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PurposeThis study was to investigate the frequency of mismatch repair deficiency/high microsatellite instability (MMRd/MSI-H) in gynecologic malignancies and the efficacy of immune checkpoint inhibitors (ICIs) in patients with recurrent gynecologic cancers according to MMR/MSI status.Materials and MethodsWe conducted a multi-center retrospective review on the patients who were diagnosed with gynecologic cancers between 2015 and 2020. Their clinicopathologic information, results of immunohistochemistry staining for MLH1/MSH2/MSH6/PMS2 and MSI analysis, tumor response to treatment with ICIs were investigated.ResultsAmong 1,093 patients included in the analysis, MMRd/MSI-H was most frequent in endometrial/uterine cancers (34.8%, 164/471), followed by ovarian, tubal, and peritoneal cancers (12.8%, 54/422) and cervical cancer (11.3%, 21/186). When assessed by histology without regard for cancer types, the frequency of MMRd/MSI-H was 11.0% (38/345) in high-grade serous adenocarcinoma, 38.6% (117/303) in endometrioid adenocarcinoma, and 30.2% (16/53) in carcinosarcoma. A total of 114 patients were treated with ICIs at least once. The objective response rate (ORR) was 21.6% (8/37) in cervical cancer, 4.7% (2/43) in ovarian cancer, and 25.8% (8/31) in endometrial/uterine cancers. Univariate regression analysis identified MMRd/MSI-H as the only significant factor associated with the ORR (28.9% [11/38] vs. 11.8% [9/76]; odds ratio, 3.033; 95% confidence interval, 1.129–8.144; p=0.028).ConclusionThe frequency of MMRd/MSI-H is moderate to high in gynecologic cancers in the Korean population. MMRd/MSI-H could be effective predictive biomarkers in gynecologic cancers of any type.

Colorectal cancer is the second leading cause of cancer-related deaths in women and men in Algeria. Lynch syndrome (LS) is an autosomal dominant disease caused by heterozygous germline pathogenic variants in mismatch repair genes (MMR) and frequently predisposes to colorectal cancer. However, data about MMR germline pathogenic variants in Algerian patients are limited. This first nationwide study aims to describe clinicopathologic features and germline variants in MMR genes in Algerian families with suspected LS. Sixty-four (64) families with suspected LS were studied. Index cases with LS who fulfilled Amsterdam criteria were screened by PCR-direct sequencing for germline variants in MMR genes: MLH1 (exons 1, 9, 10, 13, 16), MSH2 (exons 5, 6, 7, 12), MSH6 (exons 4 and 8) and PMS2 (exons 6 and 10). We selected these specific risk exons genes since they have a higher probability of harboring pathogenic variants. In addition, two unrelated LS patients were screened by next-generation sequencing using a cancer panel of 30 hereditary cancer genes. Six germline pathogenic variants and one germline likely pathogenic variant were identified in 19 (29.68%) families (4 MLH1, 2 MSH2 and 1 MSH6). Of index cases and relatives who underwent genetic testing (n = 76), 30 (39.47%) had MMR pathogenic gene variants, one (0.13%) had MMR gene likely pathogenic variant and three had MMR variant of uncertain significance, respectively. Two novel germline pathogenic variants in MLH1 (2) and one germline likely pathogenic variant in MSH6 (1) never published in individuals with LS have been detected in the present study. The recurrent MLH1 germline pathogenic variant c.1546CT has been found in nine LS families, six of them related with two large kindreds, from four North central provinces of Algeria. In addition, the common MSH2 germline pathogenic variant c.942+3AT has been detected in five unrelated patients with a strong LS family history. The accumulative knowledge about clinicopathological and genetic characteristics of LS in Algerian patients will impact clinical management in the areas of both prevention and treatment.

Adult granulosa cell tumors (AGCTs) are rare ovarian malignant neoplasms; their etiopathogenetic mechanisms remain largely unelucidated. Lately, defects in mismatch repair (MMR) have been implicated in the pathogenesis of AGCTs. Demonstration of MMR deficiency in these tumors can help identify patients potentially eligible for immune checkpoint inhibition therapy. The present study was done to explore the role of MMR deficiency in the etiopathogenesis of AGCTs.This was a retrospective study conducted on histopathologically confirmed AGCT cases. MMR protein expression was evaluated by immunohistochemistry (IHC) on tissue microarrays using an antibody panel of MSH2, MSH6, MLH1, and PMS2.Of a total of 40 ovarian AGCTs evaluated for MMR deficiency, none demonstrated loss of expression of any of the 4 MMR proteins.The results of our preliminary study show that there is no association between MMR deficiency with AGCT. Nevertheless, larger multicenter studies are needed to confirm or refute this observation.

Objective: To review the effect of universal screening of newly diagnosed upper tract urothelial carcinomas (UTUC) for mismatch repair (MMR) protein loss to aid in Lynch syndrome diagnostics. Materials and methods: Studies were identified through PubMed on December 1, 2021. Eligibility criteria were universal immunohistochemical analyses for at least 2 MMR proteins in unselected, consecutively collected UTUC cohorts. Exclusion criteria included reviews, case-reports, non-English language, and non-humans. Risk of bias was assessed using a modified Newcastle-Ottawa scale. Meta-analyses were performed to compare the association between clinical criteria and Lynch syndrome diagnoses. Results: From 12 included studies, 1628 surgically removed UTUC from 1626 patients were screened for MMR protein loss. In 11 studies, 140 of the 1559 patients had tumors with loss (9.0%) with 80.7% showing loss of MSH2, MSH6, or both. In 7 studies, genetic testing confirmed Lynch syndrome diagnosis for 20 of 970 patients (2.1%). In 8 studies, 31 patients were given a clinical Lynch syndrome diagnosis (2.6%). In total, 51 assumed or verified Lynch syndrome patients were identified among 1087 patients (4.7%). Meta-analyses of 3 studies showed significant association between previous cancer diagnosis and Lynch syndrome-associated UTUC (P = .038). Conclusion: Despite the few studies conducted and lack of genetic testing, current data suggests that universal screening for MMR protein loss in UTUC may result in Lynch syndrome diagnoses in 4.7%. However, for the screening to be effective for Lynch syndrome diagnostics, follow-up investigations, such as genetic testing for MMR variants, are needed.

Purpose In endometrial cancer, the incidence of mutations in mismatch repair genes (MMR) is estimated at 17–30%. Patients with alterations at this level (MSI) are known to have different clinical and anatomopathological characteristics than those without this genetic alteration (MSS). In this study, we aim to identify the MSI phenotype in patients who underwent hysterectomy for endometrial cancer. We assessed the correlation of this phenotype with anatomoclinical parameters such as obesity and histological subtype. Methods/patients Clinical and anatomopathological data were collected from 147 patients diagnosed with endometrial cancer and an immunohistochemical study of MMR system proteins was performed. PMS2 and MSH6 proteins were evaluated as primary screening and subsequent evaluation of MLH1 and MSH6, respectively, if the former were negative. Statistical association between the anatomopathological data and the immunohistochemical result was analyzed. Results and conclusions 22.4% of our patients were MSI phenotype. We obtained statistically significant differences by multivariate analysis between endometrioid subtype and higher FIGO classification grade with MSI phenotype and obesity with MSS phenotype. Given these statistical results, we propose a function for predicting the probability of being MSI phenotype taking into account the histological subtype (endometrioid/non-endometrioid carcinoma) and FIGO grade as well as obesity. This prediction may be useful prior to hysterectomy, for genetic study of the MLH1 promoter and subsequent genetic counseling.

Universal screening for Lynch syndrome (LS) on resected colorectal carcinomas (CRCs) and endometrial carcinomas (ECs) was implemented in Iceland in 2017 using immunohistochemistry (IHC) for mismatch repair (MMR) proteins. We examined the efficacy of the universal screening algorithm to detect LS and the diagnostic accuracy of MMR IHC by comparing results with a population-based genotype database.All patients diagnosed with CRC or EC per the Icelandic Cancer Registry from 2017 to 2019 who had tumor MMR IHC performed were included. Pathology reports and patient charts were reviewed. MMR IHC stains were crossmatched with genotyping results obtained from the deCODE database.IHC staining was done on 404 patients with CRC and 74 patients with EC. A total of 61 (15.1%) patients with CRC and 15 (20.3%) patients with EC were MMR-deficient. MMR IHC had 88.9% sensitivity in identifying patients with LS and a positive predictive value of 10.7%. Only 50% of individuals were appropriately referred for genetic testing, leading to underdiagnosis of LS.Universal screening for LS using MMR protein IHC in CRC and EC accurately identified patients appropriate for genetic testing in a population with MSH6 and PMS2 LS predominance. Because of lack of referral to genetic counseling, only 50% of patients with LS were identified through the screening algorithm.

The Lynch syndrome (LS) is an autosomal dominant disorder characterized by a strongly increased risk of developing colorectal cancer and several extra-colonic malignancies, such as carcinomas of the endometrium, ovary, ureter, stomach, and small intestine [1]. Lynch syndrome is caused by germline mutations in mismatch repair genes (MMR)[2], mainly in MLH1 and MSH2, rarely in MSH6 and PMS2 [3,4]. Tumors usually develop at a relatively young age (G mutation (rs267607760)in MLH1gene.

Lynch Syndrome (LS) is an autosomal dominant genetic condition that causes a high risk of colorectal cancer. The hallmark of LS is genetic instability as a result of mismatch repair (MMR) deficiency, particularly in repetitive low complexity regions called microsatellites (MS). MLH1

The presence of mismatch repair deficiency is frequently assessed in gastrointestinal and gynecologic neoplasms by surgical pathologists using immunohistochemical methods. Targeted next-generation sequencing (NGS) covering some genes in the mismatch repair complex is used with increasing frequency, however, the percent positive and negative agreement of immunohistochemical methods and NGS of mismatch repair genes is not well-described in the literature. We sought to compare performance of immunohistochemistry (IHC) and NGS of mismatch repair genes on our institutional targeted panel. We evaluated the concordance of immunohistochemical and panel-based gene sequencing methods in a retrospective cohort study of patients evaluated at our center with both immunohistochemical and panel-based sequencing. Our NGS panel covers only MLH1 and MSH2, whereas our immunohistochemical panel assesses for expression of MLH1, PMS2, MSH2, and MSH6. We identified 68 unique patients with both immunohistochemical evaluation of mismatch repair protein expression and NGS panel sequencing, of which 67 were suitable for analysis given the patterns of immunohistochemical loss of expression observed. The percent positive agreement for NGS with IHC was 50%, albeit with very rare positive cases (n=2/4). Percent negative agreement was also high at 100% (n=63/63). One case with loss of MLH1, PMS2, and MSH6 expression by IHC and no pathogenic variants by NGS exhibited MLH1 promoter hypermethylation. Percent negative agreement between immunohistochemical and NGS gene sequencing is high, although firm conclusions regarding percent positive agreement between NGS and IHC are limited by low numbers of positive cases in our cohort. In general, we consider the findings to support continued use of immunohistochemical methods to screen for the presence of mismatch repair deficiency and consider additional testing by NGS likely to add little diagnostic value in the context of intact immunohistochemical expression of mismatch repair proteins.

Immunohistochemistry (IHC) and/or MSI-PCR (microsatellite instability-polymerase chain reaction) tests are performed routinely to detect mismatch repair deficiency (MMR-D). Classical MMR-D tumors present a loss of MLH1/PMS2 or MSH2/MSH6 with MSI-High. Other profiles of MMR-D tumors have been described but have been rarely studied. In this study, we established a classification of unusual MMR-D tumors and determined their frequency and clinical impact. All MMR-D tumors identified between 2007 and 2017 were selected. Any profile besides the classical MMR-D phenotype was defined as unusual. For patients with unusual MMR-D tumors, IHC, and PCR data were reviewed, the tumor mutation burden (TMB) was evaluated and clinical and genetic features were collected. Of the 4948 cases of MMR testing, 3800 had both the available IHC and MSI-PCR results and 585 of these had MMR-D. After reviewing the IHC and PCR, 21% of the cases initially identified as unusual MMR-D were reclassified, which resulted in a final identification of 89 unusual MMR-D tumors (15%). Unusual MMR-D tumors were more often associated with non-CRC than classical MMR-D tumors. Unusual MMR-D tumors were classified into four sub-groups: i) isolated loss of PMS2 or MSH6, ii) classical loss of MLH1/PMS2 or MSH2/MSH6 without MSI, iii) four MMR proteins retained with MSI and, iv) complex loss of MMR proteins, with clinical characteristics for each sub-group. TMB-high or -intermediate was shown in 96% of the cancers studied (24/25), which confirmed MMR deficiency. Genetic syndromes were identified in 44.9% (40/89) and 21.4% (106/496) of patients with unusual and classical MMR-D tumors, respectively (P

Highlights • Colorectal carcinoma associated with proficient MLH1 gene are more aggressive cancers. • MLH1 influences the chemoresistance by mediating nuclear autophagy, nucleophagy. • Interaction among MLH1 and autophagy marker LC3 facilitated nucleophagy induction. • MLH1 mediated nucleophagy determined chemoresistance in colorectal cancer cells., Graphical abstract Image, graphical abstract, Colorectal Cancer (CRC) with Microsatellite instability (MSI) and mutLhomolog-1 (MLH1) gene deficiency are less aggressive than MLH1 proficient cancers. MLH1 is involved in several cellular processes, but its connection with the autophagy-dependent cellular response towards anticancer drugs remains unclear. In this study, we aimed to investigate the interaction between MLH1 and the autophagy marker LC3, which facilitated nucleophagy induction, and its potential role in determining sensitivity to 5-Fluorouracil (5-FU) induced cell death. To examine the role of MLH1 in DNA-damage-induced nucleophagy in CRC cells, we utilized a panel of MLH1 deficient and MLH1 proficient CRC cell lines. We included a parental HCT116 cell line (MLH1−/−) and its isogenic cell line HCT116 MLH1+/− in which a single allele of the MLH1 gene was introduced using CRISPR-Cas9 gene editing. We observed that MLH1 proficient cells were less sensitive to the 5-FU-induced cytotoxic effect. The 5-FU induced DNA damage led to LC3 up-regulation, which was dependent on MLH1 overexpression. Moreover, immunofluorescence and immunoprecipitation data showed LC3 and MLH1 were colocalized in CRC cells. Consequently, MLH1 dependent 5-FU-induced DNA damage contributed to the formation of micronuclei. These micronuclei colocalize with autolysosome, indicating a cytoprotective role of MLH1 dependent nucleophagy. Interestingly, siRNA knockdown of MLH1 in HCT116 MLH1+/− prevented LC3 upregulation and micronuclei formation. These novel data are the first to show an essential role of MLH1 in mediating the chemoresistance and survival of cancer cells by increasing the LC3 expression and inducing nucleophagy in 5-FU treated CRC cells.

Purpose: Lynch syndrome is defined by germline pathogenic mutations involving DNA mismatch repair (MMR) genes and linked with the development of MMR-deficient colon and endometrial cancers. Whether breast cancers developing in the context of Lynch syndrome are causally related to MMR deficiency (MMRd), remains controversial. Thus, we explored the morphologic and genomic characteristics of breast cancers occurring in Lynch syndrome individuals. Experimental Design: A retrospective analysis of 20,110 patients with cancer who underwent multigene panel genetic testing was performed to identify individuals with a likely pathogenic/pathogenic germline variant in MLH1, MSH2, MSH6, or PMS2 who developed breast cancers. The histologic characteristics and IHC assessment of breast cancers for MMR proteins and programmed death-ligand 1 (PD-L1) expression were assessed on cases with available materials. DNA samples from paired tumors and blood were sequenced with Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets (≥468 key cancer genes). Microsatellite instability (MSI) status was assessed utilizing MSISensor. Mutational signatures were defined using SigMA. Results: A total of 272 individuals with Lynch syndrome were identified, 13 (5%) of whom had primary breast cancers. The majority of breast cancers (92%) were hormone receptor–positive tumors. Five (42%) of 12 breast cancers displayed loss of MMR proteins by IHC. Four (36%) of 11 breast cancers subjected to tumor-normal sequencing showed dominant MSI mutational signatures, high tumor mutational burden, and indeterminate (27%) or high MSISensor scores (9%). One patient with metastatic MMRd breast cancer received anti-PD1 therapy and achieved a robust and durable response. Conclusions: A subset of breast cancers developing in individuals with Lynch syndrome are etiologically linked to MMRd and may benefit from anti-PD1/PD-L1 immunotherapy.

ObjectivesWhile ovarian cancer is the third most common Lynch syndrome-associated cancer in women, there is no established screening strategy to identify Lynch syndrome in this population. The objective of this study was to assess whether the 4-item brief Family History Questionnaire can be used as a screening tool to identify women with ovarian cancer at risk of Lynch syndrome.MethodsIn this prospective cohort study, participants with newly diagnosed non-serous, non-mucinous ovarian cancer completed the brief Family History Questionnaire, extended Family History Questionnaire, and had tumors assessed with immunohistochemistry for mismatch repair proteins, MLH1 methylation, and microsatellite instability testing. All underwent universal germline testing for Lynch syndrome. Performance characteristics were compared between the brief Family History Questionnaire, extended Family History Questionnaire, immunohistochemistry±MLH1 methylation, and microsatellite instability testing.ResultsOf 215 participants, 169 (79%) were evaluable with both the brief Family History Questionnaire and germline mutation status; 12 of these 169 were confirmed to have Lynch syndrome (7%). 10 of 12 patients (83%) with Lynch syndrome were correctly identified by the brief Family History Questionnaire, compared with 6 of 11 (55%) by the extended Family History Questionnaire, 11 of 13 (85%) by immunohistochemistry±MLH1 methylation, and 9 of 11 (82%) by microsatellite instability testing. The sensitivity, specificity, positive predictive values, and negative predictive values of the brief Family History Questionnaire were 83%, 65%, 15%, and 98%, respectively. A combined approach with immunohistochemistry and the brief Family History Questionnaire correctly identified all 12 patients with Lynch syndrome. The brief Family History Questionnaire was more sensitive than the extended Family History Questionnaire and took ConclusionsThe brief Family History Questionnaire alone or combined with immunohistochemistry may serve as an adequate screening strategy, especially in centers without access to universal tumor testing.

Identifying individuals who have Lynch syndrome involves a complex diagnostic workup that includes taking a detailed family history and a combination of various tests such as immunohistochemistry and/or molecular which may be germline and/or somatic. The National Society of Genetic Counselors and the Collaborative Group of the Americas on Inherited Gastrointestinal Cancer have come together to publish this practice resource for the evaluation of Lynch syndrome. The purpose of this practice resource was to provide guidance and a testing algorithm for Lynch syndrome as well as recommendations on when to offer testing. This practice resource does not replace a consultation with a genetics professional. This practice resource includes explanations in support of this and a summary of background data. While this practice resource is not intended to serve as a review of Lynch syndrome, it includes a discussion of background information and cites a number of key publications which should be reviewed for a more in-depth understanding. This practice resource is intended for genetic counselors, geneticists, gastroenterologists, surgeons, medical oncologists, obstetricians and gynecologists, nurses, and other healthcare providers who evaluate patients for Lynch syndrome.

Context.— The current College of American Pathologists reporting guideline for mismatch repair protein (MMRP) immunohistochemistry for Lynch syndrome (LS) screening considers the presence of any positive nuclear staining as intact MMRP expression. This would include tumors with combined areas of subclonal retention and loss of MMRP staining. Objective.— To evaluate the clinical significance of reporting subclonal staining patterns of MMRP immunohistochemistry in endometrial carcinoma. Design.— We retrospectively reviewed 455 consecutive MMRP immunohistochemistry results of endometrial carcinoma in hysterectomy specimens from 2012 through 2017 and identified cases with subclonal MMRP staining. These results were correlated with the patient's personal and family history of LS-associated carcinoma, MLH1 promoter methylation status, and LS genetic testing. Results.— Subclonal staining of MMRP was seen in 48 of 455 cases (10.5%) on review. Thirty cases demonstrated isolated subclonal staining and were reported by pathologists as follows: subclonal (n = 5), complete MMRP loss (n = 4), and intact MMRP (n = 21). Eighteen cases had subclonal staining in combination with complete loss of other MMRP. Cases reported as subclonal or complete MMRP loss had appropriate clinical follow-up. Two of 2 cases with isolated subclonal MSH6 loss tested positive for LS. One of 3 cases with isolated subclonal MLH1/PMS2 loss was negative for MLH1 promoter methylation; LS genetic testing was not performed because of cost. Conclusions.— Our study reveals that LS germline mutation can be detected in endometrial carcinoma patients whose tumors display sole subclonal MMRP staining. Our results stress the importance of reporting subclonal staining patterns to ensure appropriate clinical follow-up.

Lynch syndrome (LS) is an autosomal dominant hereditary cancer syndrome caused by pathogenic germline variants (PGV) in any of the 4 DNA mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2, or deletions in EPCAM. LS leads to an increased risk of intestinal and extraintestinal cancers, of which colorectal and endometrial cancers are the most common. Individuals at risk for LS can be identified by using clinical criteria, prediction models, and universal tumor testing. Understanding each of these tools, including limitations and mimics of LS, is essential to the early identification of at-risk individuals.

BackgroundGermline variants in the DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2) cause Lynch syndrome, an autosomal dominant hereditary cancer susceptibility syndrome. The risk for endometrial cancer is significantly higher in women with MSH6 pathogenic/likely pathogenic (P/LP) variants compared with that for MLH1 or MSH2 variants.MethodsThe proband was tested via a clinical testing, Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT). RT-PCR was performed using patient's blood DNA and cDNA was analyzed by DNA sequencing and a cloning approach.ResultsWe report a 56-year-old female with endometrial cancer who carries a germline variant, MSH6 c.4001G > C, located at the last nucleotide of exon 9. While the pathogenicity of this variant was previously unknown, functional studies demonstrated that this variant completely abolished normal splicing and caused exon 9 skipping, which is expected to lead to a prematurely truncated or abnormal protein.ConclusionOur results indicate that this variant likely contributes to cancer predisposition through disruption of normal splicing, and is classified as likely pathogenic.

Brain tumors affect one-third of all children with cancer. Approximately 10% of children with cancer carry variants in cancer-predisposition genes. However, germline analyses in large cohorts of Asian children have not been reported. Thirty-eight Japanese patients with pediatric brain tumors were included in this study (19 boys, 19 girls). DNA was extracted from the patients’ peripheral blood, and cancer-associated genes were analyzed using targeted resequencing. Rare variants with allele frequencies CHEK2 and FANCI; 2 frameshift deletions in SMARCB1 and PTCH1; and 3 missense variants of TSC1, WRN, and MLH1. The median age at diagnosis was 9.1 years, and three of the 7 patients had a family history of cancer. One patient diagnosed with basal cell nevus syndrome, also called Gorlin syndrome, developed a second neoplasm, and another with an SMARCB1 variant and an atypical teratoid/rhabdoid tumor developed a thyroid adenomatous nodule. This is the first cancer-related germline analysis with detailed clinical information reported in Japanese children with brain tumors. The prevalence was almost equivalent to that in white children.

BACKGROUND. Microsatellite instability (MSI-H)/mismatch repair-deficiency (dMMR) is a biomarker for response to immune-checkpoint inhibitors (ICIs). Whether mechanisms underlying MSI alter responses to ICIs is unclear. We report data from a prospective phase II pilot study (NCT02899793) of pembrolizumab in recurrent MSI-H endometrial cancer (EC) patients analyzed by whole exome sequencing (WES) and potential mechanisms of primary/secondary ICI resistance. METHODS. Patients with measurable, MSI-H/dMMR EC confirmed by polymerase chain reaction/immunohistochemistry were evaluated by WES and received pembrolizumab 200 mg every 3 weeks for ≤2 years. The primary end point was objective response rate (ORR). Secondary endpoints included progression-free survival (PFS) and overall survival (OS). RESULTS. Twenty-five patients (24 evaluable) were treated. Six (25%) patients harbored Lynch/Lynch-like tumors while 18 (75%) had sporadic EC. Tumor mutational burden (TMB) was higher in Lynch-like (median 2939 mutations/megabase [Mut/Mb], IQR:867-5108) versus sporadic tumors (median 604 Mut/Mb, IQR:411-798) (P=0.0076). ORR was 100% in Lynch/Lynch-like but only 44% in sporadic patients (P=0.024). The 3-year PFS/OS proportions were 100% versus 30% (P=0.017) and 100% versus 43% (P=0.043), respectively. CONCLUSIONS. Our study suggests prognostic significance of Lynch-like versus sporadic MSI-H/dMMR EC on ORR, PFS and OS when treated with pembrolizumab. Larger, confirmatory studies in EC and other MSI-H/dMMR tumors are necessary. Defective antigen processing/presentation and deranged induction in interferon responses served as mechanisms of resistance in sporadic MSI-H EC. Oligo-progression in MSI-H/dMMR patients appeared salvageable by surgical resection and/or local treatment and continuation of pembrolizumab off-study. Clinical studies evaluating separate subtypes of MSI-H/dMMR EC treated with ICIs are warranted.

Mut L homolog-1 (MLH1) is a key DNA mismatch repair protein which participates in the sensitivity to DNA damaging agents. However, its role in the radiosensitivity of tumor cells is less well characterized. In this study, we investigated the role of MLH1 in cellular responses to ionizing radiation (IR) and explored the signaling molecules involved. The isogenic pair of MLH1 proficient (MLH1+) and deficient (MLH1–) human colorectal cancer HCT116 cells was exposed to IR for 24 h at the dose of 3 cGy. The clonogenic survival was examined by the colony formation assay. Cell cycle distribution was analyzed with flow cytometry. Changes in the protein level of MLH1, DNA damage marker γH2AX, and protein kinase A catalytic subunit (PRKAC), a common target for anti-tumor drugs, were examined with Western blotting. The results showed that the HCT116 (MLH1+) cells demonstrated increased radio-resistance with increased S population, decreased G2 population, a low level of γH2AX, a reduced ratio of phosphorylated PRKACαβ to total PRKAC, and an elevated level of total PRKAC and phosphorylated PRKACβII following IR compared with the HCT116 (MLH1–) cells. Importantly, silencing PRKAC in HCT116 (MLH1+) cells increased the cellular radiosensitivity. In conclusion, MLH1 may increase cellular resistance to IR by activating PRKAC. Our finding is the first to demonstrate the important role of PRKAC in MLH1-mediated radiosensitivity, suggesting that PRKAC has potential as a biomarker and a therapeutic target for increasing radio-sensitization.

We report the first pedigree of Lynch syndrome bearing a germ-line MSH2 missense mutation c.1808A>T (Asp603Val). Until now, this missense mutation, in exon 12 of MSH2, was identified as a variant of unknown significance in the International Society for Gastrointestinal Hereditary Tumours database. In vitro induction mutagenesis experiments indicated that the MSH2 mutant protein (Asp603Val) is easily degraded in embryonic stem cells, albeit there is no clinical information concerning this mutant. Our pedigree includes four patients with Lynch syndrome-associated malignancies and clinically matches the Amsterdam II criteria. The proband, a female, first had an endometrial cancer at the age of 49 and then mantle cell lymphoma, colonic and gastric adenocarcinomas and neuroendocrine carcinoma, successively. Her mother also had Lynch syndrome-associated malignancies, including colonic, uterine and gastric cancers, and her elder son had rectal cancer. In the germline of the proband and her son, an MSH2 missense mutation c.1808A>T was discovered. Immunohistochemical analyses indicated that the expression of the MSH2 protein was decreased in the tumors, such as gastric cancer and neuroendocrine carcinoma, due to the missense mutation c.1808A>T. This study showed that the MSH2 missense mutation c.1808A>T (Asp603Val) is a likely pathogenic mutation and is responsible for typical Lynch syndrome-associated malignancies, including neuroendocrine carcinoma.

There are limited data regarding the correlation of clinical and pathologic parameters with mismatch repair (MMR) protein-deficient subgroups and methylation status. In this study, we analyzed the status of MMR proteins in resection specimens of 198 consecutive endometrial carcinomas and the methylation status in tumors with MLH1 and PMS2 deficiency. We, therefore, assessed the correlation of clinical and pathologic parameters with MMR protein-deficient subgroups. Univariate analysis revealed that deeper myometrial invasion and the presence of tumor-associated lymphocytes were more frequently observed in tumors with MMR protein deficiency ( P =0.023 and 0.001, respectively). The multivariate logistic regression analysis revealed that only the presence of tumor-associated lymphocytes was significantly associated with MMR protein deficiency ( P =0.002, odds ratio=2.674, 95% confidence interval=1.418-5.045). We also compared MLH1 and PMS2 deficiency with other protein deficiency regarding clinical and pathologic parameters. Furthermore, we compared MLH1 methylated tumors with MMR protein-deficient nonmethylated tumors regarding clinical and pathologic parameters. MLH1 was methylated in 51 of 54 tumors with MLH1 and PMS2 deficiency. In univariate analysis, a larger tumor size was significantly associated with MLH1 and PMS2 deficiency and with MLH1 methylation ( P =0.004 and 0.005, respectively). The multivariate logistic regression analysis revealed that a larger tumor size was significantly associated with MLH1 and PMS2 deficiency and MLH1 methylation ( P =0.002, odds ratio=14.222, 95% confidence interval=2.560-79.026, P =0.008, odds ratio=22.222, 95% confidence interval=2.220-222.395, respectively). Our results showed a slightly higher rate of MLH1 and PMS2 deficiency (34.3%) than in previous studies. This may likely be due to ethnic differences in frequency of various mutations.

Eukaryotic DNA mismatch repair (MMR) initiates through mispair recognition by the MutS homologs Msh2-Msh6 and Msh2-Msh3 and subsequent recruitment of the MutL homologs Mlh1-Pms1 (human MLH1-PMS2). In bacteria, MutL is recruited by interactions with the connector domain of one MutS subunit and the ATPase and core domains of the other MutS subunit. Analysis of the S. cerevisiae and human homologs have only identified an interaction between the Msh2 connector domain and Mlh1. Here we investigated whether a conserved Msh6 ATPase/core domain-Mlh1 interaction and an Msh2-Msh6 interaction with Pms1 also act in MMR. Mutations in MLH1 affecting interactions with both the Msh2 and Msh6 interfaces caused MMR defects, whereas equivalent pms1 mutations did not cause MMR defects. Mutant Mlh1-Pms1 complexes containing Mlh1 amino acid substitutions were defective for recruitment to mispaired DNA by Msh2-Msh6, did not support MMR in reconstituted Mlh1-Pms1-dependent MMR reactions in vitro, but were proficient in Msh2-Msh6-independent Mlh1-Pms1 endonuclease activity. These results indicate that Mlh1, the common subunit of the Mlh1-Pms1, Mlh1-Mlh2, and Mlh1-Mlh3 complexes, but not Pms1, is recruited by Msh2-Msh6 through interactions with both of its subunits.

The aim of the study was to correlate the expression of mismatch repairs proteins (MMR), programmed-death-ligand1 (PDL-1), and estro-progestinic receptors (ER/PgR) in tissue samples from a series of cervical adenocarcinoma (ADC) patients with their clinicopathological features.Thirty-nine ADC specimens were retrospectively retrieved from the Division of Pathology of the University Hospital of Pisa from 2015 to 2021. Histological subtype, grade (G), Silva pattern, presence of lymph vascular space invasion (LVI), and perineural invasion (PNI) were annotated. On representative samples, immunostaining for ER/PgR, MLH1, PMS2, MSH2, MSH6, and PDL-1(sp142) was performed.Thirty-five ADCs were HPV-associated usual type (24 invasive and 11 in situ), 2 were clear cell type, one was a minimal deviation adenocarcinoma (MDA), and one was an invasive stratified mucin-producing carcinoma (iSMC). ADC associated with LVI were mostly G2-3, whereas those associated also with PNI were G3 with Silva pattern C. No difference in the expression of ER/PgR was observed with a dichotomic age stratification (51 years) of patients. Only 6 ADCs were MMR-deficient, all of them were of the usual type (4 invasive and 2 in situ). The heterodimer MLH-1/PMS2 was the one most frequently altered (5/6), whereas only one case had MSH6 loss. None of ADCs express PDL-1, except iSMC which showed PDL-1 expression1% in neoplastic cells.Both invasive and in situ usual type ADCs indicate MMR deficiency, highlighting how this could be an early event in tumorigenesis. None of the cases, except for iSMC, express PDL-1.

Eukaryotic DNA mismatch repair (MMR) depends on recruitment of the Mlh1-Pms1 endonuclease (human MLH1-PMS2) to mispaired DNA. Both Mlh1 and Pms1 contain a long unstructured linker that connects the N- and carboxyl-terminal domains. Here, we demonstrated the Mlh1 linker contains a conserved motif ( Saccharomyces cerevisiae residues 391–415) required for MMR. The Mlh1-R401A,D403A-Pms1 linker motif mutant protein was defective for MMR and endonuclease activity in vitro, even though the conserved motif could be >750 Å from the carboxyl-terminal endonuclease active site or the N-terminal adenosine triphosphate (ATP)-binding site. Peptides encoding this motif inhibited wild-type Mlh1-Pms1 endonuclease activity. The motif functioned in vivo at different sites within the Mlh1 linker and within the Pms1 linker. Motif mutations in human cancers caused a loss-of-function phenotype when modeled in S. cerevisiae . These results suggest that the Mlh1 motif promotes the PCNA-activated endonuclease activity of Mlh1-Pms1 via interactions with DNA, PCNA, RFC, or other domains of the Mlh1-Pms1 complex.

Objectives Sessile serrated adenomas with dysplasia (SSADs) of the colon are transitional lesions between sessile serrated adenomas (SSAs) and a subset of colorectal adenocarcinomas. We wished to gain insight into the relative percentages and significance of SSAD subtypes. Methods Retrospective (2007-2012) clinicopathologic review of colorectal polyps initially regarded as having mixed serrated and dysplastic elements. SSADs were subdivided into those with cap-like adenomatous dysplasia (ad1), non-cap-like adenomatous dysplasia (ad2), serrated dysplasia (ser), minimal dysplasia (min), and dysplasia not otherwise specified (nos). MLH1 immunostaining was performed on many. Results SSADser (7.7%) had a greater propensity for right colon, women, and MLH1 loss vs the entire cohort. SSAad1 (11.6%) had the least female preponderance, was least likely to have MLH1 loss, and was most likely to affect the left colorectum. SSAD with MLH1 loss was associated with an increased burden of SSAs in the background colon (P = .0003) but not tubular adenomas or hyperplastic polyps. Most SSADs (ad2 and nos groups, 80% combined) showed difficult-to-classify dysplasia, intermediate MLH1 loss rates, and intermediate clinical features. Conclusions While some trends exist, morphologically subclassifying SSADs is probably not justified in routine clinical practice. MLH1 loss portends a greater burden of SSAs in the background colon.

The diagnosis of endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (AH/EIN) remains challenging and subjective in some cases, with variable histologic criteria and differences of opinion among gynecologic pathologists, potentially leading to under/overtreatment. There has been growing interest in the use of specific immunohistochemical markers as adjuncts in AH/EIN diagnosis. For example, the World Health Organization 2020 Classification specifies that loss of Pten, Pax2, or mismatch repair proteins are desirable diagnostic criteria. Other markers, most notably β-catenin and Arid1a, are also aberrantly expressed in some AH/EIN. However, the performance of some markers individually-and more importantly as a group-has not been rigorously explored, raising questions as to which marker(s) or combination(s) is the most effective in practice. Formalin-fixed paraffin-embedded tissue sections from AH/EIN cases (n=111) were analyzed by immunohistochemistry for 6 markers: Pax2, Pten, Mlh1, β-catenin, Arid1a, and p53. Aberrant expression was tabulated for each case and marker. An additional set of normal endometria (n=79) was also analyzed to define optimal diagnostic criteria for marker aberrance. The performance characteristics of each marker, the entire panel, and subsets thereof were quantitatively and statistically analyzed. In order of number of cases detected, the most frequently aberrant markers in AH/EIN were Pax2 (81.1% of cases), Pten (50.5%), β-catenin (47.7%), Arid1a (7.2%), Mlh1 (4.5%), and p53 (2.7%). The majority of cases showed aberrant expression of ≥2 markers. All 6 markers together identified 92.8% of cases. Arid1a, Mlh1, and p53 were robust and readily scored markers, but all cases showing aberrant expression of these 3 markers were also detected by Pax2, Pten, or β-catenin. A focused panel of only 3 markers (Pax2, Pten, and β-catenin) showed optimal performance characteristics as a diagnostic adjunct in the histopathologic diagnosis of AH/EIN. Use of this panel is practicable and robust, with at least 1 of the 3 markers being aberrant in 92.8% of AH/EIN.

Lynch syndrome (LS), Lynch-like syndrome (LLS), and familial colorectal cancer type X (FCCX) are different entities of familial cancer predisposition leading to an increased risk of colorectal cancer (CRC). The aim of this prospective study was to characterize and to compare the risks for adenoma and CRC in these three risk groups. Data was taken from the registry of the German Consortium for Familial Intestinal Cancer. Patients were prospectively followed up in an intensified colonoscopic surveillance programme that included annual examinations. Cumulative risks for adenoma and CRC were calculated separately for LS, LLS, and FCCX, and then for males and females. Multivariate Cox regression was used to analyse the independent contributions of risk group, mismatch repair gene (within LS), sex, and previous adenoma. The study population comprised 1448 individuals (103 FCCX, 481 LLS and 864 LS). The risks were similar for colorectal adenomas, but different for first and metachronous CRC between the three risk groups. CRC risk was highest in LS, followed by LLS, and lowest in FCCX. Male sex and a prevalent adenoma in the index colonoscopy were associated with a higher risk for incident adenoma and CRC. In patients with LS, CRC risks were particularly higher in female MSH2 than MLH1 carriers. This study may support the development of risk-adapted surveillance policies in LS, LLS and FCCX. This article is protected by copyright. All rights reserved.

Lynch syndrome (LS) is a condition which predisposes individuals primarily to early-onset colorectal and endometrial cancer. LS is characterized by a germline pathogenic variant in one of the MMR (MisMatch Repair) gene, inducing a phenotype of microsatellite instability in the tumor, which may be associated with a loss of expression of MMR proteins detected by standard immunohistochemistry on tumor tissue. Most of the time, LS is inherited from a parent in whom the condition may not be known due to incomplete penetrance, but de novo pathogenic variant is a rare occurrence. Here, we describe the case of a 52-year-old woman with no family history of LS, referred to the genetics department for colorectal cancer at the age of 50. Genetic analysis revealed a de novo germline pathogenic variant in the MSH6 gene. To date, this case is only the second report of a de novo pathogenic variant in the MSH6 gene in Lynch syndrome. De novo mutations have been extensively studied over the past years, but little is known about their origin and mechanism of occurrence in MMR genes. However, knowledge of mutation status allows better cancer risk management for the patient and an appropriate genetic testing and counseling for her family.

Background Endometrial carcinoma represents a sentinel cancer for Lynch syndrome (LS) identification. It is crucial to highlight how other types of tumors can arise in the gynecological tract acting as sentinel tumors in LS patients.Up to now, no established LS patient management strategy has incorporated the presence of these additional candidate sentinel tumors to improve the prevention and management of LS tumors. Methods In order to investigate the involvement of the most frequent gynecological cancers in gynecological cancers, we studied different subsets of gynecological cancers using both somatic approaches, including mismatch repair (MMR) gene immunohistochemical expression, microsatellite instability, and germline analyses ofMSH2, MSH6, MLH1, PMS2 and EPCAM genes.A total of 261 patients referring to the Cancer Genetic Counselling Service of our institution were included in the study. In detail, our series was composed of 131 patients affected by uterus cancers including endometrial, isthmus and non-HPV endocervical carcinomas, 113 patients affected by ovarian cancers and 17 patients affected by synchronous endometrial/ovarian carcinomas (SEOC).In addition, we studied 115 cases of endometrial cancers identified by 2 years of universal testing (endometrial cancers/UTs) using IHC analysis of four MMR proteins. Results and conclusions The incidence of MMR defective gynecological cancers ranged from 7.1 to 47.1% depending on cancer site and selection. LS patients carriers of pathogenetic MMR variants were identified in 19.8% of uterus cancers, 35.3% of SEOC, 4.4% of ovarian cancers. In addition, pathogenetic MMR variants were identified in 4.3% of endometrial cancers/universal testing investigated with universal screening.In conclusion, gynecological cancers are heavily involved in LS and our study shows that MMR screening using immunohistochemical pattern and MSI analysis of endometrial and ovarian cancers as well as of rare entities such as non-HPV related endocervical cancers and synchronous endometrial and ovarian cancers are sentinels for LS.Tumor testing approach improves early identification of MMR defective gynecological cancers and this is an effective strategy to detect high-risk patients and to offer them and their relatives personalized cancer prevention.

Mixed neuroendocrine/non-neuroendocrine neoplasms (MiNEN) are rare mixed epithelial neoplasms in which a neuroendocrine component is combined with a non-neuroendocrine component. Here, we provide the clinical, pathologic, and molecular report of a 73-year-old-man presenting with an intestinal MiNEN. The lesion was composed of a well-differentiated G3 neuroendocrine tumor and a colloid adenocarcinoma. The molecular characterization was performed using a multigene next-generation sequencing panel. The neoplasm displayed microsatellite instability due to MLH1 promoter methylation. The extended molecular profile documented the same mutations affecting ARID1A, ASXL1, BLM, and RNF43 genes in both components, indicating a monoclonal origin of the tumor. Regarding component-specific gene mutations, BRCA2 was specifically altered in the neuroendocrine area. It may represent a new actionable target for precision oncology in MiNEN, but the lack of its alteration in the colloid component calls for further considerations on intratumor heterogeneity. The most important finding with potential immediate implications regards the presence of microsatellite instability: it indicates that this molecular alteration should become part of the diagnostic algorithm for these rare neoplasms.

Objectives To describe clinicopathologic characteristics and survival outcomes of endometrial adenocarcinomas stratified by mismatch repair (MMR) status. Methods Single-institution, retrospective study of all women with endometrioid adenocarcinomas treated from January 2012 through December 2017. Patients were categorized into one of three groups based on MMR testing: intact MMR expression (MMR+), probable MMR mutation (MMR–), or MLH1 hypermethylation (hMLH1+). Demographics, pathologic characteristics, recurrence rates, and survival differences were analyzed. Results In total, 316 women were included in the analysis: 235 (74.4%) patients in the MMR+ group, 10 (3.1%) in the MMR– group, and 71 (22.5%) in the hMLH1+ group. Patients with hMLH1+ were significantly older, exhibited higher-grade histology and presence of lymphovascular space invasion, and were more likely to have received adjuvant treatment. The early stage hMLH1+ patients were more likely to recur (15.3% hMLH1+ vs 2.3% MMR+ vs 12.5% MMR–, P Conclusions Women with hMLH1+ endometrial cancer have worse prognostic features and recur more frequently, even in patients traditionally considered low risk for recurrence.

There exist relatively sparse and conflicting data on high-level microsatellite instability (MSI-H) and deficient mismatch repair (dMMR) in cutaneous malignancies. We aimed to determine the expression profiles of MMR proteins (MSH2, MSH6, MLH1, and PMS2) in different progression stages of cutaneous squamous cell carcinoma (cSCC, 102 patients in total) by immunohistochemistry, and search for MSI-H in patients with low-level MMR or dMMR using multiplex-PCR. Low-level MMR protein expression was observed in five patients: One patient with primary cSCC &lt, 2 mm thickness and low-level MLH1, three patients with primary cSCC &gt, 6 mm (including one with low-level MSH2, as well as MSH6 expression, and two with low-level PMS2), and one patient with a cSCC metastasis showing low-level MSH2 as well as MSH6. Intergroup protein expression analysis revealed that MLH1 and MSH2 expression in actinic keratosis was significantly decreased when compared to Bowen’s disease, cSCC &lt, 2 mm, cSCC &gt, 6 mm, and cSCC metastasis. In cases with low-level MMR, we performed MSI-H tests revealing three cases with MSI-H and one with low-level MSI-L. We found low-level MMR expression in a small subset of patients with invasive or metastatic cSCC. Hence, loss of MMR expression may be associated with tumour progression in a small subgroup of patients with non-melanoma skin cancer.

Screening for mismatch repair (MMR) deficiency in unselected patients with endometrial carcinoma (EC) and the clinicopathologic descriptions of ECs with MMR deficiency have been well demonstrated in Western populations, but studies on Asian populations are relatively scarce. In this study, we described the clinicopathologic features of ECs according to MMR status in unselected Taiwanese patients. We also conducted subgroup analysis of MMR-deficient (dMMR) cases according to the presence or absence of MLH1. Patients diagnosed with ECs between January 2017 and February 2020 at our institution were included. Immunohistochemistry analysis of MLH1, PMS2, MSH2, and MSH6 proteins on endometrial primary tumors and clinicopathologic variables were assessed retrospectively. A total of 231 EC patients were enrolled, of whom 50 (21.6%) had dMMR tumors. Of these 50 cases, 39 had tumors that lacked MLH1 expression and 11 were positive for MLH1. The overall dMMR group was significantly related to older age, parity, and high histologic grade compared with the MMR-proficient (pMMR) group. ECs with MLH1 deficiency were obviously associated with several poor pathologic features, including high histologic grade, lymph node metastasis, and lymphovascular space invasion. Moreover, we first reported that parity and the late age at menopause are strongly correlated with MLH1-related dMMR EC group compared with pMMR group. In conclusion, triaging EC patients into pMMR, MLH1-related dMMR and non-MLH1-related dMMR groups by immunohistochemistry analysis may help clinicians to predict disease behavior and guide further management. The strong association between parity and MLH1-related dMMR ECs warrants further investigation on the underlying mechanism.

Summary Targetable kinase fusions are extremely rare ( We searched for NTRK1, NTRK2, NTRK3, ALK, ROS1, BRAF, RET, and NRG1 kinase fusions in CRCs using methods easy-to-implement in pathology laboratories: immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and fully automated real-time PCR targeted analyses. RNA-sequencing analyses were used for confirmation. Among 84 selected MLH1 deficient (IHC) CRCs cases, MLH1loss MSI-High wild-type CRCs consisted first in 19 cases after Idylla™ analyses and finally in 18 cases (21%) after RNA-sequencing (detection of one additional KRASG12D mutation). FISH (and when relevant, IHC) analyses concluded in 5 NTRK1, 3 NTRK3, 1 ALK, 2 BRAF, and 2 RET FISH positive tumors. ALK and NTRK1 rearranged tumors were IHC positive, but pan-TRK IHC was negative in the 3 NTRK3 FISH positive tumors. RNA-sequencing analyses confirmed 12 of 13 fusions with only one false positive RET FISH result. Finally, 12/18 (67%) of MLH1loss MSI-High wild-type CRCs contained targetable kinase fusions. Our study demonstrates the feasibility, but also the cost-effectiveness, of a multistep but rapid diagnostic strategy based on nonsequencing methods to identify rare and targetable kinase fusions in patients with advanced CRCs, as well as the high prevalence of these kinase fusions in MLH1loss MSI-High wild-type CRCs. Nevertheless, confirmatory RNA-sequencing analyses are necessary in case of low FISH positive nuclei percentage to rule out FISH false-positive results.

Hereditary endometrial cancer (EC) is most commonly attributed to pathogenic variants in mismatch repair (MMR) genes. Evidence supports existence of additional genetic risk factors in the context of multiple cancer diagnoses and/or family history of EC. EC patients (n=5292) referred for diagnostic multi-gene cancer panel testing were annotated for: presence of a pathogenic gene variant; personal history of prior, concurrent or subsequent cancer of another type; reported family history of Lynch syndrome or endometrial cancer. The Pearson χ2 test was used to assess differences in gene variant prevalence between case sub-groups defined by personal and/or family history of cancer/s, using cases with no family history of Lynch/EC as reference. Another cancer diagnosis was reported for 55% of EC cases. EC cases with prior and reported family history of Lynch cancer were enriched for variants in MLH1 (p=3.5x10-7 ), MSH2 (p=3.1x10-7 ), and PMS2 (p=0.02). Consistent with expectations for a breast cancer (BC) gene also predisposing to EC, variant frequency was increased in EC patients with prior BC and family history of EC for BRCA1 (p=1.7x10-5 ) and PALB2 (p=0.0002). Strategic case-case analyses to address cohort ascertainment bias has provided rationale to direct future studies of candidate hereditary EC genes. This article is protected by copyright. All rights reserved.

In mismatch repair (MMR) immunohistochemistry, four MMR proteins’ staining pattern reveals which particular gene may be defective. However, in the null phenotype, four MMR proteins are lost; consequently, it will be challenging to assume the target gene by immunohistochemistry and to determine whether deficient MMR was sporadic or germline. A 70-year-old man underwent right hemicolectomy with the diagnosis of ascending colon cancer. The postoperative histopathology revealed the diagnosis of medullary carcinoma and the loss of all four MMR expressions in immunohistochemistry. The mutation analysis using a peripheral blood sample showed no germline mutations in the four genes. This clinical case presents an unusual colon carcinoma that showed a MMR protein immunohistochemistry null phenotype. The cause of expression loss of MMR proteins can be explained by the loss of MLH1 and MSH2 functions associated with somatic loss of function mutations, functional loss in all four MMR proteins associated with somatic loss of function mutations, or Lynch-like syndrome. Correct interpretation and accumulation of relevant cases are necessary to unveil unusual cases in the era of universal screening.

Mismatch repair (MMR) immunohistochemistry (IHC) is frequently used to inform prognosis, select (immuno-)therapy, and identify patients for heritable cancer syndrome testing. However, false-negative and false-positive MMR IHC interpretations have been described.Following identification of discordant MMR IHC and DNA-based microsatellite instability testing in a patient with colorectal carcinoma, we retrospectively reviewed institutional archives to identify patient samples with similar discrepancies.We report a patient with metastatic colorectal carcinoma who initially received immunotherapy on the basis of apparent isolated loss of MLH1 by IHC; notably,This study confirms that rare germline polymorphisms can result in incorrect IHC results, potentially affecting selection of optimal therapy and the decision to pursue germline testing. This case further highlights the need for expert molecular pathologic review and communication between clinical and molecular oncology teams.

Lynch syndrome is a heritable condition caused by a heterozygous germline inactivating mutation of the DNA mismatch repair (MMR) genes, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants, for which the clinical significance is unclear in many cases. We have identified three MLH1 missense alterations (p.(Glu736Lys), p.(Pro640Thr) and p.(Leu73Pro)) in six individuals from large Tunisian families. For none of these alterations, a classification of pathogenicity was available, consequently diagnosis, predictive testing and targeted surveillance in affected families was impossible. We therefore performed functional laboratory testing using a system testing stability as well as catalytic activity that includes clinically validated reference variants. Both p.(Leu73Pro) and p.(Pro640Thr) were found to be non-functional due to severe defects in protein stability and catalytic activity. In contrast, p.(Glu736Lys) was comparable to the wildtype protein and therefore considered a neutral substitution. Analysis of residue conservation and of the structural roles of the substituted residues corroborated these findings. In conjunction with the available clinical data, two variants fulfil classification criteria for class 4 "likely pathogenic". The findings of this work clarify the mechanism of pathogenicity of two unclear MLH1 variants and enables predictive testing and targeted surveillance in members of carrier families worldwide.

AIMS Screening all patients newly diagnosed with colorectal cancer (CRC) for possible Lynch syndrome (LS) has been recommended in the United Kingdom since the National Institute for Health and Care Excellence (NICE) released new diagnostics guidance in February 2017. We sought to validate the NICE screening pathway through a prospective regional programme throughout a 5.2-million population during a 2-year period. METHODS AND RESULTS Pathology departments at 14 hospital trusts in the Yorkshire and Humber region of the United Kingdom were invited to refer material from patients with newly diagnosed CRC aged 50 years or over between 1 April 2017 and 31 March 2019 for LS screening. Testing consisted of immunohistochemistry for MLH1, PMS2, MSH2 and MSH6 followed by BRAF mutation analysis ± MLH1 promoter methylation testing in cases showing MLH1 loss. A total of 3141 individual specimens were submitted for testing from 12 departments consisting of 3061 unique tumours and 2791 prospectively acquired patients with CRC. Defective mismatch repair (dMMR) was observed in 15% of cases. In cases showing MLH1 loss, 76% contained a detectable BRAF mutation and, of the remainder, 77% showed MLH1 promoter hypermethylation. Of the patients included in the final analysis, 81 (2.9%) had an indication for germline testing. CONCLUSION LS screening using the NICE diagnostics guidance pathway is deliverable at scale identifying significant numbers of patients with dMMR. This information is used to refer patients to regional clinical genetics services in addition to informing treatment pathways including the use of adjuvant/neoadjuvant chemotherapy and immunotherapy.

Meiotic recombination is essential for reciprocal exchange of genetic information between homologous chromosomes and their subsequent proper segregation in sexually reproducing organisms. MLH1 and MLH3 belong to meiosis-specific members of the MutL-homolog family, which are required for normal level of crossovers (COs) in some eukaryotes. However, their functions in plants need to be further elucidated. Here, we report the identification of OsMLH1 and reveal its functions during meiosis in rice. Using CRISPR-Cas9 approach, two independent mutants, Osmlh1-1 and Osmlh1-2, are generated and exhibited significantly reduced male fertility. In Osmlh1-1, the clearance of PAIR2 is delayed and partial ZEP1 proteins are not loaded into the chromosomes, which might be due to the deficient in resolution of interlocks at late zygotene. Thus, OsMLH1 is required for the assembly of synapsis complex. In Osmlh1-1, CO number is dropped by ~53% and the distribution of residual COs is consistent with predicted Poisson distribution, indicating that OsMLH1 is essential for the formation of interference-sensitive COs (class I COs). OsMLH1 interacts with OsMLH3 through their C-terminal domains. Mutation in OsMLH3 also affects the pollen fertility. Thus, our experiments reveal that the conserved heterodimer MutLγ (OsMLH1-OsMLH3) is essential for the formation of class I COs in rice.

DNA mismatch repair (MMR) is an essential physiological process for correcting mutations occurring during DNA replication. Deficient MMR (dMMR) leads to increased tumour mutational burden, with a heightened risk of neoplasia. Demonstration of dMMR via the immunohistochemical assessment of MMR proteins is useful when screening for inherited cancer syndromes (especially Lynch syndrome) and for the prediction of clinical cancer response to conventional chemotherapy and novel immunotherapies. Identification of dMMR may also be helpful in other situations e.g. when considering a diagnosis of conventional dysplasia in sessile serrated lesions of the large intestine. This article provides a practical illustrated guide to DNA MMR interpretation, using a series of clinical examples.

Endometrial carcinoma is one of the prototypical malignancies associated with Lynch syndrome, an inherited cancer syndrome most commonly caused by germline mutations in DNA mismatch repair (MMR) genes, although rare alternative mechanisms also exist. In this report, we describe a patient first diagnosed with colorectal cancer at age 33, then vulvar squamous cell carcinoma, facial sebaceous adenoma/sebaceoma, and finally endometrial carcinoma at age 52. All tumors were MLH1/PMS2-deficient by immunohistochemistry, and MLH1 promoter methylation was identified in the endometrial cancer. Germline MLH1 testing was negative for pathogenic variants, but she was subsequently diagnosed with Lynch syndrome secondary to a germline monoallelic constitutional epimutation of the MLH1 promoter. Identification of patients displaying a Lynch syndrome phenotype but lacking germline MMR mutations is important to avoid delays in the diagnosis of Lynch syndrome as well as the initiation of appropriate cancer screening and genetic counseling.

Background and purpose It is well known that microsatellite instability-high (MSI-H) is associated with 5-fluorouracil (5-FU) resistance in colorectal cancer. MSI-H is the phenotype of DNA mismatch repair deficiency (MMR-D), mainly occurring due to hypermethylation of MLH1 promoter CpG island. However, the mechanisms of MMR-D/MSI-H are unclear. We aim to investigate the pathway of MMR-D/MSI-H involved in 5-FU resistance. Experimental approach Human colorectal cancer specimens were diagnosed for MSI-H by immunohistochemistry and western blotting. Proteome microarray interactome assay was performed to screen nuclear proteins interacting with ATG5. Nuclear ATG5 and ATG5-Mis18α overexpression were analysed in ATG5high colorectal cancer bearing mice. The methylation assay determined the hypermethylation of hMLH1 promoter CpG island in freshly isolated human colorectal cancer tissue samples and HT29atg5 and SW480atg5 cancer cells. Key results In ATG5high colorectal cancer patients, 5-FU-based therapy resulted in nuclear translocation of ATG5, leading to MSI-H. Colorectal cancer in Atg5 Tg mice demonstrated 5-FU resistance, compared to Atg5+/- and WT mice. Proteome microarray assay identified Mis18α, a protein localized on the centromere and a source for methylation of the underlying chromatin, which responded to the translocated nuclear ATG5 leading to ATG5-Mis18α conjugate overexpression. This resulted in MLH1 deficiency due to hypermethylation of hMLH1 promoter CpG island, while the deletion of nuclear Mis18α failed to induce ATG5-Mis18α complex and MMR-D/MSI-H. Conclusions and implications Nuclear ATG5 resulted in MMR-D/MSI-H through its interaction with Mis18α in ATG5high colorectal cancer cells. We suggest that ATG5-Mis18α or Mis18α may be a therapeutic target for treating colorectal cancer.

Findings on impact of mismatch repair deficiency (MMRd) on patient outcomes in endometrial cancer (EC) have been inconsistent to date. The objective of this study was to compare the oncologic outcomes and recurrence patterns between MMRd and MMR-intact (MMRi) endometrioid EC (EEC).Between 2015 and 2018, we prospectively recruited 492 EEC cases from three cancer centers in Ontario, Canada. Tumors were reflexively assessed for MMR protein expression by immunohistochemistry (IHC). Clinicopathological, survival and recurrence patterns were compared between MMRd and MMRi cases.Of 492 EEC, 348 were MMRi (71%) and 144 were MMRd (29%) with median follow-up of 16.8 months (0-69.6). MMRd tumors tended to be grade 2 or 3 (56% vs. 29%, p 0.001), with propensity for lymphovascular space invasion (28% vs. 18%, p = 0.024), lymph node involvement (7% vs. 5%, p 0.001) and received more adjuvant treatment (46% vs. 33%, p = 0.027). This group also had significantly lower 3-year recurrence-free survival (78% vs. 90%, p = 0.014) although there was no difference in OS (p = 0.603). MMRd cases were more likely to recur in retroperitoneal lymph nodes (p = 0.045). Upon subgroup analysis, MLH1 methylated tumors had the worst prognostic features and survival outcomes.MLH1 methylated EECs exhibit more aggressive features compared to other MMRd and MMRi EECs. This may indicate an inherent difference in tumor biology, suggesting the importance of individualized management based on EC molecular phenotype.

Background Immune checkpoint inhibitor (ICI) therapy is highly effective in metastatic mismatch repair-deficient (MMR-D) colorectal cancer (CRC). In this study, we evaluated molecular and clinical predictors of ICI response in MMR-D CRC. Materials and Methods Patient databases at four cancer institutions were queried. The Fisher exact test was performed to test the association of clinical and molecular markers. The Kaplan-Meier method was used to estimate progression-free survival (PFS) and compared by the log-rank test. Twelve- and 24-month PFS rates were compared by the Z test. Results A total of 60 patients with CRC with MMR-D/microsatellite instability-high who previously received ICIs were identified. Patients with liver metastasis had a lower overall response rate as compared with other sites of metastasis (36.4% vs. 68.7%; p = .081). Patients with MLH1/PMS2 loss had worse 1-year and 2-year PFS rates compared with patients with MSH2/MSH6 loss (84.2% vs. 57.8% and 78.2% vs. 54.2%, respectively; p < .001). There were improved 1-year and 2-year PFS rates in patients with wild-type BRAF when compared with patients with BRAF V600E mutation (73.3% vs. 40%, and 73.3% vs. 26.7%; respectively; p < .001). Patients aged >65 had significantly worse PFS rates as compared with patients aged ≤65 (p < .001). Conclusion BRAF V600E mutation, MLH1 and/or PMS2 loss, as well as age >65 years and liver metastasis, may be predictive of duration of ICI response in patients with MMR-D CRC. Larger cohorts are needed to confirm our findings. Implications for Practice The results of this study reveal clinically important biomarkers that potentially predict immune checkpoint inhibitor response in patients with mismatch repair-deficient colorectal cancer.