mutL Homolog 1 Promoter Research Articles

Targeting mitochondrial DNA polymerase gamma for selective inhibition of MLH1 deficient colon cancer growth.

Synthetic lethality in DNA repair pathways is an important strategy for the selective treatment of cancer cells without harming healthy cells and developing cancer-specific drugs. The synthetic lethal interaction between the mismatch repair (MMR) protein, MutL homolog 1 (MLH1), and the mitochondrial base excision repair protein, DNA polymerase γ (Pol γ) was used in this study for the selective treatment of MLH1 deficient cancers. Germline mutations in the MLH1 gene and aberrant MLH1 promoter methylation result in an increased risk of developing many cancers, including nonpolyposis colorectal and endometrial cancers. Because the inhibition of Pol γ in MLH1 deficient cancer cells provides the synthetic lethal selectivity, we conducted a comprehensive small molecule screening from various databases and chemical drug library molecules for novel Pol γ inhibitors that selectively kill MLH1 deficient cancer cells. We characterized these Pol γ inhibitor molecules in vitro and in vivo, and identified 3,3’-[(1,1’-Biphenyl)-4’,4’-diyl)bis(azo)]bis[4-amino-1-naphthalenesulfonic acid] (congo red; CR; Zinc 03830554) as a high-affinity binder to the Pol γ protein and potent inhibitor of the Pol γ strand displacement and one-nucleotide incorporation DNA synthesis activities in vitro and in vivo. CR reduced the cell proliferation of MLH1 deficient HCT116 human colon cancer cells and suppressed HCT116 xenograft tumor growth whereas it did not affect the MLH1 proficient cell proliferation and xenograft tumor growth. CR caused mitochondrial dysfunction and cell death by inhibiting Pol γ activity and oxidative mtDNA damage repair, increasing the production of reactive oxygen species and oxidative mtDNA damage in MLH1 deficient cells. This study suggests that the Pol γ inhibitor, CR may be further evaluated for the MLH1 deficient cancers’ therapy.

Silencing of long non-coding RNA FOXD2-AS1 inhibits the progression of gallbladder cancer by mediating methylation of MLH1.

Evidence has documented the tumor-promoting properties of long non-coding RNA (lncRNA) FOXD2 adjacent opposite strand RNA 1 (FOXD2-AS1) in many cancers. However, little is known about its role in gallbladder cancer. Here, we aimed to characterize the functional relevance of lncRNA FOXD2-AS1 in gallbladder cancer and the possible mechanisms associated with methylation of MutL homolog-1 (MLH1). Initially, microarray-based gene expression profiling of gallbladder cancer was employed to identify differentially expressed lncRNAs. Next, the expression of lncRNA FOXD2-AS1 was examined, and the cell line presenting with the highest lncRNA FOXD2-AS1 expression was selected for subsequent experimentation. Then, the interaction between lncRNA FOXD2-AS1 and MLH1 was identified. The effect of lncRNA FOXD2-AS1 on proliferation, migration, invasion, and apoptosis as well as tumorigenicity of transfected GBC-SD cells was examined with gain- and loss-of-function experiments. We found that lncRNA FOXD2-AS1 was highly expressed, while MLH1 was poorly expressed in gallbladder cancer cells. Besides, lncRNA FOXD2-AS1 could promote MLH1 methylation by recruiting DNMT1 to the MLH1 promoter, and consequently inhibit MLH1 transcription. Silencing of lncRNA FOXD2-AS1 or overexpression of MLH1 inhibited gallbladder cancer cell proliferation, invasion, and migration, while facilitating cell apoptosis in vitro as well as retarding tumor growth in vivo. Thus, silencing of lncRNA FOXD2-AS1 suppressed the progression of gallbladder cancer via upregulation of MLH1 by inhibiting MLH1 promoter methylation. These findings present lncRNA FOXD2-AS1 knockdown as a potential candidate for the treatment of gallbladder cancer.

Performance characteristics of screening strategies to identify Lynch syndrome in women with ovarian cancer.

BackgroundFor women with ovarian cancer (OC), the optimal screening strategy to identify Lynch syndrome (LS) has not been determined. In the current study, the authors compared the performance characteristics of various strategies combining mismatch repair (MMR) immunohistochemistry (IHC), microsatellite instability testing (MSI), and family history for the detection of LS.MethodsWomen with nonserous and/or nonmucinous ovarian cancer were recruited prospectively from 3 cancer centers in Ontario, Canada. All underwent germline testing for LS and completed a family history assessment. Tumors were assessed using MMR IHC and MSI. The sensitivity, specificity, and positive and negative predictive values of screening strategies were compared with the gold standard of a germline result.ResultsOf 215 women, germline data were available for 189 (88%); 13 women (7%) had pathogenic germline variants with 7 women with mutS homolog 6 (MSH6); 3 women with mutL homolog 1 (MLH1); 2 women with PMS1 homolog 2, mismatch repair system component (PMS2); and 1 woman with mutS homolog 2 (MSH2). A total of 28 women had MMR‐deficient tumors (13%); of these, 11 had pathogenic variants (39%). Sequential IHC (with MLH1 promoter methylation analysis on MLH1‐deficient tumors) followed by MSI for nonmethylated and/or MMR‐intact patients was the most sensitive (92.3%; 95% confidence interval, 64%‐99.8%) and specific (97.7%; 95% confidence interval, 94.2%‐99.4%) approach, missing 1 case of LS. IHC with MLH1 promoter methylation analysis missed 2 patients of LS. Family history was found to have the lowest sensitivity at 55%.ConclusionsSequential IHC (with MLH1 promoter methylation analysis) followed by MSI was found to be most sensitive. However, IHC with MLH1 promoter methylation analysis also performed well and is likely more cost‐effective and efficient in the clinical setting. The pretest probability of LS is high in patients with MMR deficiency and warrants universal screening for LS.

Abstract 1976: Development of a liquid biopsy based purely quantitative digital droplet PCR assay for detection of MLH-1promoter methylation

Abstract Purpose Studies have shown that MutL Homolog 1 (MLH1) promotor methylation is closely associated with microsatellite instability high colorectal cancer (CRC). The strong correlation between methylation status and cancer development and progression has led to a growing interest in the use of methylation markers in circulating tumor DNA (ctDNA) for early cancer detection and longitudinal monitoring. ctDNA methylation analysis has advantages in performance over mutation analysis, such as higher sensitivity, broader dynamic range and increased stability. However, current methods for methylation analysis involving bisulfite treatment possess many challenges which limit broad clinical applications. Here, we report the development of a fit-for-purpose digital droplet PCR (ddPCR) assay to examine MLH1 promoter methylation in ctDNA in advanced CRC. Study Procedure Primers and probes were designed to amplify CpG sites of the MLH1 promoter. Methylated and unmethylated control genomic DNA were sheared to mimic ctDNA and subjected to methylation sensitive restrictive enzyme (MSRE) HpaII digestion. The performance of the MSRE ddPCR assay was evaluated as compared to MSRE qPCR assay. Plasma samples from healthy donors and CRC patients were analyzed with the optimal primer/probe sets using ddPCR. Assay performance was evaluated by determining the limit of detection (LOD), linearity of the primer/probe sets and the correlation coefficient. Receiver operating characteristic (ROC) curves were generated to determine the optimal assay sensitivity and specificity. Data Summary Using methylated and unmethylated controls, we optimized the conditions for HpaII enzyme digestion to ensure complete digestion and avoid false positives. Three sets of primer/probes were optimized for robust amplification in ddPCR. Using 0.375ng synthetic DNA input, the LOD was measured as 1 positive droplet for set A, 4 positive droplets for set B, and 3 positive droplets for set C . Based on the results of ddPCR assay using 1ng cfDNA input from healthy donors or CRC samples, ROC curves were generated for each primer/probe set with area under the curve values ranging from 0.85 to 0.95. The optimal assay sensitivity and specificity were displayed when 5 positive droplets was used as acceptance criteria (93% sensitivity and 95% specificity). The optimal cutoff value of MLH-1 promoter methylation in ctDNA associated with patient diagnosis remains to be further defined. Conclusion The liquid biopsy assay for MLH-1 promoter methylation detection using purely quantitative ddPCR is a simple and ultrasensitive procedure that provides reliable methylation detection in ctDNA. The MRSE ddPCR approach can also be applied to other genes of interest where methylation patterns could reveal clinically relevant information for future clinical biomarker and/or companion diagnostic development. Citation Format: DANYI WANG, Dennis O'Rourke, Jorge F. Sanchez-Garcia, Ti Cai, Juergen Scheuenpflug, Zheng Feng. Development of a liquid biopsy based purely quantitative digital droplet PCR assay for detection of MLH-1promoter methylation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1976.

A systematic review and meta-analysis approach on diagnostic value of MLH1 promoter methylation for head and neck squamous cell carcinoma.

Background:Head and neck squamous cell carcinoma (HNSCC) is the leading histological type among head and neck cancers. Several studies have explored an association between aberrant methylation of MutL homolog-1 (MLH1) promoter and HNSCC risk. We aimed to explore the associations between MLH1 promoter methylation and HNSCC by using a meta-analysis.Methods:Systematic literature search was conducted among PubMed, Google Scholar, Web of Science, and China National Knowledge Infrastructure, and Wanfang databases to retrieve relevant articles published up to June 30, 2018. A total of 12 studies were included in this meta-analysis (including 717 HNSCC and 609 controls).Results:The results demonstrated that MLH1 promoter methylation was notably higher in patients with HNSCC than in controls (odds ratios [ORs] = 2.52, 95% confidence intervals [CIs] = 1.33–4.79). Besides, MLH1 promoter methylation was not associated with tumor stage, lymph node status, smoking behavior, age, clinical stage, gender, and differentiation grade (all P > .05). The pooled sensitivity and specificity rates of MLH1 methylation for HNSCC were 0.23 (95% CI = 0.12–0.38) and 0.95 (95% CI, 0.82–0.99), respectively. The area under the receiver operating characteristic (ROC) curve was presented as 0.64 (95% CI = 0.60-0.68).Conclusion:The results of this meta-analysis suggested that hypermethylation of MLH1 promoter was associated with HNSCC. Methylated MLH1 could be a potential diagnostic biomarker for diagnose of HNSCC.

The polymorphic variant rs1800734 influences methylation acquisition and allele-specific TFAP4 binding in the MLH1 promoter leading to differential mRNA expression

Expression of the mismatch repair gene MutL homolog 1 (MLH1) is silenced in a clinically important subgroup of sporadic colorectal cancers. These cancers exhibit hypermutability with microsatellite instability (MSI) and differ from microsatellite-stable (MSS) colorectal cancers in both prognosis and response to therapies. Loss of MLH1 is usually due to epigenetic silencing with associated promoter methylation; coding somatic mutations rarely occur. Here we use the presence of a colorectal cancer (CRC) risk variant (rs1800734) within the MLH1 promoter to investigate the poorly understood mechanisms of MLH1 promoter methylation and loss of expression. We confirm the association of rs1800734 with MSI+ but not MSS cancer risk in our own data and by meta-analysis. Using sensitive allele-specific detection methods, we demonstrate that MLH1 is the target gene for rs1800734 mediated cancer risk. In normal colon tissue, small allele-specific differences exist only in MLH1 promoter methylation, but not gene expression. In contrast, allele-specific differences in both MLH1 methylation and expression are present in MSI+ cancers. We show that MLH1 transcriptional repression is dependent on DNA methylation and can be reversed by a methylation inhibitor. The rs1800734 allele influences the rate of methylation loss and amount of re-expression. The transcription factor TFAP4 binds to the rs1800734 region but with much weaker binding to the risk than the protective allele. TFAP4 binding is absent on both alleles when promoter methylation is present. Thus we propose that TFAP4 binding shields the protective rs1800734 allele of the MLH1 promoter from BRAF induced DNA methylation more effectively than the risk allele.

Universal screening of both endometrial and colon cancers increases the detection of Lynch syndrome.

Lynch syndrome (LS) is the most common hereditary cause of colorectal cancer (CRC) and endometrial cancer (EC). Screening of all CRCs for LS is currently recommended, but screening of ECs is inconsistent. The objective of this study was to determine the added value of screening both CRC and EC tumors in the same population. A prospective, immunohistochemistry (IHC)-based screening program for all patients with newly diagnosed CRCs and ECs was initiated in 2011 and 2013, respectively, at 2 centers (primary and tertiary). Genetic testing was recommended for those who had tumors with absent mutS homolog 2 (MSH2), MSH6, or postmeiotoic segregation increased 2 (PMS2) expression and for those who had tumors with absent mutL homolog 1 (MLH1) expression and no v-Raf murine sarcoma viral oncogene homolog B (BRAF) mutation or MLH1 promoter methylation. Amsterdam II criteria, revised Bethesda criteria, and scores from prediction models for gene mutations (the PREMM1,2,6 and PREMM5 prediction models) were ascertained in patients with LS. In total, 1290 patients with CRC and 484 with EC were screened for LS, and genetic testing was recommended for 137 patients (10.6%) and 32 patients (6.6%), respectively (P = .01). LS was identified in 16 patients (1.2%) with CRC and in 8 patients (1.7%) with EC. Among patients for whom genetic testing was recommended, the LS diagnosis rate was higher among those with EC (25.0% vs 11.7%, P = .052). The Amsterdam II criteria, revised Bethesda criteria, and both PREMM calculators would have missed 62.5%, 50.0%, and 12.5% of the identified patients with LS, respectively. Expanding a universal screening program for LS to include patients who had EC identified 50% more patients with LS, and many of these patients would have been missed by risk assessment tools (including PREMM5 ). Universal screening programs for LS should include both CRC and EC. Cancer 2018. © 2018 American Cancer Society.

Role of MLH1 methylation in esophageal cancer carcinogenesis and its clinical significance.

The mutL homolog-1 (MLH1) is a DNA mismatch repair gene and has been reported to be frequently methylated in numerous cancers. However, the association between MLH1 methylation and esophageal cancer (EC), as well as its clinical significance, remains unclear. Hence, we conducted a systematic meta-analysis based on 19 articles (including 1384 ECs, 345 premalignant lesions, and 1244 healthy controls). Our analysis revealed that the frequency of MLH1 methylation was significantly elevated during EC carcinogenesis. In addition, we observed that MLH1 promoter methylation was associated with age (odds ratio [OR]=1.79; 95% CI =1.20–2.66), advanced tumor grade (OR=3.7; 95% CI =2.37–5.77), lymph node metastasis (OR=2.65; 95% CI =1.81–3.88), distant metastasis (OR=7.60; 95% CI =1.23–47.19), advanced clinical stage (OR=4.46; 95% CI =2.88–6.91), and poor prognosis in EC patients (hazard ratio =1.64, 95% CI =1.00–2.69). The pooled sensitivity, specificity, and area under the curve of MLH1 methylation in EC patients versus healthy individuals were 0.15, 0.99, and 0.77, respectively. Our findings indicate that MLH1 methylation is involved in the carcinogenesis, progression, and metastasis of EC. Moreover, methylated MLH1 could be a potential diagnostic and prognostic biomarker for EC.

Epigenetic Silencing of the MLH1 Promoter in Relation to the Development of Gastric Cancer and its use as a Biomarker for Patients with Microsatellite Instability: a Systematic Analysis

Background/Aims: Human mutL homolog 1 (MLH1) promoter methylation was reported in gastric cancer (GC). This study determined the clinicopathological, prognostic, and diagnostic effects of MLH1 promoter methylation in GC. Methods: The combined odds ratio (OR) or hazard ratio (HR) and their corresponding 95% confidence intervals (95% CI) were calculated. The pooled sensitivity, specificity, and area under the curve (AUC) were analyzed. Results: A total of 4654 GC patients and 3669 non-malignant controls were identified in this systematic analysis. MLH1 promoter methylation was significantly higher in GC samples than in gastric adenomas, chronic gastritis, adjacent tissues, normal gastric mucosa, and normal healthy blood samples, but it exhibited a similar frequency in GC vs. intestinal metaplasia and dysplasia samples. MLH1 promoter methylation correlated with age and microsatellite instability (MSI), but it was not associated with gender, H. pylori infection, smoking, drinking behaviors, pathological histology, tumor differentiation, clinical stage, lymph node status, distant metastasis, or overall survival of GC. MLH1 promoter methylation exhibited a poor sensitivity value (< 0.5) in patients with GC compared with adjacent tissues, gastric adenomas, chronic gastritis, normal gastric mucosa, and normal healthy blood samples. The pooled sensitivity, specificity, and AUC of MLH1 promoter methylation in GC with MSI vs. GC with microsatellite stability (MSS) samples were 0.64, 0.96, and 0.90, respectively. Conclusions: Our results suggest that the detection of MLH1 promoter methylation may be a potential prognostic biomarker for GC patients with MSI.

Aberrant methylation of mutL homolog 1 is associated with increased risk of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a common malignant tumor. DNA hypermethylation in the promoter region has been served as a potential molecular marker for several tumors. The goal of the current study was to assess the diagnostic ability of mutL homolog 1 (MLH1) promoter methylation in NSCLC. A total of 111 NSCLC patients' paired tissue samples were obtained to explore the association between MLH1 promoter methylation and NSCLC by methylation-specific polymerase chain reaction (MSP) method. Public databases including The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were used to verify our findings. Our results showed a significantly higher MLH1 methylation frequency in tumor tissue samples than their paired adjacent tissues (P=.008). ROC curve indicated that MLH1MSP assay was a sensitive but not a specific method in the diagnosis for NSCLC (sensitivity=0.964, specificity=0.135, AUC=0.550). And the association between the methylation level and clinical characteristics has no statistical significance. TCGA cohort evinced a higher methylation probability in tumor group compared with nontumor group (the mean β value: -0.449 [-0.467, -0.437] vs -0.466 [-0.472, -0.437], P=.011), which was consistent with our results. Meanwhile, an inverse correlation between MLH1 methylation and MLH1 expression was detected in TCGA and GEO databases. The MSP method for MLH1 methylation was a sensitive but not a specific diagnostic method for NSCLC.

Prognostic value of MLH1 promoter methylation in male patients with esophageal squamous cell carcinoma.

The DNA mismatch repair (MMR) gene MutL homolog 1 (MLH1) is critical for the maintenance of genomic integrity. Methylation of the MLH1 gene promoter was identified as a prognostic marker for numerous types of cancer including glioblastoma, colorectal, ovarian and gastric cancer. The present study aimed to determine whether MLH1 promoter methylation was associated with survival in male patients with esophageal squamous cell carcinoma (ESCC). Formalin-fixed, paraffin-embedded ESCC tissues were collected from 87 male patients. MLH1 promoter methylation was assessed using the methylation-specific polymerase chain reaction approach. Kaplan-Meier survival curves and log-rank tests were used to evaluate the association between MLH1 promoter methylation and overall survival (OS) in patients with ESCC. Cox regression analysis was used to obtain crude and multivariate hazard ratios (HR), and 95% confidence intervals (CI). The present study revealed that MLH1 promoter methylation was observed in 53/87 (60.9%) of male patients with ESCC. Kaplan-Meier survival analysis demonstrated that MLH1 promoter hypermethylation was significantly associated with poorer prognosis in patients with ESCC (P=0.048). Multivariate survival analysis revealed that MLH1 promoter hypermethylation was an independent predictor of poor OS in male patients with ESCC (HR=1.716; 95% CI=1.008-2.921). Therefore, MLH1 promoter hypermethylation may be a predictor of prognosis in male patients with ESCC.

Methylation Analysis of DNA Mismatch Repair Genes Using DNA Derived from the Peripheral Blood of Patients with Endometrial Cancer: Epimutation in Endometrial Carcinogenesis.

Germline mutation of DNA mismatch repair (MMR) genes is a cause of Lynch syndrome. Methylation of MutL homolog 1 (MLH1) and MutS homolog 2 (MSH2) has been detected in peripheral blood cells of patients with colorectal cancer. This methylation is referred to as epimutation. Methylation of these genes has not been studied in an unselected series of endometrial cancer cases. Therefore, we examined methylation of MLH1, MSH2, and MSH6 promoter regions of peripheral blood cells in 206 patients with endometrial cancer using a methylation-specific polymerase chain reaction (MSP). Germline mutation of MMR genes, microsatellite instability (MSI), and immunohistochemistry (IHC) were also analyzed in each case with epimutation. MLH1 epimutation was detected in a single patient out of a total of 206 (0.49%)—1 out of 58 (1.72%) with an onset age of less than 50 years. The patient with MLH1 epimutation showed high level MSI (MSI-H), loss of MLH1 expression and had developed endometrial cancer at 46 years old, complicated with colorectal cancer. No case had epimutation of MSH2 or MSH6. The MLH1 epimutation detected in a patient with endometrial cancer may be a cause of endometrial carcinogenesis. This result indicates that it is important to check epimutation in patients with endometrial cancer without a germline mutation of MMR genes.

Promoter methylation of ITF2, but not APC, is associated with microsatellite instability in two populations of colorectal cancer patients.

BackgroundAberrant Wnt signaling activation occurs commonly in colorectal carcinogenesis, leading to upregulation of many target genes. APC (adenomatous polyposis coli) is an important component of the β-catenin destruction complex, which regulates Wnt signaling, and is often mutated in colorectal cancer (CRC). In addition to mutational events, epigenetic changes arise frequently in CRC, specifically, promoter hypermethylation which silences tumor suppressor genes. APC and the Wnt signaling target gene ITF2 (immunoglobulin transcription factor 2) incur hypermethylation in various cancers, however, methylation-dependent regulation of these genes in CRC has not been studied in large, well-characterized patient cohorts. The microsatellite instability (MSI) subtype of CRC, featuring DNA mismatch repair deficiency and often promoter hypermethylation of MutL homolog 1 (MLH1), has a favorable outcome and is characterized by different chemotherapeutic responses than microsatellite stable (MSS) tumors. Other epigenetic events distinguishing these subtypes have not yet been fully elucidated.MethodsHere, we quantify promoter methylation of ITF2 and APC by MethyLight in two case-case studies nested in population-based CRC cohorts from the Ontario Familial Colorectal Cancer Registry (n = 330) and the Newfoundland Familial Colorectal Cancer Registry (n = 102) comparing MSI status groups.ResultsITF2 and APC methylation are significantly associated with tumor versus normal state (both P < 1.0×10-6). ITF2 is methylated in 45.8 % of MSI cases and 26.9 % of MSS cases and is significantly associated with MSI in Ontario (P = 0.002) and Newfoundland (P = 0.005) as well as the MSI-associated feature of MLH1 promoter hypermethylation (P = 6.72×10-4). APC methylation, although tumor-specific, does not show a significant association with tumor subtype, age, gender, or stage, indicating it is a general tumor-specific CRC biomarker.ConclusionsThis study demonstrates, for the first time, MSI-associated ITF2 methylation, and further reveals the subtype-specific epigenetic events modulating Wnt signaling in CRC.

Efficient screening strategy for Lynch syndrome in Japanese endometrial cancer.

Lynch syndrome (LS) is an inherited disorder caused by a germline mutation in the DNA mismatch repair (MMR) genes and is associated with increased risk of various cancers, particularly colorectal cancer and endometrial cancer (EC). It is significant to identify LS in EC patients for prediction and prevention of the succeeding other associated cancers. However, useful LS screening guidelines for EC have not been established. The purpose of our study is to devise an efficient and practical screening strategy for LS in EC. We designed original criteria, named "APF criteria," with lenient terms (Age of onset < 50, or Personal or Family history of associated cancers) and applied it to unselected EC patients. We performed immunohistochemistry (IHC) and the methylation assay of MutL homolog 1 (MLH1) gene promoter using the tumors of patients who met our criteria, and thus selected "suspected LS" as the candidates for genetic analyses. Of 360 EC patients, 187 (51.9%) met the APF criteria, and the tumor specimens were available from 182 out of the 187 patients. IHC revealed that expression of at least one MMR protein was absent in cell nuclei of 54 (29.6%) tumors. Of 20 tumors lacking MLH1 protein expression, 14 cases were judged sporadic EC because of the hypermethylated MLH1 promoter. We thus selected 40 (11.1%) of 360 EC patients as "suspected LS." Our strategy that consists of clinical triage and the molecular analyses is expected to improve the screening efficiency and reduce the cost of LS identification in EC.

Prevalence of somatic mutl homolog 1 promoter hypermethylation in Lynch syndrome colorectal cancer.

Colorectal cancers (CRCs) that have microsatellite instability (MSI) and mutL homolog 1 (MLH1) immunoloss are observed in 3 clinical scenarios: Lynch syndrome (LS), sporadic MSI CRC, and Lynch-like syndrome (LLS). v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutational analysis is used to differentiate LS from sporadic MSI CRC. The role of MLH1 promoter methylation status for the differential diagnosis of these clinical forms is not well established. The objectives of this study were: 1) to analyze MLH1 promoter methylation in MLH1-deficient CRCs by pyrosequencing, and 2) to assess its role in the differential diagnosis of MLH1-deficient CRCs. In total, 165 CRCs were analyzed, including LS (n = 19), MSI BRAF-mutated CRC (n = 37), MSI BRAF wild-type CRC (n = 60), and a control group of CRCs without MSI (microsatellite stable [MSS] CRC; n = 49). MLH1 promoter methylation status was analyzed by pyrosequencing, and the ability of different strategies to identify LS was assessed. The average ± standard deviation methylation in LS (9% ± 7%) was significantly lower than that in MSI BRAF-mutated CRC (42% ± 17%; P < .001) and in MSI BRAF wild-type CRC (25% ± 19%; P = .002). Somatic MLH1 hypermethylation was detected in 3 patients (15.8%) with LS, in 34 patients (91.9%) with MSI BRAF-mutated CRC, and in 37 patients (61.7%) with MSI BRAF wild-type tumors. Patients with MSI BRAF wild-type, unmethylated tumors (ie, LLS) had a stronger family history of CRC than those who had tumors with MLH1 methylation (P < .05). The sensitivity for ruling out LS was 100% for BRAF analysis, 84.2% for MLH1 methylation analysis, and 84.2% for the combination of both analyses. Somatic MLH1 promoter methylation occurs in up to 15% of LS CRCs. Somatic BRAF analysis is the most sensitive strategy for ruling out LS. Patients who have CRCs with loss of MLH1 protein expression and neither BRAF mutation nor MLH1 methylation resemble patients with LS.

Immunohistochemical detection of ARID1A in colorectal carcinoma: loss of staining is associated with sporadic microsatellite unstable tumors with medullary histology and high TNM stage.

AT-rich interactive domain-containing protein 1A (ARID1A), a chromatin remodeling gene recently discovered to be a tumor suppressor in ovarian cancers, has been found to be mutated at low frequencies in many other tumors including colorectal carcinoma (CRC). An association between ARID1A alteration and DNA mismatch repair (MMR) deficiency has been implicated; understanding this association may facilitate the understanding of the role of ARID1A in the various tumors. In this pilot study, we analyzed the immunohistochemical expression of ARID1A in a consecutive series of 257 CRCs that fulfilled a set of relaxed criteria for Lynch syndrome screening; 59 (23%) were MMR deficient by immunohistochemistry (44 MLH1/PMS2 deficient, 9 MSH2/MSH6 deficient, 4 MSH6 deficient, and 2 PMS2 deficient). ARID1A loss was observed in 9% (22/257) of the cohort: 24% of MMR-deficient tumors (14/59, 13 of the 14 being MLH1/PMS2 deficient) and 4% of MMR-normal tumors (8/198) (P < .05). MLH1 (mutL homolog 1) promoter hypermethylation was observed in 10 of the 13 MLH1/PMS2-deficient/ARID1A-loss tumors, indicating an association between ARID1A loss and sporadic microsatellite unstable CRCs. Among the MMR-deficient cases, ARID1A loss correlated with old age (P = .04), poor tumor differentiation (P < .01), medullary histology (P < .01), and an increased rate of nodal and distant metastasis (P = .03); these patients also trended toward a worse 5-year overall survival. Among MMR-normal tumors, no differences in clinicopathological features were detected between the groups stratified by ARID1A. In conclusion, our results suggest that ARID1A loss may be linked to a specific subset of sporadic microsatellite unstable CRCs that may be medullary but is more likely to present with metastatic disease, warranting further investigation.

Vitamin and antioxidant rich diet increases MLH1 promoter DNA methylation in DMT2 subjects

BackgroundOxidative stress may lead to an increased level of unrepaired cellular DNA damage, which is discussed as one risk for tumor initiation. Mismatch repair (MMR) enzymes act as proofreading complexes that maintain the genomic integrity and MMR-deficient cells show an increased mutation rate. One important gene in the MMR complex is the MutL homolog 1 (MLH1) gene. Since a diet rich in antioxidants has the potential to counteract harmful effects by reactive oxygen species (ROS), we investigated the impact of an antioxidant, folate, and vitamin rich diet on the epigenetic pattern of MLH1. These effects were analyzed in individuals with non-insulin depended diabetes mellitus type 2 (NIDDM2) and impaired fasting glucose (IFG).MethodsIn this post-hoc analysis of a randomized trial we analyzed DNA methylation of MLH1, MSH2, and MGMT at baseline and after 8 weeks of intervention, consisting of 300 g vegetables and 25 ml plant oil rich in polyunsaturated fatty acids per day. DNA methylation was quantified using combined bisulfite restriction enzyme analysis (COBRA) and pyrosequencing. MLH1 and DNMT1 mRNA expression were investigated by qRT-PCR. DNA damage was assessed by COMET assay. Student’s two-tailed paired t test and one-way ANOVA with Scheffé corrected Post hoc test was used to determine significant methylation and expression differences. Two-tailed Pearson test was used to determine correlations between methylation level, gene expression, and DNA strand break amount.ResultsThe intervention resulted in significantly higher CpG methylation in two particular MLH1 promoter regions and the MGMT promoter. DNA strand breaks and methylation levels correlated significantly. The expression of MLH1, DNMT1, and the promoter methylation of MSH2 remained stable. CpG methylation levels and gene expression did not correlate.ConclusionThis vitamin and antioxidant rich diet affected the CpG methylation of MLH1. The higher methylation might be a result of the ROS scavenging antioxidant rich diet, leading to lower activity of DNA demethylating enzymes. Our results suggest the hypothesis of CpG demethylation via DNA repair enzymes under these circumstances. NIDDM2 and IFG patients benefit from this simple dietary intervention involving epigenetic and DNA repair mechanisms.

Microsatellite instability, MLH1 promoter methylation, and BRAF mutation analysis in sporadic colorectal cancers of different ethnic groups in Israel

The molecular mechanisms that underlie colorectal cancer (CRC) include microsatellite instability (MSI), chromosomal instability, and the CpG island methylator phenotype. There is evidence to suggest that CRC incidence varies among different ethnic populations worldwide. The authors of this report hypothesized that environmental factors and lifestyle differences among various ethnic groups may differentially influence the epigenetic regulation of tumor suppressor genes in CRC. In the current study, microdissection and DNA extraction were performed on 128 samples of CRC from Israeli patients (85 Jews and 43 Arabs). MSI analysis, mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2) protein expression levels, and MLH1 promoter methylation were investigated by combined bisulfite restriction analysis. The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) valine-to-glutamic acid mutation at residue 600 was investigated by direct DNA sequencing. High MSI (MSI-H), MLH1 methylation, and BRAF mutations were observed in 11.6%, 9.4%, and 23.5% of Jews, respectively, and in 16.2%, 17.6%, and 20.9% of Arabs, respectively (P value nonsignificant). MLH1 promoter methylation was observed in 22.6% of microsatellite-stable (MSS) tumors and in 53.8% of MSI-H tumors (P < .015). Extensive methylation (covering both 5' and 3' promoter regions) was present in all MSI-H tumors with loss of MLH1 expression. BRAF mutation was observed in 15.6% and 46.1% of MSS tumors and MSI-H tumors, respectively (P < .007). BRAF mutation was observed in 66%, 22.2%, and 14.7% of patients who had tumors with extensive MLH1 promoter methylation, methylation of the 5' region alone, or without methylation, respectively (P < .006). There was no difference in molecular signatures examined between Jewish and Arab patients with CRC in Israel. Extensive promoter methylation was associated with MLH1 inactivation, MSI, and BRAF mutation.

Immunohistochemistry versus Microsatellite Instability Testing for Screening Colorectal Cancer Patients at Risk for Hereditary Nonpolyposis Colorectal Cancer Syndrome: Part II. The Utility of Microsatellite Instability Testing

Germline mutations in the mismatch repair genes mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2), MSH6, and postmeiotic segregation increased 2 (PMS2) lead to the development of hereditary nonpolyposis colorectal cancer (HNPCC). Diagnosis of HNPCC relies on the compilation of a thorough family history of cancer, documentation of pathological findings, tumor testing for microsatellite instability (MSI) and immunohistochemistry (IHC), and germline mutation analysis of the suspected genes. As a hallmark of HNPCC, microsatellite instability is widely accepted as a primary method for identifying individuals at risk for HNPCC. It serves as an excellent, easy-to-evaluate marker of mismatch repair deficiency. Recent improvements in MSI testing have significantly enhanced the accuracy and reduced its cost. Proficiency testing for MSI is available, and laboratory-to-laboratory reproducibility of such testing can be easily evaluated. In addition, the combination of microsatellite instability testing, MLH1 promoter methylation analysis, and BRAF (V600E) mutation analysis can distinguish a sporadic colorectal cancer from one associated with HNPCC, helping to avoid costly molecular genetic testing for germline mutations in mismatch repair genes. In this article, we discuss the development of MSI markers used for HNPCC screening and focus on the advantages and disadvantages of MSI testing in screening for HNPCC patients. We conclude that MSI is as sensitive and specific as IHC, given its excellent reproducibility and its potential capability to indicate mutations not be detected by IHC. MSI has been used and will continue to prevail as the primary screening tool for identifying HNPCC patients.

A review of the clinical relevance of mismatch‐repair deficiency in ovarian cancer

Ovarian cancer ranks fifth in both cancer incidence and mortality among women in the United States. Defects in the mismatch-repair (MMR) pathway that arise through genetic and/or epigenetic mechanisms may be important etiologically in a reasonable proportion of ovarian cancers. Genetic mechanisms of MMR dysfunction include germline and somatic mutations in the MMR proteins. Germline mutations cause hereditary nonpolyposis colorectal cancer (HNPCC), which is the third most common cause of inherited ovarian cancer after BRCA1 and BRCA2 mutations. An epigenetic mechanism known to cause inactivation of the MMR system is promoter hypermethylation of 1 of the MMR genes, mutL homolog 1 (MLH1). Various laboratory methods, in addition to clinical and histopathologic criteria, can be used to identify MMR-deficient ovarian cancers. Such methods include microsatellite instability analysis, immunohistochemistry, MLH1 promoter hypermethylation testing, and germline mutation analysis. In this review, the authors describe the existing literature regarding the molecular, clinical, and histologic characteristics of MMR-deficient ovarian cancers along with the possible effect on survival and treatment response. By further defining the profile of MMR-deficient ovarian cancers and their associated etiologic mechanisms, there may be a greater potential to distinguish between those of hereditary and sporadic etiology. The ability to make such distinctions may be of diagnostic, prognostic, and therapeutic utility.