Surviving without BRCA2: MLH1 gets R-looped in to curtail genomic instability.

While breast cancer 2 (BRCA2) loss of heterozygosity (LOH) promotes cancer initiation, it can also induce death in nontransformed cells. In contrast, mismatch repair gene mutL homolog 1 (MLH1) is a tumor-suppressor gene that protects cells from cancer development through repairing mismatched base pairs during DNA mismatch repair (MMR). Sengodan et al., in this issue of the JCI, reveal an interplay between the 2 genes: MLH1 promoted the survival of BRCA2-deficient cells independently of its MMR function. MLH1 protected replication forks from degradation, while also resolving R-loops, thereby reducing genomic instability. Moreover, MLH1 expression was regulated directly by estrogen, shedding light into the hormone-responsive nature of many BRCA2 mutant breast cancers. These results provide important insight into the genetics that drive the initiation of BRCA2-mutated breast cancers.

Association between colorectal cancer, the frequency of Bacteroides fragilis, and the level of mismatch repair genes expression in the biopsy samples of Iranian patients.

BACKGROUND: Deficient DNA mismatch repair (MMR) can cause microsatellite instability (MSI) and is more common in colorectal cancer (CRC) patients. Understanding the carcinogenic mechanism of bacteria and their impact on cancer cells is crucial. Bacteroides fragilis (B. fragilis) has been identified as a potential promoter of tumorigenesis through the alteration of signaling pathways. This study aims to assess the expression levels of msh2, msh6, mlh1, and the relative frequency of B. fragilis in biopsy samples from CRC patients. MATERIALS AND METHODS: Based on the sequence of mlh1, msh2, and msh6 genes, B. fragilis specific 16srRNA and bacterial universal 16srRNA specific primers were selected, and the expression levels of the target genes were analyzed using the Real-Time PCR method. RESULTS: Significant increases in the expression levels of mlh1, msh2, and msh6 genes were observed in the cancer group. Additionally, the expression of these MMR genes showed a significant elevation in samples positive for B. fragilis presence. The relative frequency of B. fragilis in the cancer group demonstrated a significant rise compared to the control group. CONCLUSION: The findings suggest a potential correlation between the abundance of B. fragilis and alterations in the expression of MMR genes. Since these genes can play a role in modifying colon cancer, investigating microbial characteristics and gene expression changes in CRC could offer a viable solution for CRC diagnosis.

The relationship between DNA mismatch repair gene and other prognostic parameters in pancreatic adenocarcinoma.

The aim of the study was to determine DNA mismatch repair (MMR) proteins by immunohistochemically using MLH1, MSH2, MSH6, and PMS2 antibodies in patients diagnosed as pancreatic ductal adenocarcinoma and to assess its relationship with histopathological and clinical prognostic parameters. Fifty cases with a diagnosis of pancreatic ductal adenocarcinoma who underwent surgical resection, were included in the study. Demographic and histopathological features of the patients were collected from the medical records. The relationships between microsatellite status and prognostic parameters were determined. The mean age of the patients was 66.5 ± 9.5 years (range: 47-87) and male/female ratio was 1.63 (31/19). No errors were detected in DNA MMR proteins in any of the cases, and were classified as microsatellite stable. The mean tumor diameter was 4.01 ± 1.77 cm and 74% of the tumors were localized in the pancreatic head. All of the cases had lymphatic invasion, whereas vascular invasion was detected in only 78% and perineural invasion in 98% of the patients. When the relationship between prognostic parameters and survival was evaluated, statistically significant correlation was observed in patient age and histopathological parameters such as tumor diameter, status of surgical margins, and vascular invasion (p < 0.05). Age, tumor size, presence of tumor at surgical margins, vascular invasion, and adjuvant treatment were correlated with survival. Although microsatellite instability was not detected in our cases, it is important to determine the microsatellite status by immunohistochemistry for predicting the chemotherapy response and determining the immunotherapy option in pancreatic adenocarcinomas.

Mismatch repair protein MLH1 suppresses replicative stress in BRCA2-deficient breast tumors.

Loss of BRCA2 (breast cancer 2) is lethal for normal cells. Yet it remains poorly understood how, in BRCA2 mutation carriers, cells undergoing loss of heterozygosity overcome the lethality and undergo tissue-specific neoplastic transformation. Here, we identified mismatch repair gene mutL homolog 1 (MLH1) as a genetic interactor of BRCA2 whose overexpression supports the viability of Brca2-null cells. Mechanistically, we showed that MLH1 interacts with Flap endonuclease 1 (FEN1) and competes to process the RNA flaps of Okazaki fragments. Together, they restrained the DNA2 nuclease activity on the reversed forks of lagging strands, leading to replication fork (RF) stability in BRCA2-deficient cells. In these cells, MLH1 also attenuated R-loops, allowing the progression of stable RFs, which suppressed genomic instability and supported cell viability. We demonstrated the significance of their genetic interaction by the lethality of Brca2-mutant mice and inhibition of Brca2-deficient tumor growth in mice by Mlh1 loss. Furthermore, we described estrogen as inducing MLH1 expression through estrogen receptor α (ERα), which might explain why the majority of BRCA2 mutation carriers develop ER-positive breast cancer. Taken together, our findings reveal a role of MLH1 in relieving replicative stress and show how it may contribute to the establishment of BRCA2-deficient breast tumors.

Molecular and Clinicopathologic Characterization of Mismatch Repair-Deficient Endometrial Carcinoma Not Related to MLH1 Promoter Hypermethylation.

Universal tumor screening in endometrial carcinoma (EC) is increasingly adopted to identify individuals at risk of Lynch syndrome (LS). These cases involve mismatch repair-deficient (MMRd) EC without MLH1 promoter hypermethylation (PHM). LS is confirmed through the identification of germline MMR pathogenic variants (PV). In cases where these are not detected, emerging evidence highlights the significance of double-somatic MMR gene alterations as a sporadic cause of MMRd, alongside POLE/POLD1 exonuclease domain (EDM) PV leading to secondary MMR PV. Our understanding of the incidence of different MMRd EC origins not related to MLH1-PHM, their associations with clinicopathologic characteristics, and the prognostic implications remains limited. In a combined analysis of the PORTEC-1, -2, and -3 trials (n = 1254), 84 MMRd EC not related to MLH1-PHM were identified that successfully underwent paired tumor-normal tissue next-generation sequencing of the MMR and POLE/POLD1 genes. Among these, 37% were LS associated (LS-MMRd EC), 38% were due to double-somatic hits (DS-MMRd EC), and 25% remained unexplained. LS-MMRd EC exhibited higher rates of MSH6 (52% vs 19%) or PMS2 loss (29% vs 3%) than DS-MMRd EC, and exclusively showed MMR-deficient gland foci. DS-MMRd EC had higher rates of combined MSH2/MSH6 loss (47% vs 16%), loss of >2 MMR proteins (16% vs 3%), and somatic POLE-EDM PV (25% vs 3%) than LS-MMRd EC. Clinicopathologic characteristics, including age at tumor onset and prognosis, did not differ among the various groups. Our study validates the use of paired tumor-normal next-generation sequencing to identify definitive sporadic causes in MMRd EC unrelated to MLH1-PHM. MMR immunohistochemistry and POLE-EDM mutation status can aid in the differentiation between LS-MMRd EC and DS-MMRd EC. These findings emphasize the need for integrating tumor sequencing into LS diagnostics, along with clear interpretation guidelines, to improve clinical management. Although not impacting prognosis, confirmation of DS-MMRd EC may release patients and relatives from burdensome LS surveillance.

MLH1 Promotor Hypermethylation in Colorectal and Endometrial Carcinomas from Patients with Lynch Syndrome.

Screening for Lynch syndrome (LS) in colorectal cancer (CRC) and endometrial cancer patients generally involves immunohistochemical staining of the mismatch repair (MMR) proteins. In case of MLH1 protein loss, MLH1 promotor hypermethylation (MLH1-PM) testing is performed to indirectly distinguish the constitutional MLH1 variants from somatic epimutations. Recently, multiple studies have reported that MLH1-PM and pathogenic constitutional MMR variants are not mutually exclusive. This study describes 6 new and 86 previously reported MLH1-PM CRCs or endometrial cancers in LS patients. Of these, methylation of the MLH1 gene promotor C region was reported in 30 MLH1, 6 MSH2, 6 MSH6, and 3 PMS2 variant carriers at a median age at diagnosis of 48.5 years [interquartile range (IQR), 39-56.75 years], 39 years (IQR, 29-51 years), 58 years (IQR, 53.5-67 years), and 68 years (IQR, 65.6-68.5 years), respectively. For 31 MLH1-PM CRCs in LS patients from the literature, only the B region of the MLH1 gene promotor was tested, whereas for 13 cases in the literature the tested region was not specified. Collectively, these data indicate that a diagnosis of LS should not be excluded when MLH1-PM is detected. Clinicians should carefully consider whether follow-up genetic MMR gene testing should be offered, with age <60 to 70 years and/or a positive family history among other factors being suggestive for a potential constitutional MMR gene defect.

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.

Characterization of mismatch-repair/microsatellite instability-discordant endometrial cancers.

BACKGROUND: Mismatch-repair (MMR)/microsatellite instability (MSI) status has therapeutic implications in endometrial cancer (EC). The authors evaluated the concordance of testing and factors contributing to MMR expression heterogeneity. METHODS: Six hundred sixty-six ECs were characterized using immunohistochemistry (IHC), MSI testing, and mut-L homolog 1 (MLH1) methylation. Select samples underwent whole-transcriptome analysis and next-generation sequencing. MMR expression of metastatic/recurrent sites was evaluated. RESULTS: MSI testing identified 27.3% of cases as MSI-high (n = 182), MMR IHC identified 25.1% cases as MMR-deficient (n = 167), and 3.8% of cases (n = 25) demonstrated discordant results. A review of IHC staining explained discordant results in 18 cases, revealing subclonal loss of MLH1/Pms 1 homolog 2 (PMS2) (n = 10) and heterogeneous MMR IHC (mut-S homolog 6 [MSH6], n = 7; MLH1/PMS2, n = 1). MSH6-associated Lynch syndrome was diagnosed in three of six cases with heterogeneous expression. Subclonal or heterogeneous cases had a 38.9% recurrence rate (compared with 16.7% in complete MMR-deficient cases and 9% in MMR-proficient cases) and had abnormal MMR IHC results in all metastatic recurrent sites (n = 7). Tumors with subclonal MLH1/PMS2 demonstrated 74 differentially expressed genes (determined using digital spatial transcriptomics) when stratified by MLH1 expression, including many associated with epithelial-mesenchymal transition. CONCLUSIONS: Subclonal/heterogeneous MMR IHC cases showed epigenetic loss in 66.7%, germline mutations in 16.7%, and somatic mutations in 16.7%. MMR IHC reported as intact/deficient missed 21% of cases of Lynch syndrome. EC with subclonal/heterogeneous MMR expression demonstrated a high recurrence rate, and metastatic/recurrent sites were MMR-deficient. Transcriptional analysis indicated an increased risk for migration/metastasis, suggesting that clonal MMR deficiency may be a driver for tumor aggressiveness. Reporting MMR IHC only as intact/deficient, without reporting subclonal and heterogeneous staining, misses opportunities for biomarker-directed therapy. PLAIN LANGUAGE SUMMARY: Endometrial cancer is the most common gynecologic cancer, and 20%-40% of tumors have a defect in DNA proofreading known as mismatch-repair (MMR) deficiency. These results can be used to guide therapy. Tests for this defect can yield differing results, revealing heterogeneous (mixed) proofreading capabilities. Tumors with discordant testing results and mixed MMR findings can have germline or somatic defects in MMR genes. Cells with deficient DNA proofreading in tumors with mixed MMR findings have DNA expression profiles linked to more aggressive characteristics and cancer spread. These MMR-deficient cells may drive tumor behavior and the risk of spreading cancer.

[A Case of Transverse Colon Cancer Associated with Lynch Syndrome with Excellent Response to Pembrolizumab].

A 47-year-old woman diagnosed with transverse colon cancer with liver, peritoneal, and lymph node metastases was admitted. Modified FOLFOX6(mFOLFOX6)regimen was given as a first line chemotherapy and was followed by pembrolizumab after 1 cycle of the mFOLFOX6, because microsatellite instability(MSI)test of the tumor showed high-frequency MSI. Because of the transverse colon obstruction after 2 cycles of pembrolizumab, she underwent right hemicolectomy. Histological examination of the resected specimen revealed no residual tumor cells in the primary tumor and reginal lymph nodes. Immunohistochemistry for mismatch repair proteins(IHC-MMR)showed loss of MSH2 and MSH6 expression. Genetic test identified a MSH2 pathogenic variant leading to the diagnosis of Lynch syndrome. The present case shows the importance of MSI test or IHC-MMR before the treatment of metastatic colorectal cancer.

Prevalence and characteristics of patients with upper urinary tract urothelial carcinoma having potential Lynch syndrome identified by immunohistochemical universal screening and Amsterdam criteria II.

BACKGROUND: This study aimed to identify patients with upper urinary tract urothelial carcinoma (UTUC) having potential Lynch syndrome (pLS) by immunohistochemistry (IHC) of DNA mismatch repair gene-related proteins (MMRPs) and Amsterdam criteria II and explore their clinical characteristics. METHODS: We retrospectively collected the clinical data of 150 consecutive patients with UTUC who underwent surgical resection at our institution between February 2012 and December 2020, and immunohistochemistry (IHC) of four MMRPs (MLH1, MSH2, MSH6, and PMS2) on all UTUC specimens was performed. Patients who tested positive for Amsterdam criteria (AMS) II and/or IHC screening were classified as having pLS and others as non-pLS, and their characteristics were explored. RESULTS: In this study, 5 (3%) and 6 (4%) patients were positive for AMS II and IHC screening, respectively. Two patient were positive for both AMS II and IHC screening, resulting in 9 (6%) patients with pLS. The pLS group was predominantly female (67% vs. 36%; p = 0.0093) and had more right-sided tumors (100% vs. 43%; p = 0.0009) than the non-pLS group. Of the 6 patients who were positive for IHC screening, 4 showed a combined loss of MSH2/MSH6 (n = 3) and MLH1/PMS2 (n = 1). Other two patients showed single loss of MSH6 and PSM2. CONCLUSIONS: AMS II and IHC screening identified pLS in 6% of patients with UTUC. The IHC screening-positive group tends to have relatively high rate of combined loss, but some patients have single loss. AMS II may overlook patients with LS, and a universal screening may be required for patients with UTUC as well as those with colorectal and endometrial cancer.

Impact of formalin fixation on mismatch repair protein evaluation by immunohistochemistry.

Mismatch repair/microsatellite instability (MMR/MSI) status in colorectal cancer (CRC) has become fundamental as a diagnostic, prognostic, and predictive factor. MMR immunohistochemistry (IHC) is considered a simple and reliable approach; however, its effectiveness depends on pre-analytic factors. Aim of this study was to investigate the impact of different fixation times/protocols on MMR protein IHC quality. Left over tissue from surgically resected CRC samples (cold ischemia time < 30 min) where fixed as follows: standard formalin fixation (24-48 h); hypo-fixation (<20 h); hyper-fixation (>90 h); cold (4°C) fixation (24-48 h); standard fixation for small sample size (0.5×0.5 cm). Samples for each group were collected from 30 resected CRC and the following parameters were evaluated on 600 immunohistochemical stains: intensity of expression; patchiness of staining; presence of central artefact. Forty-six immunoreactions were inadequate (score 0 intensity), the majority regarding MLH1 or PMS2 in the hypo-fixation group (47.8%), followed by the hyper-fixation group (28.1%); cold formalin fixation showed the least inadequate cases. Patchiness and central artefact were more frequent in hypo-fixation and standard fixation group compared to the others. MLH1 (closely followed by PMS2) performed worse with regard to immunostaining intensity (p=0.0002) in the standard and in the hypo-fixation group (p< 0.00001). Using a small sample size improved patchiness/central artefacts. This is the first study specifically created to evaluate the impact of fixation on MMR protein IHC, showing that both formalin hypo- and hyper-fixation can cause problems; 24-h formalin fixation as well as cold (4°C) formalin fixation are recommended for successful IHC MMR evaluation.

Mismatch Repair Protein Deficiency Does Not Affect Disease Free Survival in Type I Endometrial Carcinoma.

BACKGROUND: This study aimed to analyze the correlation between the 3-year disease-free survival (DFS) and mismatch repair (MMR) protein levels in patients with type 1 endometrial carcinoma. Many studies have reported different results regarding the role of MMR in the prognosis of endometrial carcinoma; therefore, we aimed to identify this association in our hospital. METHODS: This observational study employed a historical cohort design and included patients with type 1 endometrial carcinoma who underwent surgery at Dr. Soetomo Hospital between January 2017 and December 2019. Medical records and paraffin blocks meeting these criteria were obtained. MMR proteins (MLH1 and MSH2) were assessed using immunohistochemistry. RESULTS: A total of 46 patients with type 1 endometrial carcinoma were analyzed. We observed MMR deficiency (dMMR) in 12 patients (26.1%) and MMR proficiency (pMMR) in 34 patients (73.9%). Of the 12 patients with dMMR, nine cases (75%) were diagnosed as stage I and 7 (58.33%) as low grade. The 3-year DFS in patients with dMMR and pMMR was 83.3% and 67.6%, respectively (Hazard Ratio 2.31, 95% CI 0.5135-10.475, p=0.27). Higher stages had a 5.42 times increased risk of recurrence (95% CI 1.3378-21.9358, p=0.018). Higher histopathological grades were also associated with 8.65 times increased risk of recurrence (95% CI 2.5020-29.8738, p=0.001). CONCLUSION: Patients with dMMR had a better DFS compared to those with pMMR; however, the difference was not statistically significant. The tumor stage and histopathological grade were independent risk factors for recurrence.

Role of MLH1 and MSH2 deficiency in the development of tumorigenesis and chemo-tolerance of cervical Carcinoma: Clinical implications.

Cervical cancer (CACX) is one of the top causes of cancer death in women globally. The involvement of several cellular pathways in carcinogenesis is still poorly understood. Here, we focused to evaluate the contributory role of Mismatch Repair (MMR) pathway genes-MLH1 and MSH2 in CACX and their association with chemo-tolerance of the disease. For this purpose, molecular profiles (expression/promoter methylation/deletion) of the genes were analysed in both normal cervical epithelium and tumour tissue, also validated in in-silico dataset as well. Later on, prognostic importance of the genes was identified through analysis of their methylation/expression status in plasma DNA of circulating tumour cells (CTCs) and cisplatin-tolerant CACX cell lines respectively. It was found that the expression profile of MLH1 and MSH2 genes was considerably reduced from undifferentiated basal-parabasal layers of normal cervical epithelium towards progression of the disease. Further analysis showed that frequent deletion [34-48%] and promoter methylation events [28-46%] of the genes were the plausible reasons for their reduced expression during tumorigenesis. Incidentally, the prevalence of MLH1 [32%] and MSH2 [27%] promoter methylation found in CTCs of plasma of the clinically advanced CACX patients implicated their prognostic importance of the disease. In addition, the patients having high alterations of those genes resulted in poor patient outcomes even after the therapy. In in-depth analysis of this result in cisplatin-tolerant CACX cell lines, we discovered that increased promoter methylation frequency of those genes at higher concentrations of cisplatin and gradual accumulation of the cells in the G2/M phase of the cell cycle were the rational causes for their reduced expression and MMR deficiency in the system. Hence, it is possible to conclude that the gradual down-regulation of MLH1 and MSH2 proteins may be a key event for MMR pathway inactivation in CACX. This might also be associated with chemo-tolerance and overall poor survival among the patients.

Key role of phosphorylation sites in ATPase domain and Linker region of MLH1 for DNA binding and functionality of MutLα.

MutLα is essential for human DNA mismatch repair (MMR). It harbors a latent endonuclease, is responsible for recruitment of process associated proteins and is relevant for strand discrimination. Recently, we demonstrated that the MMR function of MutLα is regulated by phosphorylation of MLH1 at serine (S) 477. In the current study, we focused on S87 located in the ATPase domain of MLH1 and on S446, S456 and S477 located in its linker region. We analysed the phosphorylation-dependent impact of these amino acids on DNA binding, MMR ability and thermal stability of MutLα. We were able to demonstrate that phosphorylation at S87 of MLH1 inhibits DNA binding of MutLα. In addition, we detected that its MMR function seems to be regulated predominantly via phosphorylation of serines in the linker domain, which are also partially involved in the regulation of DNA binding. Furthermore, we found that the thermal stability of MutLα decreased in relation to its phosphorylation status implying that complete phosphorylation might lead to instability and degradation of MLH1. In summary, we showed here, for the first time, a phosphorylation-dependent regulation of DNA binding of MutLα and hypothesized that this might significantly impact its functional regulation during MMR in vivo.

Insights into DNA cleavage by MutL homologs from analysis of conserved motifs in eukaryotic Mlh1.

MutL family proteins contain an N-terminal ATPase domain (NTD), an unstructured interdomain linker, and a C-terminal domain (CTD), which mediates constitutive dimerization between subunits and often contains an endonuclease active site. Most MutL homologs direct strand-specific DNA mismatch repair by cleaving the error-containing daughter DNA strand. The strand cleavage reaction is poorly understood; however, the structure of the endonuclease active site is consistent with a two- or three-metal ion cleavage mechanism. A motif required for this endonuclease activity is present in the unstructured linker of Mlh1 and is conserved in all eukaryotic Mlh1 proteins, except those from metamonads, which also lack the almost absolutely conserved Mlh1 C-terminal phenylalanine-glutamate-arginine-cysteine (FERC) sequence. We hypothesize that the cysteine in the FERC sequence is autoinhibitory, as it sequesters the active site. We further hypothesize that the evolutionary co-occurrence of the conserved linker motif with the FERC sequence indicates a functional interaction, possibly by linker motif-mediated displacement of the inhibitory cysteine. This role is consistent with available data for interactions between the linker motif with DNA and the CTDs in the vicinity of the active site.

Improving genetic testing following abnormal mismatch repair immunohistochemistry results in endometrial cancer.

OBJECTIVES: Although universal mismatch repair (MMR) immunohistochemistry (IHC) in endometrial cancer began at our institution in July 2015, not all eligible patients were referred for genetic testing (GT). In April 2017, genetic counselors obtained IHC data and contacted physicians to approve genetic counseling referrals (GCRs) for Lynch Syndrome (LS) in eligible patients. We assessed if this protocol increased frequency of GCRs and GT in patients with abnormal MMR IHC. METHODS: We retrospectively (7/2015-5/2022) identified patients with abnormal MMR IHC at a large urban hospital. GCRs and GT were compared between cases from 7/2015-4/2017 (pre-protocol) and 5/2017-5/2022 (post-protocol) with chi-square and Fisher's exact tests. RESULTS: Of 794 patients with IHC testing, 177 (22.3%) had abnormal MMR results with 46 (26.0%) meeting criteria for LS screening with GT. Of 46 patients, 16 (34.8%) were identified prior to and 30 (65.2%) after the protocol initiation. GCRs significantly increased from 11/16 (68.8%) to 29/30 (96.7%) in the pre-protocol versus post-protocol groups, p = 0.02. There was no statistically significant difference in GT between groups (10/16, 62.5% vs 26/30, 86.7%, p = 0.07). Of 36 patients who underwent GT, 16 (44.4%) had LS: MSH6, 9; MSH2, 4; PMS2, 2; MLH1, 1. CONCLUSIONS: Increased frequency of GCRs was observed following the change in protocol, which is important as LS screening has clinical implications for patients and their families. Despite this additional effort, approximately 15% who met criteria did not undergo GT; further efforts such as universal germline testing in patients with endometrial cancer should be considered.

Context-dependent environmental associations with endometrial cancer histotype and genotype.

OBJECTIVE: MLH1 loss due to MLH1 methylation, detected during Lynch syndrome screening, is one of the most common molecular changes in endometrial cancer. It is well established that environmental influences such as nutritional state can impact gene methylation, both in the germline and in a tumor. In colorectal cancer and other cancer types, aging is associated with changes in gene methylation. The objective of this study was to determine if there was an association between aging or body mass index on MLH1 methylation in sporadic endometrial cancer. METHODS: A retrospective review of patients with endometrial cancer was performed. Tumors were screened for Lynch syndrome via immunohistochemistry, with MLH1 methylation analysis performed when there was loss of MLH1 expression. Clinical information was abstracted from the medical record. RESULTS: There were 114 patients with mismatch repair deficient tumors associated with MLH1 methylation, and 349 with mismatch repair proficient tumors. Patients with mismatch repair deficient tumors were older than those whose tumors were proficient. Mismatch repair deficient tumors had a higher incidence of lymphatic/vascular space invasion. When stratified by endometrioid grade, associations with body mass index and age became apparent. Patients with endometrioid grades 1 and 2 tumors and somatic mismatch repair deficiency were significantly older, but body mass index was comparable with that of the mismatch repair intact group. For endometrioid grade 3, patient age did not significantly vary between the somatic mismatch repair deficient group and the mismatch repair intact group. In contrast, body mass index was significantly higher in the patients with grade 3 tumors with somatic mismatch repair deficiency. CONCLUSION: The relationship of MLH1 methylated endometrial cancer with age and body mass index is complex and somewhat dependent on tumor grade. As body mass index is modifiable, it is possible that weight loss induces a 'molecular switch' to alter the histologic characteristics of an endometrial cancer.

MLH1 Loss in Primary Prostate Cancer.

PURPOSE: Among mismatch repair-deficient (MMRd) prostate cancers, loss of MLH1 is relatively uncommon and few cases have been reported in detail. METHODS: Here, we describe the molecular features of two cases of primary prostate cancer with MLH1 loss detected by immunohistochemistry, and in one case, confirmed via transcriptomic profiling. RESULTS: Both cases were microsatellite stable on standard polymerase chain reaction (PCR)-based microsatellite instability (MSI) testing, but showed evidence of MSI on a newer PCR-based long mononucleotide repeat (LMR) assay and by next-generation sequencing. Germline testing was negative for Lynch syndrome-associated mutations in both cases. Targeted or whole-exome tumor sequencing using multiple commercial/academic platforms (Foundation, Tempus, JHU, and UW-OncoPlex) showed modestly elevated, though variable, tumor mutation burden estimates (2.3-10 mutations/Mb) consistent with MMRd, but without identifiable pathogenic single-nucleotide or indel mutations in MLH1. Copy-number analysis confirmed biallelic MLH1 loss in one case and monoallelic MLH1 loss in the second case, without evidence of MLH1 promoter hypermethylation in either. The second patient was treated with single-agent pembrolizumab and demonstrated a short-lived prostate-specific antigen response. CONCLUSION: These cases highlight the challenges in identifying MLH1-deficient prostate cancers using standard MSI testing and commercial sequencing panels, and support the utility of immunohistochemical assays and LMR- or sequencing-based MSI testing for detection of MMRd prostate cancers.

Somatic Sequencing and Microsatellite Instability Results From Mismatch Repair-deficient Endometrial Carcinoma Patients Without Lynch Syndrome ("Lynch-like" tumors): Implications for Heritable Cancer Screening, Molecular Prognostication, and Immunotherapeutic Vulnerability.

Immunostaining of endometrial carcinomas for mismatch repair (MMR) protein loss is standard-of-care for Lynch syndrome screening, but also identifies MMR-deficient tumors without germline pathogenic variants. While the majority show MLH1 hypermethylation ( MLH1hm ), somatic MMR pathogenic variants are increasingly recognized drivers of immunohistochemistry-germline discordance. Because MMR abnormalities with both germline and somatic origins have prognostic significance and impart susceptibility to immune checkpoint inhibitors, it is important to understand how frequently tumors with MMR immunohistochemical loss and normal germline testing ("Lynch-like" tumors) have underlying somatic MMR pathogenic variants. Somatic tumor sequencing±microsatellite instability (MSI) testing was performed on 18 endometrial cancers with MMR immunohistochemical loss but negative MMR germline results and negative MLH1hm where relevant. Tumor sequencing and MSI testing were successful in 94%. Where successful, 80% were MSI-high and 94% had a molecular correlate for the initial immunohistochemical interpretation. The single case without an identified somatic pathogenic variant was MSI-low and initially showed loss of MSH6 by immunohistochemistry but with extremely limited internal control staining. On review, MSH6 immunohistochemistry was reclassified as equivocal, and repeat staining revealed improved control expression with intact MSH6. Following reclassification of this case, 100% tumors with MMR deficiency by immunohistochemistry had at least 1 confirmed somatic MMR pathogenic variant, and 86% were MSI-high. These results demonstrate that when correctly interpreted immunohistochemistry is a strong surrogate for somatic MMR pathogenic variants and support its use as the frontline MMR biomarker in endometrial cancer for heritable screening, molecular prognostic classification, and immunotherapeutic biomarker testing purposes.

A conserved motif in the disordered linker of human MLH1 is vital for DNA mismatch repair and its function is diminished by a cancer family mutation.

DNA mismatch repair (MMR) is essential for correction of DNA replication errors. Germline mutations of the human MMR gene MLH1 are the major cause of Lynch syndrome, a heritable cancer predisposition. In the MLH1 protein, a non-conserved, intrinsically disordered region connects two conserved, catalytically active structured domains of MLH1. This region has as yet been regarded as a flexible spacer, and missense alterations in this region have been considered non-pathogenic. However, we have identified and investigated a small motif (ConMot) in this linker which is conserved in eukaryotes. Deletion of the ConMot or scrambling of the motif abolished mismatch repair activity. A mutation from a cancer family within the motif (p.Arg385Pro) also inactivated MMR, suggesting that ConMot alterations can be causative for Lynch syndrome. Intriguingly, the mismatch repair defect of the ConMot variants could be restored by addition of a ConMot peptide containing the deleted sequence. This is the first instance of a DNA mismatch repair defect conferred by a mutation that can be overcome by addition of a small molecule. Based on the experimental data and AlphaFold2 predictions, we suggest that the ConMot may bind close to the C-terminal MLH1-PMS2 endonuclease and modulate its activation during the MMR process.