaceous neoplasms would indicate the likely presence of otherwise unsuspected LS or MTS. 164 specimens of sporadic cutaneous sebaceous neoplasms were obtained. IHC was performed for expression of the DNA mismatch repair (MMR) genes MSH2 and MLH1. A 5-marker mononucleotide repeat microsatellite panel was analyzed to detect MSI, and two or more mutated markers were required for MSI. 164 sebaceous neoplasms were obtained from 162 patients. IHC data was successfully obtained from 162 samples and MSI data was obtained from 138 samples. 50/162 (31%) had abnormal IHC with loss of staining for either MSH2 (37/162, 23%), MLH1 (9/162, 5%) or both (4/162, 2%). 37% (52/138) of the tumors had MSI. 82% (111/136) of those with both IHC and MSI results correlated as expected. 18% (25/136) showed discordance between IHC and MSI. 69/163 (42%) had either abnormal IHC or MSI, indicating deficient DNA MMR activity. Given the substantial proportion of DNA MMR deficiency in these sebaceous neoplasms, screening for DNA MMR defects in sebaceous neoplasms would not appear to be an effective way to distinguish patients with LS or MTS from those with sporadic skin lesions and an ordinary risk of cancer.

Quality of colonoscopy in Lynch syndrome.

Lynch syndrome (LS) accounts for 2 – 4 % of all colorectal cancers. Affected family members have a germline mutation in one of the DNA mismatch repair genes MLH1, PMS2, MSH2, or MSH6, and a lifetime risk for development of colorectal cancer of 25 – 75 %. Current guidelines recommend annual to biannual surveillance colonoscopy in mutation carriers. Several factors may predict failure to prevent interval cancer in LS: more lesions in the right colon; more flat (“non polypoid”) and lateral growing polyps; small adenomas may already harbor high grade dysplasia or a high percentage of villous component and become advanced adenomas; there is a short duration of the adenoma – carcinoma sequence; synchronous lesions have high prevalence; patients are younger and less tolerant to colonoscopy (need more sedation); and repeated colonoscopies are needed for lifelong surveillance (patient experience is important for compliance). In order to prevent cancer in LS patients, surveillance colonoscopy should be performed in an endoscopic unit experienced with LS, every 1 – 2 years, starting at age 20 – 25 years, or 10 years younger than the age of first diagnosis in the family (whichever is first), and yearly after the age of 40 years. Colonoscopy in LS patients should be a very meticulous and precise procedure (i. e. taking sufficient withdrawal time, documentation of such warranted), with removal of all of the polyps, special attention to the right colon and alertness to flat lesions. Following quality indicators such as successful cleansing of the colon and removal of every polyp will probably improve prevention of interval cancers. At this moment, none of the new endoscopic techniques have shown convincing superiority over conventional high resolution white light colonoscopy.

Silencing of the DNA Mismatch Repair Gene MLH1 Induced by Hypoxic Stress in a Pathway Dependent on the Histone Demethylase LSD1

Silencing of MLH1 is frequently seen in sporadic colorectal cancers. We show here that hypoxia causes decreased histone H3 lysine 4 (H3K4) methylation at the MLH1 promoter via the action of the H3K4 demethylases LSD1 and PLU-1 and promotes durable long-term silencing in a pathway that requires LSD1. Knockdown of LSD1 or its corepressor, CoREST, also prevents the resilencing (and associated cytosine DNA methylation) of the endogenous MLH1 promoter in RKO colon cancer cells following transient reactivation by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). The results demonstrate that hypoxia is a driving force for silencing of MLH1 and that the LSD1/CoREST complex is necessary for this process. The results reveal a mechanism by which hypoxia promotes cancer cell evolution to drive malignant progression through epigenetic modulation. Our findings suggest that LSD1/CoREST acts as a colon cancer oncogene by epigenetically silencing MLH1 and also identify the LSD1/CoREST complex as a potential target for therapy.

Lynch syndrome: an updated review.

Lynch syndrome is one of the most common cancer susceptibility syndromes. Individuals with Lynch syndrome have a 50%–70% lifetime risk of colorectal cancer, 40%–60% risk of endometrial cancer, and increased risks of several other malignancies. It is caused by germline mutations in the DNA mismatch repair genes MLH1, MSH2, MSH6 or PMS2. In a subset of patients, Lynch syndrome is caused by 3' end deletions of the EPCAM gene, which can lead to epigenetic silencing of the closely linked MSH2. Relying solely on age and family history based criteria inaccurately identifies eligibility for Lynch syndrome screening or testing in 25%–70% of cases. There has been a steady increase in Lynch syndrome tumor screening programs since 2000 and institutions are rapidly adopting a universal screening approach to identify the patients that would benefit from genetic counseling and germline testing. These include microsatellite instability testing and/or immunohistochemical testing to identify tumor mismatch repair deficiencies. However, universal screening is not standard across institutions. Furthermore, variation exists regarding the optimum method for tracking and disclosing results. In this review, we summarize traditional screening criteria for Lynch syndrome, and discuss universal screening methods. International guidelines are necessary to standardize Lynch syndrome high-risk clinics.

A comparison of right versus left-sided colorectal carcinoma for DNA mismatch repair and presence of putative CD133-stem cells

Right and left-sided colorectal carcinoma (CRC) are generally observed to differ in pathogenetic mechanisms, with deficiency of DNA mismatch repair (dMMR) leading to microsatellite instability occurring more commonly on the right. A study to assess the DNA mismatch repair status and presence of CD133-putative stem cells in 40 consecutive right and 40 left-sided formalin-fixed, paraffin-embedded CRC was carried out at the Department of Pathology, University of Malaya Medical Centre. Immunohistochemical staining for DNA mismatch repair gene products, MLH1, MSH2, MSH6 and PMS2 and CD133 were carried out on a Ventana XT autostainer. dMMR was significantly increased in right-sided (35%) compared with left-sided (0%) CRC. Of the dMMR cases, 10 had concomitant loss of MLH1 and PMS2, two concomitant loss of MSH2 and MSH6, one isolated loss of MSH6 and another of PMS2. CD133 immunoexpression score, arbitrarily computed as the semi-quantitative estimation of proportion of CRC immunopositive cells multiplied by intensity of staining was significantly lower (<i>p</i><0.05) in right (mean score=7.0) compared with left-sided tumours (mean score=9.9). The observations of a higher frequency of dMMR and lower CD133 expression in right-sided CRC further support the notion of dissimilar mechanisms involved in aetiology and evolution of right and left-sided tumours.

Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients

BackgroundGerm-line mutations in the DNA mismatch repair genes MLH1, MSH2, and MSH6 predispose to the development of colorectal cancer (Lynch syndrome or hereditary nonpolyposis colorectal cancer). These mutations include disease-causing frame-shift, nonsense, and splicing mutations as well as large genomic rearrangements. However, a large number of mutations, including missense, silent, and intronic variants, are classified as variants of unknown clinical significance.MethodsIntronic MLH1, MSH2, or MSH6 variants were investigated using in silico prediction tools and mini-gene assay to asses the effect on splicing.ResultsWe describe in silico and in vitro characterization of nine intronic MLH1, MSH2, or MSH6 mutations identified in Danish colorectal cancer patients, of which four mutations are novel. The analysis revealed aberrant splicing of five mutations (MLH1 c.588 + 5G > A, MLH1 c.677 + 3A > T, MLH1 c.1732-2A > T, MSH2 c.1276 + 1G > T, and MSH2 c.1662-2A > C), while four mutations had no effect on splicing compared to wild type (MLH1 c.117-34A > T, MLH1 c.1039-8 T > A, MSH2 c.2459-18delT, and MSH6 c.3439-16C > T).ConclusionsIn conclusion, we classify five MLH1/MSH2 mutations as pathogenic, whereas four MLH1/MSH2/MSH6 mutations are classified as neutral. This study supports the notion that in silico prediction tools and mini-gene assays are important for the classification of intronic variants, and thereby crucial for the genetic counseling of patients and their family members.

The MLH1 c.-27C&gt;A and c.85G&gt;T variants are linked to dominantly inherited MLH1 epimutation and are borne on a European ancestral haplotype

Germline mutations of the DNA mismatch repair genes MLH1, MSH2, MSH6 or PMS2, and deletions affecting the EPCAM gene adjacent to MSH2, underlie Lynch syndrome by predisposing to early-onset colorectal, endometrial and other cancers. An alternative but rare cause of Lynch syndrome is constitutional epimutation of MLH1, whereby promoter methylation and transcriptional silencing of one allele occurs throughout normal tissues. A dominantly transmitted constitutional MLH1 epimutation has been linked to an MLH1 haplotype bearing two single-nucleotide variants, NM_000249.2: c.-27C>A and c.85G>T, in a Caucasian family with Lynch syndrome from Western Australia. Subsequently, a second seemingly unrelated Caucasian Australian case with the same MLH1 haplotype and concomitant epimutation was reported. We now describe three additional, ostensibly unrelated, cancer-affected families of European heritage with this MLH1 haplotype in association with constitutional epimutation, bringing the number of index cases reported to five. Array-based genotyping in four of these families revealed shared haplotypes between individual families that extended across ≤2.6-≤6.4 megabase regions of chromosome 3p, indicating common ancestry. A minimal ≤2.6 megabase founder haplotype common to all four families was identified, which encompassed MLH1 and additional flanking genes and segregated with the MLH1 epimutation in each family. Our findings indicate that the MLH1 c.-27C>A and c.85G>T variants are borne on a European ancestral haplotype and provide conclusive evidence for its pathogenicity via a mechanism of epigenetic silencing of MLH1 within normal tissues. Additional descendants bearing this founder haplotype may exist who are also at high risk of developing Lynch syndrome-related cancers.