Mutations In Mismatch Repair Genes Research Articles

Mismatch repair enzymes regulate telomere recombination in Saccharomycescerevisiae

DNA mismatch repair (MMR) is a crucial mechanism that ensures chromosome stability and prevents the development of various human cancers. Apart from its role in correcting mismatches during DNA replication, MMR also plays a significant role in regulating recombination between non-identical sequences, a process known as homeologous recombination. Telomeres, the protective ends of eukaryotic chromosomes, possess sequences that are not perfectly homologous. While telomerase primarily maintains telomere length in the yeast Saccharomyces cerevisiae, recombination between telomeres becomes a major pathway for length maintenance in cells lacking telomerase. This study investigates the participation of MMR in telomere recombination. Our findings reveal that mutations in MMR genes activate type I recombination. Notably, among the MMR proteins, MutSα (Msh2 and Msh6) and MutLα (Mlh1 and Pms1) exerted the most pronounced effects on telomere recombination. We also found that yeast cells containing simple human telomeric TTAGGG DNA sequences preferentially utilize type II recombination to maintain their telomeres, highlighting the influence of the heterogeneous nature of yeast telomeric sequences on type II recombination. Furthermore, our observations indicate that MMR activity is indispensable for its impact on telomere recombination. Collectively, these results contribute to a more comprehensive understanding of the role of MMR in telomere recombination.

Frameshift mutations in peripheral blood as a biomarker for surveillance of Lynch syndrome.

Lynch syndrome is a hereditary cancer predisposition syndrome caused by germline mutations in DNA mismatch repair genes, which lead to high microsatellite instability and frameshift mutations at coding mononucleotide repeats in the genome. Recurrent frameshift mutations in these regions are thought to play a central role in the increased risk of various cancers, but no biomarkers are currently available for the surveillance of high microsatellite instability-associated cancers. A frameshift mutation-based biomarker panel was developed and validated by targeted next-generation sequencing of supernatant DNA from cultured high microsatellite instability colorectal cancer cells. This panel supported selection of 122 frameshift mutation targets as potential biomarkers. This biomarker panel was then tested using matched tumor, adjacent normal tissue, and buffy coat samples (53 samples) and blood-derived cell-free DNA (cfDNA) (38 samples) obtained from 45 high microsatellite instability and mismatch repair-deficient patients. We also sequenced cfDNA from 84 healthy participants to assess background noise. Recurrent frameshift mutations at coding mononucleotide repeats were detectable not only in tumors but also in cfDNA from high microsatellite instability and mismatch repair-deficient patients, including a Lynch syndrome carrier, with a varying range of target detection (up to 85.2%), whereas they were virtually undetectable in healthy participants. Receiver operating characteristic curve analysis showed high sensitivity and specificity (area under the curve = 0.94) of the investigated panel. We demonstrated that frameshift mutations can be detected in cfDNA from high microsatellite instability and mismatch repair-deficient patients and asymptomatic carriers. The 122-target frameshift mutation panel described here has promise as a tool for improved surveillance of high microsatellite instability and mismatch repair-deficient patients, with the potential to reduce the frequency of invasive screening methods for this high-cancer-risk cohort.

Abstract IA013: Unique metabolic changes in Lynch syndrome-associated endometrial cancer

Abstract Lynch syndrome-associated endometrial cancers (ECs) are defined by germline mutations in DNA mismatch repair genes, including MSH2, MSH6, PMS2, and MLH1. Lynch syndrome (LS) tumors have received increased attention in the therapeutic context as these tumors are microsatellite instable and often reflect increased immune cell infiltration and enhanced response to immunotherapy. However, relatively little attention has been paid to pathogenesis of LS ECs. Women with LS represent an at-risk population who could benefit from cancer interception strategies due to a 60% cumulative lifetime risk of developing EC. We developed a mouse model of MSH2 loss targeted to the uterus to evaluate EC pathogenesis, with the goal of elucidating mechanisms that alter disease penetrance for women with hereditary risk for EC development. Our Progesterone Receptor-Cre Msh2f lox/flox mice (abbreviated Msh2KO) develop ECs that closely mimic human EC. Whole transcriptome profiling of Msh2KO EC and Msh2KO normal endometrium compared to wild-type endometrium identified “Mitochondrial dysfunction” as a significantly altered pathway in Msh2 deficiency, prompting our in-depth evaluation of mitochondrial alterations in Msh2KO mice, MSH2-deficient cell lines, and LS-associated EC patient specimens. We will discuss the impact of MSH2 deficiency across multiple measures of mitochondrial content, integrity, function, and metabolomic analyses in ECs using preclinical models and human tissue studies. Overall, our studies reveal that MSH2-deficient EC is characterized by mitochondrial DNA damage, mitochondrial content reduction, and decreased mitochondrial function. In addition, decreased mitochondrial respiration for energy production facilitates metabolic reprogramming toward glycolysis. We are conducting further studies to define mechanisms underlying mitochondrial defects due to MSH2 loss and opportunities for intervention. Mitochondrial and metabolic aberrations could serve as novel biomarkers for EC development and targets for cancer prevention in women with LS. Citation Format: Melinda S. Yates. Unique metabolic changes in Lynch syndrome-associated endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA013.

Abstract A010: Mitochondrial depletion and metabolic reprogramming is a novel phenotype of Lynch syndrome-related endometrial carcinogenesis

Abstract Background: Lynch syndrome (LS), defined by mutations in DNA mismatch repair genes including MSH2, carries a 60% lifetime risk of developing endometrial cancer (EC). Our recent studies in Msh2-deficient mice revealed mitochondrial dysfunction in EC pathogenesis. We aimed to define mechanisms of mitochondrial dysfunction and impact on metabolism to identify targets for EC prevention. Methods: We assessed the effects of MSH2 loss on EC pathogenesis using a novel mouse model (PR-Cre Msh2flox/flox, abbreviated Msh2KO), primary cell lines from this model, and human EC cells with isogenic MSH2-knockdown. mtDNA damage was measured using RT-PCR. Mitochondrial content was quantified in vitro by immunofluorescence (IF) for TOM20 and in vivo by anti-TOM20 immunohistochemistry (IHC). Mitochondrial stress tests (MSTs) assessed mitochondrial function in vitro. Metabolite profiling via high resolution ion chromatography mass spectrometry was conducted on Msh2KO EC and MSH2-intact EC from the Pten +/− mouse model. Glycolysis was suppressed in vitro by replacing glucose with galactose in culture media. Results: Baseline mtDNA damage was elevated in MSH2-deficient MFE280 and RL95-2 EC cell lines by 4.8- to 7.9-fold compared to MSH2-intact KLE and Hec50 cells (p<0.001). MSH2 knockdown increased mtDNA damage in KLE and Hec50 cells by 2.5- to 5.7-fold (p<0.05). MSH2-deficient mouse and human cell lines had lower mitochondrial content compared to MSH2-intact counterparts as indicated by TOM20 staining, with over 60% reductions in mitochondrial volume in MSH2-deficient mouse cell lines and over 95% reductions in MSH2-deficient human EC cells compared to respective controls (p<0.0001). IHC showed reduced mitochondrial content during EC development in Msh2KO mice and in human LS EC tissues. MSTs revealed decreased baseline and induced mitochondrial function in Msh2KO cells relative to controls and in MSH2-deficient human cells (MFE280, RL95-2, and Hec59) compared to MSH2-intact human cells (KLE, Hec1a, Hec50), with p<0.0001. Within the top 10 differentially modulated metabolites in Msh2KO EC compared to Pten +/− EC were glucose 6-phosphate (log2 fold-change (FC) -1.79), mannose 6-phosphate (log2FC -1.77), fructose 6-phosphate (log2FC -1.54), and lactate (log2FC 0.68), padj<0.05. Galactose reduced viability in MSH2-deficient mouse and human cells more than in their MSH2-intact counterparts (p<0.01). Conclusions: We identified mitochondrial depletion as a novel phenotype of MSH2-deficient EC. mtDNA damage in MSH2-deficient cells suggests a role for MSH2 in mtDNA integrity. MSH2-deficient EC is characterized by mitochondrial DNA damage, mitochondrial content reduction, and decreased mitochondrial function. Decreased mitochondrial respiration for energy production facilitates metabolic reprogramming toward glycolysis, signified by increased usage of glycolytic intermediates and dependence on glycolysis for survival. Mitochondrial and metabolic aberrations could be evaluated as novel biomarkers for EC development and targets for prevention in women with LS. Citation Format: Mikayla B. Bowen, Brenda Melendez, Anna Zal, Collene Jeter, Wai Kin Chan, Lin Tan, Philip Lorenzi, Diana Moreno, Leah Peralta, Qian Zhang, Nisha Gokul, Karen Lu, Hyun-Eui Kim, Melinda S. Yates. Mitochondrial depletion and metabolic reprogramming is a novel phenotype of Lynch syndrome-related endometrial carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A010.

A tale of two pathways: Review of immune checkpoint inhibitors in DNA mismatch repair-deficient and microsatellite instability-high endometrial cancers.

The DNA mismatch repair (MMR) pathway is critical for correcting DNA mismatches generated during DNA replication. MMR-deficiency (MMR-D) leads to microsatellite instability (MSI) associated with an increased mutation rate, driving cancer development. This is particularly relevant in endometrial cancer (EC) as 25%-30% of tumors are of MMR-D/MSI-high (MSI-H) phenotype. Comprehensive assessment using immunohistochemistry (IHC) and sequencing-based techniques are necessary to fully evaluate ECs given the importance of molecular subtyping in staging and prognosis. This also influences treatment selection as clinical trials have demonstrated survival benefits for immune checkpoint inhibitors (ICIs) alone and in combination with chemotherapy for MMR-D/MSI-H EC patients in various treatment settings. As a portion of MMR-D/MSI-H ECs are driven by Lynch syndrome, an inherited cancer predisposition syndrome that is also associated with colorectal cancer, this molecular subtype also prompts germline assessment that can affect at-risk family members. Additionally, heterogeneity in the tumor immune microenvironment and tumor mutation burden (TMB) have been described by MMR mechanism, meaning MLH1 promoter hypermethylation versus germline/somatic MMR gene mutation, and this may affect response to ICI therapies. Variations by ancestry in prevalence and mechanism of MMR-D/MSI-H tumors have also been reported and may influence health disparities given observed differences in tumors of Black compared to White patients which may affect ICI eligibility. These observations highlight the need for additional prospective studies to evaluate the nuances regarding MMR-D heterogeneity as well as markers of resistance to inform future trials of combination therapies to further improve outcomes for patients with EC.

A164 OPTIMIZING A PROVINCIAL SCREENING PROGRAM FOR DETECTION OF LYNCH SYNDROME

Abstract Background Up to 30% of those with colorectal cancer (CRC) have an affected family member. Lynch syndrome (LS) is the most common inherited condition predisposing patients to CRC. LS is an autosomal dominant condition associated with microsatellite instability (MSI) and mutations in DNA mismatch repair (MMR) genes, and screening CRC cases ≤ age 70 can identify LS in a cost-effective manner. Manitoba launched Canada's first provincial reflex screening program for all CRC cases diagnosed ≤ age 70 using MMR immunohistochemistry (MMR-IHC) in 2017. Our prior 2022 study evaluating the program showed compliance rates of 89.4% for MMR-IHC reflex testing and a 75.8% overall referral rate of screen-positive cases to genetics. However, only 50% were referred directly by pathologists. Several modifications have since been implemented to enhance the program. These included providing feedback to pathologists, creating an “additional testing comment section” on synoptic pathology reports, and regular review of the pathology reports by a pathology assistant. Information materials were developed to encourage genetic testing among relatives. Aims Assess whether modifications to Manitoba’s universal screening protocol improved LS patient outcomes and determine any remaining factors to optimize. We specifically aimed to identify current compliance rates for MMR-IHC testing, the overall referral rate of screen-positive cases to genetics, uptake of genetic referrals and genetic testing as well as rate of cascade testing among relatives. Methods Data has been obtained by searching the provincial pathology database for “adenocarcinoma” in the colorectal specimen pathology reports. All specimens from 2022 and Q1/Q2 of 2023 were reviewed, meeting the criteria of: a) specimen from colon biopsy/excision containing the term “adenocarcinoma”, b) patient age ≤70 years, and c) missing MMR centralized by the IT/Pathology department. Additionally, a random sample of cases categorized as “MMR performed” will be audited. We are in the process of reviewing genetic referrals, genetic uptake and the frequency of reminders triggered to each pathologist. Results Currently, a total of 1,308 colonic adenocarcinoma specimens (811 from 2022, 497 for 2023) have been reviewed. Appropriate MMR testing was missed in only 1.5% of cases (13/811 =1.6% missed in 2022, 7/497= 1.4% missed in 2023). Conclusions Current MMR testing rates for colonic adenocarcinoma specimens within the Manitoba LS screening program have reached a current compliance rate of 98.5%, a substantial improvement from the prior audit (89.4% previously). Further data collection and analysis is ongoing and will also be presented. The reported rates of compliance are the highest that have been reported from any jurisdiction. This study highlights the sustained efforts required to improve detection of LS. Funding Agencies None

Establishing CRISPRi for Programmable Gene Repression and Genome Evolution in Cupriavidus necator.

Cupriavidus necator H16 is a "Knallgas" bacterium with the ability to utilize various carbon sources and has been employed as a versatile microbial cell factory to produce a wide range of value-added compounds. However, limited genome engineering, especially gene regulation methods, has constrained its full potential as a microbial production platform. The advent of CRISPR/Cas9 technology has shown promise in addressing this limitation. Here, we developed an optimized CRISPR interference (CRISPRi) system for gene repression in C. necator by expressing a codon-optimized deactivated Cas9 (dCas9) and appropriate single guide RNAs (sgRNAs). CRISPRi was proven to be a programmable and controllable tool and could successfully repress both exogenous and endogenous genes. As a case study, we decreased the accumulation of polyhydroxyalkanoate (PHB) via CRISPRi and rewired the carbon fluxes to the synthesis of lycopene. Additionally, by disturbing the expression of DNA mismatch repair gene mutS with CRISPRi, we established CRISPRi-Mutator for genome evolution, rapidly generating mutant strains with enhanced hydrogen peroxide tolerance and robustness in microbial electrosynthesis (MES) system. Our work provides an efficient CRISPRi toolkit for advanced genetic manipulation and optimization of C. necator cell factories for diverse biotechnology applications.

Abstract A015: Mutations in mismatch repair genes that alter subcellular localization of the protein predict resistance to standard therapy but response to cell cycle inhibitors

Abstract Hormone positive breast cancer is one of the most common cancer types in U.S. Every year, over 150,000 patients are detected with this cancer type of which, >40,000 patients will go on to die of their disease. We recently identified defects in a principal mismatch repair (MMR) gene, MLH1, as a driver of resistance to standard-care treatment and poor patient outcomes. Moreover, we demonstrated increased sensitivity to palbociclib, a CDK4/6 inhibitor, in experimental model systems in vitro and in vivo and in clinical trial data. MLH1 is mutated in 5-10% of treatment-resistant tumors, i.e., 4,000 patient tumors every year. However, most of these mutations are missense and variants of unknown significance (VUS). The impact of these mutations on mismatch repair capability and on treatment resistance remains uncertain. Here, we use experimental model systems (ER+/HER2- breast cancer cells, organoid models, and patient tumors) to study how variants of MLH1 and its subcellular localization predicts resistance to standard endocrine therapy but sensitivity to cell cycle inhibitors. We also assessed the ability of nuclear MLH1 to induce activation of G1/S cell cycle checkpoints by activating Chk2. Currently, there are few tests available in the clinic to predict response to therapies early in the timeline of hormone positive breast cancer patients. Due to the nature of the disease, this can result in years of overtreatment. Overtreatment comes at great financial, physical, and emotional cost to patients and their families. We for the first time show that stalling nuclear translocation of MLH1 inhibits Chk2 activation which counterbalances CDK4/6 activity. Therefore, cytoplasmic Mlh1 may be first in-class biomarker for CDK4/6 response in cancer types. Citation Format: Aloran Mazumder, Svasti Haricharan. Mutations in mismatch repair genes that alter subcellular localization of the protein predict resistance to standard therapy but response to cell cycle inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A015.

Colonoscopy and Upper Endoscopy Surveillance in Lynch Syndrome: A Longitudinal Study From a Large Tertiary Healthcare System

Background and AimsLynch syndrome (LS) is caused by pathogenic mutations in mismatch repair (MMR) genes. There is limited data on differences in colorectal cancer (CRC) surveillance by MMR genes, and an international consensus on surveillance based on genes is not established. We aimed to evaluate colonoscopy and EGD surveillance outcomes and compare CRC surveillance findings by the mutated gene. Methods101 patients with LS were included and colonoscopy results were compared by MMR mutation. Primary outcomes included the development and recurrence of adenoma, CRC, high-grade dysplasia (HGD), advanced adenoma (AA), and sessile serrated lesions (SSL). Logistic regressions evaluated the relationship between genes and the development or recurrence of primary outcomes. Survival analysis evaluated primary outcomes in patients with > 2 colonoscopies. EGD results were summarized. Results327 colonoscopies were reviewed. Compared to PMS2, MLH1 was associated with a higher risk of AA/HGD/CRC development (OR 9.85, 95% CI: 1.97-77.24) and MSH2 was associated with a higher risk of adenoma development (OR 4.17, 95% CI: 1.11-17.61). Among those with >2 colonoscopies, MLH1 (HR 18.98, 95% CI: 1.31-274.51) and MSH6 (HR 15.03, 95% CI: 1.16-194.65) had a higher risk of SSL compared to MSH2. Among patients who had adenoma detected once, MLH1 had a higher risk of adenoma recurrence compared to MSH6 (OR 14.59, 95% CI: 1.53-244.30) and PMS2 (OR 47.15, 95% CI: 4.26-984.28). MSH2 had a higher risk of adenoma recurrence compared to PMS2 (OR 11.89, 95% CI: 1.38-164.78). Of 170 EGDs, an actionable finding was identified in 16% of patients during their first three EGDs. ConclusionSurveillance colonoscopy outcomes differed in patients with Lynch Syndrome and suggest the need to guide surveillance based on MMR gene mutation.

“De novo replication repair deficient glioblastoma, IDH-wildtype” is a distinct glioblastoma subtype in adults that may benefit from immune checkpoint blockade

Glioblastoma is a clinically and molecularly heterogeneous disease, and new predictive biomarkers are needed to identify those patients most likely to respond to specific treatments. Through prospective genomic profiling of 459 consecutive primary treatment-naïve IDH-wildtype glioblastomas in adults, we identified a unique subgroup (2%, 9/459) defined by somatic hypermutation and DNA replication repair deficiency due to biallelic inactivation of a canonical mismatch repair gene. The deleterious mutations in mismatch repair genes were often present in the germline in the heterozygous state with somatic inactivation of the remaining allele, consistent with glioblastomas arising due to underlying Lynch syndrome. A subset of tumors had accompanying proofreading domain mutations in the DNA polymerase POLE and resultant “ultrahypermutation”. The median age at diagnosis was 50 years (range 27–78), compared with 63 years for the other 450 patients with conventional glioblastoma (p < 0.01). All tumors had histologic features of the giant cell variant of glioblastoma. They lacked EGFR amplification, lacked combined trisomy of chromosome 7 plus monosomy of chromosome 10, and only rarely had TERT promoter mutation or CDKN2A homozygous deletion, which are hallmarks of conventional IDH-wildtype glioblastoma. Instead, they harbored frequent inactivating mutations in TP53, NF1, PTEN, ATRX, and SETD2 and recurrent activating mutations in PDGFRA. DNA methylation profiling revealed they did not align with known reference adult glioblastoma methylation classes, but instead had unique globally hypomethylated epigenomes and mostly classified as “Diffuse pediatric-type high grade glioma, RTK1 subtype, subclass A”. Five patients were treated with immune checkpoint blockade, four of whom survived greater than 3 years. The median overall survival was 36.8 months, compared to 15.5 months for the other 450 patients (p < 0.001). We conclude that “De novo replication repair deficient glioblastoma, IDH-wildtype” represents a biologically distinct subtype in the adult population that may benefit from prospective identification and treatment with immune checkpoint blockade.

PATH-40. “DE NOVO REPLICATION REPAIR DEFICIENT GLIOBLASTOMA, IDH-WILDTYPE” IS A DISTINCT GLIOBLASTOMA SUBTYPE IN ADULTS THAT MAY BENEFIT FROM IMMUNE CHECKPOINT BLOCKADE

Abstract Glioblastoma is clinically and molecularly heterogeneous, and new predictive biomarkers are needed to identify those patients most likely to respond to specific treatments. Through prospective genomic profiling of 459 primary treatment-naïve IDH-wildtype glioblastomas in adults, we identified a subgroup (2%, 8/459) defined by somatic hypermutation and DNA replication repair deficiency due to mismatch repair gene inactivation and/or proofreading domain mutation in the DNA polymerase POLE. The deleterious mutations in mismatch repair genes were often present in the germline in the heterozygous state with somatic inactivation of the remaining wildtype allele, consistent Lynch syndrome-associated glioblastomas. All tumors were hypermutated ( &amp;gt;20 somatic nonsynonymous mutations/Mb), while the three tumors with POLE mutations were “ultrahypermutated” with &amp;gt;150 mutations/Mb. The median age at diagnosis was 52 years (range 27-78), compared with median age of 63 years for the other 451 patients with conventional glioblastoma. All tumors were diffuse astrocytic gliomas of the giant cell histologic subtype. They lacked EGFR amplification and only rarely had TERT promoter mutation (1/8), CDKN2A homozygous deletion (1/8), or the combination of trisomy 7 and monosomy 10 (1/8). Instead, they harbored frequent inactivating mutations in the TP53, NF1, PTEN, ATRX, and SETD2 genes ( &amp;gt;50% each) and recurrent activating mutations in PDGFRA (4/8). DNA methylation profiling revealed they did not align with known glioblastoma methylation classes, but instead mostly classified as “Diffuse pediatric-type high grade glioma, RTK1 subtype, subclass A (novel)”. Some patients were treated with immune checkpoint blockade (nivolumab or pembrolizumab) and survived over three years. The median overall survival for the cohort was 37.4 months (95% CI: 22-not reached), compared to 15.4 months (95% CI: 14.7-17.9) for the other 451 patients. We speculate that “de novo replication repair deficient glioblastoma, IDH-wildtype” represents a biologically distinct subtype that may benefit from prospective identification and treatment with immune checkpoint blockade.

PATH-37. PROGNOSTIC IMPACT OF TUMOR MUTATIONAL BURDEN AT BASELINE IN IDH-WILDTYPE GLIOBLASTOMA

Abstract Tumor mutational burden (TMB) has emerged as a potential biomarker for survival and response to immune checkpoint therapy. However, limited data are available regarding the prognostic impact of TMB at baseline in glioblastoma (GBM). This study aimed to evaluate the somatic mutation status and survival outcomes of isocitrate dehydrogenase (IDH) wildtype GBM using a dataset of GBM samples from cBioPortal and single tertiary hospital. A total of 840 samples were extracted from cBioPortal, including 410 samples from Memorial Sloan Kettering Cancer Center (MSKCC), 234 samples from The Cancer Genome Atlas (TCGA), 106 samples from Glioma Longitudinal Analysis Consortium (GLASS), and 90 samples from Clinical Proteomic Tumor Analysis Consortium (CPTAC). Additionally, we enrolled 341 GBM patients with targeted sequencing data (OncoPanel AMC) from Asan Medical Center. The TMBHigh group was defined as the top 10% value of TMB within each cohort. Among the total of 1,081 IDH-wt GBM samples, 104 samples were classified as TMBHigh. No significance difference in overall survival (OS) was observed between the TMBHigh group and the non-TMBHigh group across the 5 cohorts. The mutations of DNA mismatch repair genes (MLH1, MSH2, MSH6, PMS2, and POLE) was significantly higher in the TMBHigh group compared with the non-TMBHigh group in 3 of 5 cohorts (pASAN=1.02x10-4, pMSKCC=1.5x10-2, and pCPTAC=1.26x10-2). However, when we calculated the indel to total mutation ratio (I-index) to dedect microsatellite Instability, only the MSKCC cohort showed a higher I-index in the TMBHigh group compared to the non-TMBHigh group (16.7% vs 13.7%, p=2.67x10-3). In summary, there is no significant association between TMB at baseline and poor survival outcomes in IDH-wildtype GBM. Further research is needed to explore the potential implications of TMB as a prognostic marker and its relevance for personalized treatment strategies in GBM patients.

Diffuse Large B-Cell Lymphoma Has a Low Frequency of dMMR and High Frequencies of DNA Mismatch Repair Protein High Expression Associated with Lower T-Cell Infiltration

Background: DNA mismatch repair (MMR) deficiency (dMMR), which leads to genomic and microsatellite instability, is a biomarker that predicts response to immunotherapies and has variable prognostic effects for chemotherapies in solid tumors. The dMMR can be detected using gene sequencing or immunohistochemistry for four essential MMR proteins: MSH6, MSH2, MLH1, and PMS2. Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma. Previous studies have suggested the role of MMR gene variants in DLBCL lymphomagenesis, yet the prognostic role of dMMR in DLBCL has not been well studied. Methods: We performed targeted next-generation sequencing and immunohistochemistry for MSH6, MSH2, MLH1, and PMS2 in a large cohort of DLBCL patients treated with standard chemoimmunotherapy. Gene expression profiling (GEP) was performed using the Affymetrix GeneChip Human Genome HG-U133 Plus 2.0 microarray (data in GSE31312). Fluorescent multiplex immunohistochemistry (mIHC) was performed using MultiOmyx multiplexing immunofluorescence staining protocols and antibodies against 13 immune markers. We investigated the frequencies of dMRR and MMR protein expression and correlated the mutation status and expression levels with patient survival and DLBCL biology, including the number of mutated genes, GEP data, previously analyzed DLBCL biomarkers, and immune cell immunophenotypes. Results: MMR gene mutations and loss of MMR protein expression were infrequent in DLBCL (Figure A) and did not show a significant prognostic impact. MMR proteins were commonly expressed in DLBCL samples and the germinal centers of reactive tonsil controls (Figure B), with higher mean and median percentages of tumor cells expressing MSH6 and MLH1 proteins than in the solid tumor samples used for comparison. High expression of MMR proteins was associated with Ki-67, MYC, and p53 overexpression as determined by immunohistochemistry. GEP analysis identified significantly upregulated genes involved in the mitotic cell cycle and DNA metabolism and prominent downregulated immune gene signatures in DLBCL with high MMR protein expression. Fluorescent mIHC confirmed the associations with decreased T cell abundance in MSH6/MSH2/MLH1/PMS2 highly expressing DLBCL (Figure C) independent of p53 and MYC expression status, whereas MSH6/ MLH1 mutations were associated with increased T cell frequencies. High versus low expression of MSH6, MLH1, and PMS2 showed significant unfavorable prognostic effects in DLBCL when the optimal cutoffs were used for high expression. However, when the median percentages were used as cutoffs, the prognostic effects were not significant; nonetheless, MSH6 and PMS2 high expression showed significant adverse prognostic effects in the MYC¯ DLBCL subset. Interestingly, when we used Ki-67, MYC + or BCL2 + percentages in DLBCL assessed by immunohistochemistry to subtract MMR protein percentages and then correlated the differences to survival, we found significant favorable prognostic impact by higher percentage expression differences between MMR proteins and BCL2, or by intermediately higher differences between MMR proteins and MYC. For MSH2/MLH1/PMS2, the higher percentages compared with MYC/BCL2 showed no significant correlations with T cell infiltration levels, whereas the higher differences between MSH6 and MYC/BCL2 were associated with significantly lower T cell frequencies (Figure D). Conclusions: This study revealed that high expression of MMR proteins is a common feature of DLBCL associated with lower tumor-infiltrating T cells, MYC and p53 overexpression, and context-dependent prognostic effects. In contrast, dMMR and loss of MMR proteins are infrequent and have no significant prognostic effects in DLBCL treated with standard chemoimmunotherapy. These results may have implications for understanding DLBCL biology and the low efficacy of PD-1 blockade immunotherapy in DLBCL.

Novel MSH2 and TSC2 variants in a Chinese family with Lynch syndrome and their synergistic impact in urothelial carcinoma

Lynch syndrome, an autosomal dominant hereditary disease arising from mutations in mismatch repair genes, is linked to the development of multiple tumor types, notably colorectal cancer, endometrial carcinoma and upper urinary tract urothelial carcinoma. In this study, we present the case of a young patient diagnosed with upper urinary tract urothelial carcinoma, notable for a familial history of diverse malignancies. By employing genetic analysis, we verified the presence of Lynch syndrome within the family and detected novel variants, MSH2 p.A604D and TSC2 p.C738Y, utilizing NGS technology. Subsequently, we conducted validation experiments to assess the pathogenicity of the MSH2 and TSC2 variants. We illustrated that the MSH2 variant can result in diminished MSH2 expression, compromised mismatch repair function, and induce resistance to cisplatin in urothelial carcinoma. Furthermore, we substantiated the promotional impact of the identified TSC2 variant on urothelial carcinoma, encompassing proliferation, invasion, and migration. Significantly, we found that the MSH2 p.A604D variant and TSC2 p.C738Y variant synergistically enhance the promotion of urothelial carcinoma.

Tumor Testing and Genetic Analysis to Identify Lynch Syndrome Patients in an Italian Colorectal Cancer Cohort.

Lynch syndrome (LS) is an inherited cancer susceptibility syndrome caused by germline mutations in a DNA mismatch repair (MMR) gene or in the EPCAM gene. LS is associated with an increased lifetime risk of colorectal cancer (CRC) and other malignancies. The screening algorithm for LS patient selection is based on the identification of CRC specimens that have MMR loss/high microsatellite instability (MSI-H) and are wild-type for BRAFV600. Here, we sought to clinically and molecularly characterize patients with these features. From 2017 to 2023, 841 CRC patients were evaluated for MSI and BRAFV600E mutation status, 100 of which showed MSI-H. Of these, 70 were wild-type for BRAFV600. Among these 70 patients, 30 were genetically tested for germline variants in hereditary cancer predisposition syndrome genes. This analysis showed that 19 of these 30 patients (63.3%) harbored a germline pathogenic or likely pathogenic variant in MMR genes, 2 (6.7%) harbored a variant of unknown significance (VUS) in MMR genes, 3 (10%) harbored a VUS in other cancer-related genes, and 6 (20%) were negative to genetic testing. These findings highlight the importance of personalized medicine for tailored genetic counseling, management, and surveillance of families with LS and other hereditary cancer syndromes.

The prevalence of lynch syndrome (DNA mismatch repair protein deficiency) in patients with primary localized prostate cancer using immunohistochemistry screening

BackgroundProstate cancer is one of the most heritable human cancers. Lynch syndrome is an autosomal dominant inheritance caused by germline mutations in DNA mismatch repair (MMR) genes, which are also associated with an increased incidence of prostate cancer. However, prostate cancer has not been defined as a Lynch syndrome-associated cancer. The proportion of Lynch syndrome patients in primary prostate cancers is unclear. In this study, we investigated MMR protein loss using universal immunohistochemical screening to determine the prevalence of Lynch syndrome in patients with localized prostate cancer who underwent radical prostatectomy.MethodsOne hundred twenty-nine surgical specimens from radical prostatectomy performed at Toranomon Hospital between 2012 and 2015 were retrospectively tested using universal screening with immunohistochemistry staining for expression of the MMR proteins MLH1, PMS2, MSH2, and MSH6. For all suspected MMR-deficient patients, germline genetic tests focusing on MMR genes were performed.ResultsMMR protein loss was found in only one patient (0.8%) who showed dual MSH2/MSH6 loss. This patient showed a single nucleotide pathogenic germline mutation from c.1129 C to T (p.Gln377*) at exon 7 in the MSH2 gene. He was diagnosed with a primary prostate cancer at 66 years of age. He had a documented history of Lynch syndrome (Muir–Torre syndrome) with previous colon cancer, sebaceous tumor, and keratoacanthoma as well as subsequent bladder cancer, all of which also showed dual MSH2/MSH6 loss. He also had a strong family history of colorectal and other Lynch syndrome-associated cancers. The pathological stage was pT3aN0M0, and the pathological grade was Gleason 7(4 + 3) with tertiary pattern 5.ConclusionsIn this study, immunohistochemical screening of MMR proteins for Lynch syndrome was performed in a series of prostate cancer cases. The prevalence of Lynch syndrome in localized prostate cancer was 0.8%, which is low compared with other Lynch syndrome-associated cancers.

Relationship among DDR gene mutations, TMB and PD-L1 in solid tumour genomes identified using clinically actionable biomarker assays

The DNA damage response (DDR) pathway regulates DNA repair and cell survival, and inactivating mutations in DDR genes can increase tumour mutational burden (TMB), a predictive biomarker of treatment benefit from anti-PD-1/PD-L1 immunotherapies. However, a better understanding of the relationship among specific DDR mutations, TMB and PD-L1 expression is needed to improve translational strategies. Here, we determined genomic alteration frequencies in selected DDR genes that are clinically actionable biomarkers and investigated their association with TMB and PD-L1 in bladder, colorectal, non-small cell lung, ovarian and prostate cancers using the FoundationInsights® web portal. Our results not only confirm known associations, such as mismatch repair and POLE gene mutations with high TMB, but also identify significant associations between mutations in the SWI/SNF chromatin remodelling genes ARID1A and SMARCA4 and high TMB in multiple tumour types. Mutations in the ATR gene were associated with high TMB in colorectal and prostate cancers; however, associations between individual DDR mutations and high PD-L1 expression were uncommon and tumour-type specific. Finally, we found that high TMB and high PD-L1 expression were poorly associated, emphasising their independence as predictive biomarkers for immune checkpoint inhibitor use.

Glioma arising in the setting of mismatch repair deficiency-rare or are we missing it?

Germline mutations in mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2) can be mono-allelic or biallelic, resulting in a Lynch syndrome (LS) or constitutional mismatch repair deficiency (CMMRD) syndrome respectively. Glioma arising in the setting of MMR deficiency is uncommon. We describe two pediatric patients with high-grade glioma (HGG) and associated MMR protein deficiency. On histomorphology both cases showed HGG with astrocytic morphology and prominent multinucleated tumor cells. On immunohistochemistry, the first case was negative for IDH1 p.R132H showed loss of ATRX and p53 positivity. The second case was positive for IDH1 p.R132H and p53, but showed retained expression of ATRX. The histomorphology in both cases and additionally IDH mutation with retained ATRX in the second case, prompted us to test for MMR protein deficiency which was carried out by immunohistochemistry (IHC). One case revealed an immunostaining pattern suggestive of CMMRD while the other was suggestive of LS. Both the cases showed good response to surgery and radio-chemotherapy in the follow-up available. Our cases highlight the importance of testing for MMR proteins by simple IHC, in the setting of appropriate clinical scenario, histopathological and immunohistochemical findings. The recognition of these tumors is extremely important to guide further treatment and prompt family screening.

Differential outcomes and immune checkpoint inhibitor response among endometrial cancer patients with MLH1 hypermethylation versus MLH1 “Lynch-like” mismatch repair gene mutation

ObjectivesTo identify differential survival outcomes and immune checkpoint inhibitor (ICI) response in MLH1 hypermethylated versus MLH1 mutated (“Lynch-like”) endometrial tumors and determine whether their molecular profiles can elucidate the differential outcomes. Methods1673 mismatch repair deficient endometrial tumors were analyzed by next-generation sequencing and whole transcriptome sequencing (Caris Life Sciences, Phoenix, AZ). PD-L1, ER, and PR were tested by immunohistochemistry and immune cell infiltrates were calculated using MCP-counter. Significance was determined using Chi-square and Mann-Whitney U tests and adjusted for multiple comparisons. Overall survival (OS) was depicted using Kaplan-Meier survival curves. ResultsThe endometrial cancer cohort comprised 89.2% patients with MLH1 hypermethylated tumors and 10.8% with MLH1 mutated tumors, with median ages of 67 and 60 years, respectively (p < 0.01). Patients with MLH1 hypermethylated tumors had significantly worse OS and trended toward worse OS following ICI treatment than patients with MLH1 mutated tumors. The immune microenvironment of MLH1 hypermethylated relative to MLH1 mutated was characterized by decreased PD-L1 positivity, immune checkpoint gene expression, immune cell infiltration, T cell inflamed scores, and interferon gamma (IFNγ) scores. MLH1 hypermethylation was also associated with decreased mutation rates in TP53 and DNA damage repair genes, but increased rates of JAK1, FGFR2, CCND1, and PTEN mutations, as well as increased ER and PR positivity. ConclusionsEndometrial cancer patients with MLH1 hypermethylation display significantly decreased survival and discrepant immunotherapy responses compared to patients with MLH1 mutated tumors, which was associated with differential mutational profiles, a more immune cold phenotype, and increased ER/PR expression in MLH1 hypermethylated tumors. Providers may consider early transition from single agent ICI to a multi-agent regimen or hormonal therapy for patients with MLH1 hypermethylated tumors.

Organoids and metastatic orthotopic mouse model for mismatch repair-deficient colorectal cancer.

Genome integrity is essential for the survival of an organism. DNA mismatch repair (MMR) genes (e.g., MLH1, MSH2, MSH6, and PMS2) play a critical role in the DNA damage response pathway for genome integrity maintenance. Germline mutations of MMR genes can lead to Lynch syndrome or constitutional mismatch repair deficiency syndrome, resulting in an increased lifetime risk of developing cancer characterized by high microsatellite instability (MSI-H) and high mutation burden. Although immunotherapy has been approved for MMR-deficient (MMRd) cancer patients, the overall response rate needs to be improved and other management options are needed. To better understand the biology of MMRd cancers, elucidate the resistance mechanisms to immune modulation, and develop vaccines and therapeutic testing platforms for this high-risk population, we generated organoids and an orthotopic mouse model from intestine tumors developed in a Msh2-deficient mouse model, and followed with a detailed characterization. The organoids were shown to be of epithelial origin with stem cell features, to have a high frameshift mutation frequency with MSI-H and chromosome instability, and intra- and inter-tumor heterogeneity. An orthotopic model using intra-cecal implantation of tumor fragments derived from organoids showed progressive tumor growth, resulting in the development of adenocarcinomas mixed with mucinous features and distant metastasis in liver and lymph node. The established organoids with characteristics of MSI-H cancers can be used to study MMRd cancer biology. The orthotopic model, with its distant metastasis and expressing frameshift peptides, is suitable for evaluating the efficacy of neoantigen-based vaccines or anticancer drugs in combination with other therapies.