MLH1 Research Articles

Implementing an integrated molecular classification for gastric cancer from endoscopic biopsies using on-slide tests.

The availability of more effective biological therapy can improve outcomes of gastric cancer (GC), but most patients do not have access to personalized treatment. GC molecular classification helps identify patients suitable for specific therapies and provides useful prognostic information. To date, only a small number of patients have access to molecular classification. We proposed a working molecular classification that can be delivered using on-slide tests available in most histopathology laboratories. We used eight on-slide tests [in situ hybridization (ISH) for Epstein-Barr virus-encoded small ribonucleic acid (EBER) and immunohistochemistry (IHC) for MutL homolog 1 (MLH1), PMS1 homolog 2 (PMS2), MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), E-cadherin, β-catenin and p53] to classify GC into one of six categories: GC associated with Epstein-Barr virus (GC-EBV), GC mismatch repair deficient (GC-dMMR), GC with epithelial-mesenchymal transition (GC-EMT), GC with chromosomal instability (GC-CIN), GC genomically stable (GC-GS) and GC not otherwise specified (GC-NOS)∕indeterminate. The classification has provision also for current and future on-slide companion diagnostic (CDx) tests necessary to select specific biological therapies and, as proof of principle, in this study we used three CDx tests currently required for the management of GC [human epidermal growth factor receptor 2 (Her2), programmed cell death-ligand 1 (PD-L1) 22C3 and Claudin18.2 (CLDN18.2)]. This paper describes the necessary tissue pathways and laboratory workflow and assesses the feasibility of using this classification prospectively on small endoscopic biopsies of gastric and gastroesophageal junction adenocarcinoma. This work demonstrates that such molecular classification can be implemented in the context of a histopathology diagnostic routine with little impact on turnaround times and laboratory capacity. The widespread adoption of a molecular classification for GC will help refine prognosis and guide the choice of more appropriate biological therapy for these patients.

Epigenetic differences in the tumor suppressor genes MLH1 and p16INK4a between Nepalese and Swedish patients with colorectal cancer

Abstract Objectives Colorectal cancer (CRC) is one of the most prevalent cancer types worldwide, exhibiting significant variance in incidence rates across different ethnicities and geographical regions. Notably, there is a rising incidence of CRC among younger adults, particularly evident in advanced stages, with a more pronounced trend observed in developing nations. Epigenetic alterations potentially play a role in the early onset of CRC and could elucidate interpopulation disparities. This study aimed to examine DNA methylation levels in the tumor suppressor genes MLH1 and p16INK4a, comparing Nepalese and Swedish patients with CRC. Methods Patients who underwent CRC surgery at Tribhuvan University Teaching Hospital, Nepal (n=39), and Sahlgrenska University Hospital, Sweden (n=39) were included. Demographic and clinicopathological data were analyzed, and pyrosequencing was employed to determine methylation levels in the MLH1 promoter region and the first exon of p16INK4a in tumor tissues and adjacent mucosa located 10 cm from the tumor site. Subsequently, methylation status was compared between Nepalese and Swedish patients and correlated with clinicopathological parameters. Results Nepalese and Swedish patients displayed equal levels of MLH1 and p16INK4a methylation in tumors, but Nepalese patients exhibited a significantly higher level of MLH1 methylation in mucosa compared to Swedish patients (p=0.0008). Moreover, a greater proportion of Nepalese patients showed MLH1 methylation in mucosa compared to Swedish patients (31 vs. 2.6 %). Aberrant methylation of p16INK4a was also observed in the mucosa of Nepalese patients, characterized by high methylation at specific sites rather than uniform methylation across CpG sites. There were no significant differences in methylation levels based on tumor location among Nepalese patients, whereas Swedish patients exhibited higher methylation in right- compared to left-sided colon tumors. Swedish patients showed an increase in p16INK4a methylation in tumors with advancing age. Conclusions Nepalese and Swedish patients displayed equal levels of MLH1 and p16INK4a methylation in tumors. In contrast, Nepalese patients had a higher level of MLH1 methylation as well as aberrant methylation of p16INK4a in mucosa compared to Swedish patients. These epigenetic differences may be linked to environmental and lifestyle factors. Ongoing research will further explore whether hypermethylation in the mucosa of Nepalese patients is associated with tumorigenesis and its potential utility in screening high-risk patients or predicting recurrence.

MLH1 Inhibits Metastatic Potential of Pancreatic Ductal Adenocarcinoma via Downregulation of GPRC5C

DNA mismatch repair gene MutL homolog-1 (MLH1) has divergent effects in many cancers; however, its impact on the metastasis of pancreatic ductal adenocarcinoma (PDAC) remains unclear. In this study, MLH1 stably overexpressed (OE) and knockdowned (KD) sublines were established. Wound healing and transwell assays were used to evaluate cell migration/invasion. In vivo metastasis was investigated in orthotopic implantation models (severe combined immunodeficiency mice). RT-qPCR and western blotting were adopted to show gene/protein expression. MLH1 downstream genes were screened by transcriptome sequencing. Tissue microarray–based immunohistochemistry was applied to determine protein expression in human specimens. In successfully generated sublines, OE cells presented weaker migration/invasion abilities, compared with controls, whereas in KD cells, these abilities were significantly stronger. The metastasis-inhibitory effect of MLH1 was also observed in mice. Mechanistically, G protein–coupled receptor, family C, group 5, member C (GPRC5C) was a key downstream gene of MLH1 in PDAC cells. Subsequently, transient GPRC5C silencing effectively inhibited cell migration/invasion and remarkably reversed the proinvasive effect of MLH1 knockdown in KD cells. In animal models and human PDAC tissues, tumoral GPRC5C expression, negatively associated with MLH1 expressions, was positively correlated with histologic grade, vessel invasion, and poor cancer-specific survival. In conclusion, MLH1 inhibits the metastatic potential of PDAC via downregulation of GPRC5C.

Secondary findings in genes related to cancer phenotypes in Turkish exome sequencing data from 2020 individuals.

Big data generated from exome sequencing (ES) and genome sequencing (GS) analyses can be used to detect actionable and high-penetrance variants that are not directly associated with the primary diagnosis of patients but can guide their clinical follow-up and treatment. Variants that are classified as pathogenic/likely pathogenic and are clinically significant but not directly associated with the primary diagnosis of patients are defined as secondary findings (SF). The aim of this study was to examine the frequency and variant spectrum of cancer-related SF in 2020 Turkish ES data and to discuss the importance of the presence of cancer-related SF in at-risk family members in terms of genetic counseling and follow-up. A total of 2020 patients from 2020 different families were evaluated by ES. SF were detected in 28 unrelated cases (1.38%), and variants in BRCA2 (11 patients) and MLH1 (4 patients) genes were observed most frequently. A total of 21 different variants were identified, with 4 of them (c.9919_9932del and c.3653del in the BRCA2 gene, c.2002A>G in the MSH2 gene, c.26_29del in the TMEM127 gene) being novel variations. In three different families, c.1189C>T (p.Gln397*) variation in BRCA2 gene was detected, suggesting that this may be a common variant in the Turkish population. This study represents the largest cohort conducted in the Turkish population, examining the frequency and variant spectrum of cancer-related SF. With the identification of frequent variations and the detection of novel variations, the findings of this study have contributed to the variant spectrum. Genetic testing conducted in family members is presented as real-life data, showcasing the implications in terms of counseling, monitoring, and treatment through case examples.

EPM2AIP1 immunohistochemistry is inadequate as a surrogate marker for MLH1 promoter hypermethylation testing in colorectal cancer

MLH1 promoter hypermethylation (MPH) analysis is an essential step in the universal tumor testing algorithm for Lynch syndrome, the most common inherited predisposition to colorectal cancer (CRC). MPH usually indicates sporadic CRC. EPM2AIP1 gene shares the same promoter as MLH1, therefore MPH should also silence EPM2AIP1 transcription leading to loss of protein expression on immunohistochemistry (IHC). It has been previously reported that EPM2AIP1 IHC can be used as a surrogate for MPH in endometrial cancer. Our goal was to evaluate the feasibility of EPM2AIP1 IHC as a surrogate for MPH in CRC. 101 microsatellite instable CRC cases were selected, including 19 cases from whole tumor sections and 82 cases from tissue microarrays. 74 cases were with MPH and 27 without MPH. All 74 cases with MPH showed absent MLH1 by IHC, but only 47 (64%) exhibited loss of expression of EPM2AIP1. Of the 27 cases without MPH, 9 (33%) cases had unexpected loss of EPM2AIP1 expression. Of note, 10 cases were MLH1-mutated Lynch syndrome without MPH, and 2 of these cases showed unexpected loss of EPM2AIP1 staining. Of the 6 cases with double somatic mutations of MLH1 gene (without MPH), only 4 cases demonstrated intact expression of EPM2AIP1 as expected. Taken together, EPM2AIP1 loss was 64% sensitive and 67% specific for MPH, with an accuracy of 64%. We conclude that, unless stain quality improves with different clones or platforms, EPM2AIP1 IHC will likely not be useful as a surrogate test for MPH in CRC.

AMPK Deficiency Increases DNA Methylation and Aggravates Colorectal Tumorigenesis in AOM/DSS Mice.

The incidence of colorectal cancer (CRC) is closely linked to metabolic diseases. Accumulating evidence suggests the regulatory role of AMP-activated protein kinase (AMPK) in cancer metabolic reprogramming. In this study, wild-type and AMPK knockout mice were subjected to azoxymethane-induced and dextran sulfate sodium (AOM/DSS)-promoted colitis-associated CRC induction. A stable AMPK-deficient Caco-2 cell line was also established for the mechanistic studies. The data showed that AMPK deficiency accelerated CRC development, characterized by increased tumor number, tumor size, and hyperplasia in AOM/DSS-treated mice. The aggravated colorectal tumorigenesis resulting from AMPK ablation was associated with reduced α-ketoglutarate production and ten-eleven translocation hydroxylase 2 (TET2) transcription, correlated with the reduced mismatch repair protein mutL homolog 1 (MLH1) protein. Furthermore, in AMPK-deficient Caco-2 cells, the mRNA expression of mismatch repair and tumor suppressor genes, intracellular α-ketoglutarate, and the protein level of TET2 were also downregulated. AMPK deficiency also increased hypermethylation in the CpG islands of Mlh1 in both colonic tissues and Caco-2 cells. In conclusion, AMPK deficiency leads to reduced α-ketoglutarate concentration and elevates the suppressive epigenetic modifications of tumor suppressor genes in gut epithelial cells, thereby increasing the risk of colorectal tumorigenesis. Given the modifiable nature of AMPK activity, it holds promise as a prospective molecular target for the prevention and treatment of CRC.

HGG-26. MLH1, MSH2, MSH6, AND PMS2 IMMUNOHISTOCHEMISTRY REPRESENTS A HIGHLY SENSITIVE SCREENING METHOD FOR MISMATCH REPAIR DEFICIENCY SYNDROMES IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract BACKGROUND Pediatric central nervous system (CNS) tumors can occur as first manifestations of cancer predisposition syndromes (CPS) resulting from pathogenic variants in DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2. Early detection of MMR deficiencies (MMRD) may warrant the therapeutic use of checkpoint inhibitors and enable genetic consulting for the patient’s families. METHODS We prospectively screened 128 pediatric high-grade gliomas (pedHGG) for MMR protein expression by immunohistochemistry (IHC) as part of the HIT-HGG-2013 clinical trial. We compared IHC results to independently collected patient information on CPS including MMRD to test the method’s efficiency in screening for Lynch syndrome and Constitutional MMRD syndrome CMMRD. RESULTS From 128 successfully tested tumors, ten cases (10/128, 7.8%), showed loss of expression of at least one of the MMR proteins. In seven of the ten affected patients (7/128, 5.5%), genetic testing uncovered heterozygous (6/10) or homozygous (1/10) pathogenic germline variants in MMR genes (MLH1, MSH2 or MSH6) confirming the diagnosis of Lynch syndrome respectively CMMRD. In three cases (3/128 2.3%), MMR alterations were restricted to tumor tissue. The group of diffuse midline gliomas, H3 K27-altered, (67/128, 52.3%) did not show MMR alterations, while either somatic or germline MMR mutations were detected in two of eight patients with diffuse hemispheric glioma, H3 G34-mutant. In 88 patients (88/128, 68.8%) either clinical or molecular-genetic information on CPS could be provided. All ten patients with signs of MMRD were successfully detected by IHC from tumor tissue. CONCLUSIONS IHC for MMR proteins represents a highly sensitive screening method for MMRD in pedHGG. Considering the occurrence of at least 11.5% (7/61) of MMRD in pediatric patients with diffuse high-grade glioma excluding DMG, MMRD IHC should be part of routine diagnostics as cost-effective, broadly available and robust screening method.

A second hereditary cancer predisposition syndrome in a patient with lynch syndrome and three primary cancers

Current National Comprehensive Cancer Network ® (NCCN ®) guidelines for Colorectal Genetic/Familial High-Risk Assessment provide limited guidance for genetic testing for individuals with already diagnosed hereditary cancer conditions. We are presenting the case of a 36-year-old woman who was diagnosed with Lynch Syndrome at age 23 after genetic testing for a familial variant (c.283del) in the MLH1 gene. The patient had a previous history of Hodgkin Lymphoma at the time of familial variant testing, and she would later develop stage IIIa cecal adenocarcinoma at age 33 and metastatic papillary thyroid carcinoma at age 35. The patient’s family history included a first-degree relative who was diagnosed with colorectal cancer at age 39, multiple second-degree relatives with colorectal, endometrial, and stomach cancer, and third and fourth-degree relatives with breast cancer. In light of her personal and family history, a comprehensive cancer panel was recommended. This panel found a second hereditary cancer predisposition syndrome: a likely pathogenic variant (c. 349 A > G) in the CHEK2 gene. This specific CHEK2 variant was recently reported to confer a moderately increased risk for breast cancer. The discovery of this second cancer predisposition syndrome had important implications for the patient’s screening and risk management. While uncommon, the possibility of an individual having multiple cancer predisposition syndromes is important to consider when evaluating patients and families for hereditary cancer, even when a familial variant has been identified.

OPTIMIZATION AND VALIDATION OF FOXP3, BRAFV600E AND MMR IMMUNOHISTOCHEMISTRY STAINING PROTOCOLS

To ensure consistent and repeatable results, t Here, we described could enhance the IHC signals of the targeted antibodies. Mismatch Repair (MMR) proteins such as MutL homolog 1 (MLH1), Mut S homolog 2 (MSH2), Mut S homolog 6 (MSH6), and postmeiotic segregation 2 (PMS2), including B-rapidly accelerated fibrosarcoma with V600E mutation (BRAFV600E) protein and forkhead box P3 (FOXP3) protein, are important for colorectal cancer diagnosis. The IHC staining was performed using different archival tissue controls and commercial antibodies. MLH1 and PMS2 showed higher staining intensity after an overnight incubation at a higher concentration of primary antibody (1:50) compared to BRAFV600E (1:100). The enhanced MSH2 signal was likewise generated at a one-hour incubation period with an equivalent antibody concentration. However, even with a shorter incubation duration of one hour, MSH6 and FOXP3 generated good IHC signals when incubated with primary antibody at a lower antibody concentration of 1:100 and 1:300, respectively. The addition of a primary antibody linker improved the IHC signals for all targeted proteins. In conclusion, when using archival tissues, modifying the aforementioned IHC staining variables produces optimal staining for the MMR, BRAFV600E, and FOXP3 proteins.

Insights into MLH1 Methylation in Endometrial Adenocarcinoma through Pyrosequencing Analysis: A Retrospective Observational Study.

Background: In cancer care, the MLH1 gene is crucial for DNA mismatch repair (MMR), serving as a vital tumor suppressor. Evaluating MLH1 protein expression status, followed by analysis of MLH1 promoter methylation, has become a key diagnostic and prognostic approach. Our study investigates the complex link between MLH1 methylation and prognosis in endometrial adenocarcinoma (EA) patients. Methodology: MLH1 methylation status was accessed by a Pyrosequencing (PSQ) assay. Qualitative positivity for methylation was established if it exceeded the 11% cut-off; as well, a quantitative methylation analysis was conducted to establish correlations with clinicopathological data, relapse-free survival, and disease-free survival. Results: Our study revealed that 33.3% of patients without MLH1 methylation experienced relapses, surpassing the 23.3% in patients with methylation. Furthermore, 16.7% of patients without methylation succumbed to death, with a slightly higher rate of 17.6% in methylated patients. Qualitative comparisons highlighted that the mean methylation rate in patients experiencing relapse was 35.8%, whereas in those without relapse, it was 42.2%. This pattern persisted in disease-specific survival (DSS), where deceased patients exhibited a higher mean methylation level of 49.1% compared to living patients with 38.8%. Conclusions: Our findings emphasize the efficacy of PSQ for evaluating MLH1 methylation. While unmethylation appears to be associated with a higher relapse rate, the survival rate does not seem to be influenced by methylation. Quantitative percentages suggest that elevated MLH1 methylation is linked to relapse and mortality, though a study with a larger sample size would be essential for statistically significant results.

A collaborative review of the microsatellite instability/deficient mismatch repair phenotype in patients with upper tract urothelial carcinoma.

To perform a collaborative review of the literature exploring the microsatellite instability/deficient mismatch repair (MSI/dMMR) phenotype in patients with upper tract urothelial carcinoma (UTUC). A collaborative review of the literature available on Medline was conducted by the Cancer Committee of the French Association of Urology to report studies describing the genetic mechanisms, investigation, prevalence and impact of the MSI/dMMR phenotype in UTUC patients. The predominant genetic mechanism leading to the MSI/dMMR phenotype in UTUC patients is related to the constitutional mutation of one allele of the MMR genes MLH1, MSH2, MSH6 and PMS2 within Lynch syndrome. Indications for its investigation currently remain limited to patients with a clinical suspicion for sporadic UTUC to refer only those with a positive testing for germline DNA sequencing to screen for this syndrome. With regard to technical aspects, despite the interest of MSIsensor, only PCR and immunohistochemistry are routinely used to somatically investigate the MSI and dMMR phenotypes, respectively. The prevalence of the MSI/dMMR phenotype in UTUC patients ranges from 1.7% to 57%, depending on the study population, investigation method and definition of a positive test. Younger age and a more balanced male to female ratio at initial diagnosis are the main specific clinical characteristics of UTUC patients with an MSI/dMMR phenotype. Despite the conflicting results available in the literature, these patients may have a better prognosis, potentially related to more favourable pathological features. Finally, they may also have lower sensitivity to chemotherapy but greater sensitivity to immunotherapy. Our collaborative review summarises the available data from published studies exploring the MSI/dMMR phenotype in UTUC patients, the majority of which are limited by a low level of evidence.

Microsatellite Instability Testing and Prognostic Implications in Colorectal Cancer.

Given the crucial predictive implications of microsatellite instability (MSI) in colorectal cancer (CRC), MSI screening is commonly performed in those with and at risk for CRC. Here, we compared results from immunohistochemistry (IHC) and the droplet digital PCR (ddPCR) MSI assay on formalin-fixed paraffin-embedded tumor samples from 48 patients who underwent surgery for colon and rectal cancer by calculating Cohen's kappa measurement (k), revealing high agreement between the methods (k = 0.915). We performed Kaplan-Meier survival analyses and univariate and multivariate Cox regression to assess the prognostic significance of ddPCR-based MSI and to identify clinicopathological features associated with CRC outcome. Patients with MSI-high had better overall survival (OS; p = 0.038) and disease-free survival (DFS; p = 0.049) than those with microsatellite stability (MSS). When stratified by primary tumor location, right-sided CRC patients with MSI-high showed improved DFS, relative to those with MSS (p < 0.001), but left-sided CRC patients did not. In multivariate analyses, MSI-high was associated with improved OS (hazard ratio (HR) = 0.221, 95% confidence interval (CI): 0.026-0.870, p = 0.042), whereas the loss of DNA mismatch repair protein MutL homolog 1 (MLH1) expression was associated with worse OS (HR = 0.133, 95% CI: 0.001-1.152, p = 0.049). Our results suggest ddPCR is a promising tool for MSI detection. Given the opposing effects of MSI-high and MLH1 loss on OS, both ddPCR and IHC may be complementary for the prognostic assessment of CRC.

Null Mismatch Repair Proteins Expression Reveals the Temporal Molecular Events in Lynch Syndrome-Related Cancers.

The immunohistochemical assessment of mismatch repair (MMR) proteins represents a pivotal screening tool for identifying Lynch syndrome (LS)-related cancers, as the loss of their expression often indicates MMR dysfunction associated with genetic or epigenetic alterations. Frequently, LS-related colorectal cancers present germline pathogenic variants in the MLH1 or MSH2 genes, which result in the simultaneous immunohistochemical loss of MLH1 and PMS2 or MSH2 and MSH6 proteins expression, respectively. Less commonly observed is the single involvement of the MSH6 or PMS2 proteins expression, indicative of the presence of germline pathogenic variants in the corresponding genes. Extremely rarely reported are the null immunohistochemistry phenotypes represented by the complete loss of expression of all MMR proteins. The molecular mechanisms contributing to the raising of this latter uncommon immunohistochemical phenotype are derived from the combination of pathogenic germline variants in MMR genes with the somatic hypermethylation of the MLH1 gene promoter. This study focuses on elucidating the molecular cascade leading to the development of the null immunohistochemical phenotype, providing valuable insights into understanding the sequential molecular events driving the LS-associated tumorigenesis, which may have pivotal implications in the clinical management of patients with LS-related cancers.

The Use of Glucagon-like Peptide-1 Receptor Agonists in Patients with Type 2 Diabetes Mellitus Does Not Increase the Risk of Pancreatic Cancer: A U.S.-Based Cohort Study.

GLP-1 RAs are widely used for T2DM treatment due to their cardiorenal and metabolic benefits. This study examines the risk of pancreatic cancer with GLP-1 RA use in patients with T2DM. We analyzed TriNetX's deidentified research database using the U.S. Collaborative Network comprising 62 healthcare organizations across the U.S.A. Patients with T2DM were split into two cohorts: one receiving GLP-1 RAs, and one not receiving GLP-1 RAs. We excluded patients with known risk factors for pancreatic cancer, including pancreatic cysts, a personal or family history of BRCA1, BRCA2, CDKN2A, KRAS, MEN1, MLH1, MSH2, NOTCH1, PALB2, PMS2, and PRSS1S genes, family history of pancreatic cancer, and VHL syndrome. Using a 1:1 propensity score-matching model based on baseline characteristics and comorbidities, we created comparable cohorts. We then compared the rate of pancreatic cancer between the two cohorts at a 7-year interval. Out of 7,146,015 identified patients with T2DM, 10.3% were on a GLP-1 RA and 89.7% were not. Post-PSM, 721,110 patients were in each group. Patients on GLP-1 RAs had a 0.1% risk compared to a 0.2% risk of pancreatic cancer in the 7-year timeframe. The use of GLP-1 RAs in patients with type 2 diabetes mellitus (T2DM) does not appear to substantially elevate the risk of pancreatic cancer; in fact, it may potentially exert a protective effect.

Associations Between Fusobacterium nucleatum and msh2, mlh1, and msh6 Gene Expression in Colorectal Cancer

Background: Colorectal cancer (CRC) is a major global health concern, and the link with Fusobacterium nucleatum has received considerable attention. Objectives: This study aimed to explore the prevalence of F. nucleatum and to assess the expression of the msh2, mlh1, and msh6 genes in CRC patients compared to a control group using real-time PCR. Methods: Forty CRC patients and twenty individuals from a control group participated in this study. Gastroenterologists collected biopsy specimens from which DNA and RNA were extracted using a specialized tissue extraction kit. Complementary DNA (cDNA) was then synthesized. Real-time PCR was employed to evaluate the expression levels of the msh2, mlh1, and msh6 genes and the presence of the F. nucleatum-specific 16srRNA gene to determine the relative prevalence of this bacterium in each group. Results: Results indicated a higher prevalence of the F. nucleatum-specific 16srRNA gene in CRC patients than in the control group. Additionally, expression levels of the msh2, mlh1, and msh6 genes were significantly higher in the cancer group, suggesting their role in CRC pathogenesis. The distribution of F. nucleatum was particularly high in the sigmoid and rectum areas of the colon. Conclusions: This study underscores the significance of F. nucleatum in CRC and provides insights into its association with altered gene expression patterns. Understanding the prevalence of F. nucleatum and its impact on msh2, mlh1, and msh6 genes may aid in developing improved diagnostic and therapeutic strategies for CRC. Further research is necessary to elucidate these relationships more comprehensively.

Clinicopathological characteristics of Lynch-like syndrome.

Lynch-like syndrome (LLS) has recently been proposed as a third type of microsatellite instability (MSI) tumor after Lynch syndrome (LS) and sporadic MSI colorectal cancer (CRC) without either a germline variant of mismatch repair (MMR) genes or hypermethylation of the MLH1 gene. The present study aimed to clarify and compare the clinicopathological characteristics of LLS with those of the other MSI CRC subtypes. In total, 2634 consecutive patients with CRC who underwent surgical resection and subsequently received universal tumor screening (UTS), including MSI analysis were enrolled between January 2008 and November 2019. Genetic testing was performed in patients suspected of having Lynch syndrome. UTS of the cohort found 146 patients with MSI CRC (5.5%). Of these, excluding sporadic MSI CRC, 30 (1.1%) had a diagnosis of LS, and 19 (0.7%) had no germline pathogenic variants of the MMR gene. The CRC type in the latter group was identified as LLS. LLS occurred significantly more often in young patients, was left-sided, involved a KRAS variant and BRAF wild-type, and had a higher concordance rate with the Revised Bethesda Guidelines than sporadic MSI CRC. No significant differences were observed in terms of the clinicopathological factors between LLS and LS-associated MSI CRC; however, LLS had a lower frequency of LS-related neoplasms compared with LS. Distinguishing clinically between LS and LLS was challenging, but the incidence of neoplasms was higher in LS than in LLS, suggesting the need for different screening and surveillance methods for the two subtypes.

Identification of a Catalytic Lysine Residue Conserved Among GHKL ATPases: MutL, GyrB, and MORC

DNA mismatch repair endonuclease MutL is a member of GHKL ATPase superfamily. Mutations of MutL homologs are causative of a hereditary cancer, Lynch syndrome. We characterized MutL homologs from human and a hyperthermophile, Aquifex aeolicus, (aqMutL) to reveal the catalytic mechanism for the ATPase activity. Although involvement of a basic residue had not been conceived in the catalytic mechanism, analysis of the pH dependence of the aqMutL ATPase activity revealed that the reaction is catalyzed by a residue with an alkaline pKa. Analyses of mutant aqMutLs showed that Lys79 is the catalytic residue, and the corresponding residues were confirmed to be critical for activities of human MutL homologs, on the basis of which a catalytic mechanism for MutL ATPase is proposed. These and other results described here would contribute to evaluating the pathogenicity of Lynch syndrome-associated missense mutations. Furthermore, it was confirmed that the catalytic lysine residue is conserved among DNA gyrases and microrchidia ATPases, other members of GHKL ATPases, indicating that the catalytic mechanism proposed here is applicable to these members of the superfamily.

Clinicopathological Implications of Mismatch Repair Status in Endometrioid Endometrial Cancer in Duhok City.

DNA mismatch repair (MMR) is a specialized system that corrects errors in DNA replication, namely, base substitution mismatches and minor insertion-deletion mismatches. The deficient mismatch repair (d-MMR) protein plays a vital role in predicting the prognosis of endometrioid carcinoma. The study aimed to determine the prevalence of MMR errors in endometrial cancer (EC) and their correlation with clinicopathological features. We examined the immunohistochemistry presence of four MMR proteins in 50 samples of EC tissues that were preserved in formalin and embedded in paraffin. The proteins identified were MutL homolog 1 (MLH1), post-meiotic segregation increased 2 (PMS2), MutS homolog 2 (MSH2), and MutS homolog 6 (MSH6). The study examined several clinicopathological characteristics and conducted MMR phenotyping. The findings revealed that among the 50 cases of EC, 40% of patients had grade I disease and 78% had stage I malignancy. Furthermore, among the 50 individuals evaluated, 56% exhibited competence in MMR, whereas 44% displayed loss in nuclear expression of MMR. The rate of MLH1 and PMS2 protein loss was recorded as the greatest, at 18%, while the loss of MSH2 and MSH6 was documented at 6%. Within the same range, the majority of patients with d-MMR were above the age of 50 years. The majority of the recruited EC patients in this study showed advanced age and a high percentage of d-MMR status.

Chronic low-dose chromium VI exposure induces oxidative stress and apoptosis with altered expressions of DNA repair genes and promoter hypermethylation in the liver of Swiss albino mice.

The present investigation dealt with harmful effects of hexavalent chromium (Cr [VI]) on liver of Swiss albino mice. This variant exhibited cytotoxicity, mutagenicity, and carcinogenicity. Our study focused on elucidating the hepatotoxic effects of chronic low-dose exposure to Cr (VI) (2, 5, and 10ppm) administered via drinking water for 4 and 8months. The observed elevation in SGPT, ALP, and SGOT and increased oxidative stress markers unequivocally confirmed the severe disruption of liver homeostasis at these low treatment doses. Noteworthy alterations in histoarchitecture, body weight, and water intake provided further evidences of the harmful effects of Cr (VI). Production of reactive oxygen species (ROS) during metabolism led to DNA damages. Immunohistochemistry and qRT-PCR analyses revealed that chronic low-dose exposure of Cr (VI) induced apoptosis in liver tissue. Our study exhibited alterations in the expression pattern of DNA repair genes (Rad51, Mutyh, Mlh1, and Ogg1), coupled with promoter hypermethylation of Mutyh and Rad51, leading to transcriptional inhibition. Our findings underscored the potential of low-dose Cr (VI) exposure on hepatotoxicity by the intricate interplay between apoptosis induction and epigenetic alterations of DNA repair genes.

Abstract 2566: Molecular characteristic profile in early colorectal cancer with V600E and non-V600E BRAF mutations

Abstract Background: The BRAF V600E mutation in colorectal cancer (CRC) is a significant indicator of poor prognosis, especially in tumors exhibiting microsatellite instability (MSI). It is more common on the right side of the colon but can occur anywhere within the colon. Although other BRAF mutations existed, current research primarily focused on BRAF V600E. Most clinical data sets were centered on advanced colorectal cancer. There was limited understanding of the molecular characteristics of early-stage colorectal cancer patients with BRAF mutations, which impacted postoperative prognosis management, including refining adjuvant therapy regimens. Therefore, we analyzed the molecular characteristics of early-stage CRC surgical patients. Methods: Tumor tissue from 767 early-stage CRC patients diagnosed between 2021 to 2022 in China were analyzed using NGS (panel on 733 gene) to validate the mutation characteristics. Tumor specimens were tested for the BRAF mutation and MSI status. Result: Among 767 early CRC cases tested, BRAF mutations accounted for 6.52% (50/767), with 62% (31/50) being BRAF V600E, followed by 14% (7/50) for BRAF D594G. Notably, there were two cases of fusion variations, TRIM24-BRAF, and one case of BRAF copy gain. Among the BRAF mutation population, 11 cases were MSI-H, with 8 of them being BRAF V600E/MSI-H. Only one MSH-H patient had a germline mutation in the MLH1 gene, with a concurrent mutation in BRAF K601T.Interestingly, when comparing the mutation profiles of the BRAF V600E population with those of the non-V600E BRAF mutation population, we found distinct co-mutated genes despite both being BRAF mutations. In the BRAF V600E group, the top three frequently mutated genes were TP53 (52%), RNF43 (32%), and SMAD4 (32%). Additionally, the BRAF V600E/MSI-H subgroup presented a distinct mutation profile, with minimal occurrences of TP53 and SMAD4 mutations. In the non-V600E BRAF mutation group, the top three frequently mutated genes were TP53 (84%), APC (63%), and KRAS (32%). Conclusions: In examining the mutation profile characteristics of early colorectal cancer with BRAF mutations, the proportion of BRAF V600E/MSI-H appeared to have been lower than that reported in advanced cases. Furthermore, in terms of the frequency of co-mutated genes, it seemed that BRAF V600E had exhibited a higher malignant driving mutation effect as a single gene, while non-V600E mutations had required more cooperative tumor suppressor genes or oncogenes. Analyzing the molecular characteristics of early colorectal cancer patients with V600E and non-V600E BRAF mutations could help improve postoperative management and further our understanding of tumor progression. Citation Format: Zhang Quan An. Molecular characteristic profile in early colorectal cancer with V600E and non-V600E BRAF mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2566.