Loss Of Mismatch Repair Protein Expression Research Articles

O09 Microsatellite-unstable sporadic sebaceous and duodenal tumours are associated with loss of mismatch protein expression

Abstract Introduction and aims Lynch syndrome (LS) is a common heritable cancer predisposition syndrome. Tumours from patients with LS demonstrate microsatellite instability (MSI-high), a genetic signature detectable by low-cost DNA sequencing assays that is currently utilized in colorectal cancer screening. The utility of MSI assays for LS detection has not been extensively studied in unselected sebaceous skin tumours or duodenal tumours, where currently immunohistochemical (IHC) expression of mismatch repair (MMR) proteins is used. In addition, it has recently been suggested that cutaneous squamous cell carcinoma (cSCC) could be linked to LS; however MSI status has not been studied in patients with early-onset cSCC. Methods We investigated MSI status in archival formalin-fixed paraffin-embedded cases with ethical approval. We examined 110 sebaceous tumours (STs) (45 female patients, 65 male patients; median age 77 years), 50 duodenal tumours (DTs) (28 female patients, 22 male patients; median age 64 years), and 148 cSCCs selected from patients under 50 years of age (69 female patients, 79 male patients; median age 49 years). Immunohistochemistry (IHC) was performed on each MSI-high sample to determine the expression of MMR proteins MLH1, MSH2, MSH6 and PMS2. Samples that passed quality control were included in the analysis. Results A total of 42 of 106 STs (39.6%) were MSI-high. All STs had loss of at least one MMR protein. Overall, 10 of 41 (24%) DTs were MSI-high. All DTs had loss of at least one MMR protein. We found that 12 of 148 (8.1%) cSCCs were MSI-high and 5 of 12 SCCs exhibited loss of at least one MMR protein expression (41%). Conclusions MSI-high status correlated well with loss of MMR protein expression in STs and DTs samples. Future studies will help establish MSI status in tumours initially selected by MMR loss on IHC, informing its use as an adjunct to MMR IHC in LS detection in patients with STs and DTs.

A Prospective Study to Evaluate the Prevalence of Microsatellite Instability in Endometrial Carcinoma by using Immunohistochemistry for Mismatch Repair Proteins as a Surrogate Marker

Abstract Aim Use of immunohistochemistry for mismatch repair (MMR) proteins to identify the prevalence of microsatellite instability (MSI) in cases of endometrial carcinoma and its subsequent correlation with various histopathological parameters. Materials and Methods The expression of MMR proteins, viz PMS2, MLH1, MSH2, and MSH6, were assessed in 114 endometrial cancer cases by immunohistochemistry using Dako EnVision FLEX system, on paraffin blocks of tumor tissue fixed in 10% formalin. Results We studied 114 endometrial cases for MMR protein expression, of which the majority were of endometrioid histologic subtype (n = 93, 81.6%), whereas the remainder comprised serous carcinoma (n = 12, 10.5%), clear cell carcinoma (n = 1, 0.9%), carcinosarcoma (n = 5, 4.4%), and dedifferentiated uterine carcinoma (n = 3, 2.6%). Twenty-one (18%) of these cases were found to be deficient for MMR proteins, of which 20 were of endometrioid histologic subtype and only 1 was dedifferentiated uterine carcinoma. Loss of MMR protein expression occurred in pairs of either PMS2 and MLH1 or MSH2 and MSH6. Conclusion MSI is one of the major molecular pathways contributing to tumorigenesis in endometrial carcinomas. Immunohistochemistry for MMR proteins is a highly sensitive and cost-effective alternative for molecular testing for MSI. It is also a great tool for screening patients for Lynch syndrome. Immunohistochemical testing for MMR should be offered to all patients of endometrial cancers.

Characterization of sessile serrated adenomas with dysplasia including intramucosal adenocarcinoma and colorectal carcinoma with a microsatellite instability phenotype

Recent studies have shown that sessile serrated lesions (SSLs) lead to the development of colorectal cancer (CRC) with a microsatellite instability (MSI) phenotype via a dysplasia-carcinoma sequence. However, the pathological and molecular mechanisms of SSL with dysplasia (SSLD) are unclear. Here, we aimed to examine the clinicopathological and molecular alterations in SSLD and to evaluate the significance of such alterations with regard to lesion progression. Fifty-four SSLDs (20 serrated dysplasia cases and 17 intestinal dysplasia cases, including 30 low-grade dysplasia [LGD] cases, 7 high-grade dysplasia [HGD] cases, and 17 intramucosal adenocarcinomas [IMAs]) were evaluated. Molecular alterations, including immunohistochemical expression of various markers, DNA methylation status, and multiple genetic mutations (using next-generation sequencing), were assessed. Additionally, such alterations were also investigated in 41 CRCs with an MSI phenotype (invasion beyond submucosa). The frequency of mismatch repair (MMR) deficiency in SSLD was 12 of 39 cases (32.4 %), whereas the MMR proficient type was observed in 17 of 39 SSLD cases. SSLD with serrated dysplasia showed a significantly higher frequency of loss of MMR protein expression and methylation status. Moreover, loss of MMR protein expression differed significantly between LGD and IMA. Furthermore, the frequency of TP53 mutation was significantly higher in IMA than in LGD. The current findings demonstrated that SSL with serrated dysplasia may be associated with an increased risk of malignant transformation compared with intestinal dysplasia. Loss of MMR proteins and mutation of TP53 may play important roles in tumor progression from dysplasia to carcinomatous lesions.

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

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

Mismatch repair deficiency testing in Lynch syndrome-associated urothelial tumors.

Lynch syndrome-associated cancer develops due to germline pathogenic variants in one of the mismatch repair (MMR) genes, MLH1, MSH2, MSH6 or PMS2. Somatic second hits in tumors cause MMR deficiency, testing for which is used to screen for Lynch syndrome in colorectal cancer and to guide selection for immunotherapy. Both MMR protein immunohistochemistry and microsatellite instability (MSI) analysis can be used. However, concordance between methods can vary for different tumor types. Therefore, we aimed to compare methods of MMR deficiency testing in Lynch syndrome-associated urothelial cancers. Ninety-seven urothelial (61 upper tract and 28 bladder) tumors diagnosed from 1980 to 2017 in carriers of Lynch syndrome-associated pathogenic MMR variants and their first-degree relatives (FDR) were analyzed by MMR protein immunohistochemistry, the MSI Analysis System v1.2 (Promega), and an amplicon sequencing-based MSI assay. Two sets of MSI markers were used in sequencing-based MSI analysis: a panel of 24 and 54 markers developed for colorectal cancer and blood MSI analysis, respectively. Among the 97 urothelial tumors, 86 (88.7%) showed immunohistochemical MMR loss and 68 were successfully analyzed by the Promega MSI assay, of which 48 (70.6%) were MSI-high and 20 (29.4%) were MSI-low/microsatellite stable. Seventy-two samples had sufficient DNA for the sequencing-based MSI assay, of which 55 (76.4%) and 61 (84.7%) scored as MSI-high using the 24-marker and 54-marker panels, respectively. The concordance between the MSI assays and immunohistochemistry was 70.6% (p = 0.003), 87.5% (p = 0.039), and 90.3% (p = 1.00) for the Promega assay, the 24-marker assay, and the 54-marker assay, respectively. Of the 11 tumors with retained MMR protein expression, four were MSI-low/MSI-high or MSI-high by the Promega assay or one of the sequencing-based assays. Our results show that Lynch syndrome-associated urothelial cancers frequently had loss of MMR protein expression. The Promega MSI assay was significantly less sensitive, but the 54-marker sequencing-based MSI analysis showed no significant difference compared to immunohistochemistry. Data from this study alongside previous studies, suggest that universal MMR deficiency testing of newly diagnosed urothelial cancers, using immunohistochemistry and/or sequencing-based MSI analysis of sensitive markers, offer a potentially useful approach to identification of Lynch syndrome cases.

Promising Therapeutic Impact of Immune Checkpoint Inhibitors in Type II Endometrial Cancer Patients with Deficient Mismatch Repair Status.

Type II endometrial cancer (EC) is responsible for most endometrial cancer-related deaths due to its aggressive nature, late-stage detection, and high tolerance to standard therapies. Thus, novel treatment strategies for type II EC are imperative. For patients with mismatch repair-deficient (dMMR) tumors, immunotherapy with immune checkpoint inhibitors represents a promising therapeutic strategy. However, the prevalence of dMMR tumors in type II EC patients remains unclear. In this study, using immunohistochemistry, we evaluated the expression of mismatch repair (MMR) proteins, tumor-infiltrating lymphocytes (CD8+), and immune checkpoint molecules (PD-L1) in 60 patients with type II EC (16, 5, 17, and 22 were endometrioid G3, serous, de-differentiated, and carcinosarcoma cases, respectively) to investigate the therapeutic effect of immune checkpoint inhibitors. Approximately 24 cases (40%) had a loss of MMR protein expression. The positivity rate of CD8+ (p = 0.0072) and PD-L1 (p = 0.0061) expression was significantly associated with the dMMR group. These results suggest immune checkpoint inhibitors (anti-PD-L1/PD-1 antibodies) could effectively treat type II EC with dMMR. The presence of dMMR might be a biomarker for a positive response to PD-1/PD-L1 immunotherapy in type II EC.

The Importance of Immunohistochemical Heterogeneous Expression of MMR Protein in Patients with Colorectal Cancer in Stage II and III of the Disease

Background and objectives: In patients with colorectal cancer (CRC), heterogeneous expression of Mismatch repair (MMR) proteins can manifest itself in several different forms and is not such a rare phenomenon. Therefore, it is very important to recognize the nuclear expression of MMR proteins of different MMR status in order to avoid false positive or false negative results. The aim of this study was to determine the frequency and distribution of heterogeneous expression of MMR proteins in patients with stages II and III of the disease as well as its association with clinical, demographic and pathological characteristics of CRC in relation to proficient and deficient expression of MMR proteins. Material and Methods: The study included 104 cases of colorectal cancer obtained from surgical colectomy material in stages II and III of the disease. Results: From a total of 104 patients with colorectal cancer, immunohistochemical analysis of the expression of all four MMR proteins showed that heterogeneous expression of MMR proteins (as well as deficient immunoreactivity of tumor cells) was present in 12 cases, while proficient expression of MMR proteins was detected in 80 tumors. Conclusions: Our study showed that the only independent predictors of the loss of MMR protein expression were younger patient age and right-sided anatomical location of the tumor. The study also established the existence of heterogeneous expression of MMR proteins in a non-negligible percentage of CRCs (11.5%), where heterogeneous nuclear expression of MMR proteins was described in several different forms.

Non-classical phenotypes of mismatch repair deficiency and microsatellite instability in primary and metastatic tumors at different sites in Lynch syndrome.

Lynch syndrome is a genetic disease characterized by abnormal DNA replication caused by germline variation in the mismatch repair (MMR) gene. There are rare non-classical phenotypes with loss of MMR protein expression and inconsistent microsatellite stability (MSS) in Lynch syndrome-related colorectal cancers. However, the difference between microsatellite instability (MSI) of extraintestinal tumors in a patient with Lynch syndrome has been closely studied. Herein, we reported the non-classical phenotypes of mismatch repair deficiency (dMMR) and MSI in four cases of Lynch syndrome in patients with colorectal cancer and other primary and metastatic tumors. A retrospective analysis was conducted on four patients diagnosed with Lynch syndrome between 2018 and 2022 in the Department of Pathology of the Rocket Forces Specialized Medical Center. A one-step immunohistochemical (IHC) assay was employed to detect loss in the expression of Lynch syndrome-associated MMR proteins (MLH1, PMS2, MSH2, and MSH6). MSI detection was performed in both primary and metastatic tumors at different sites in the four patients using NCI 2B3D (BAT25, BAT26, D2S123, D17S250, and D5S346) and single nucleotide site (BAT25, BAT26, NR21, NR24, NR27, and MONO27) methods. In addition, related MMR gene germline variation, somatic mutations, and MLH1 gene promoter methylation were analyzed using next-generation sequencing and TaqMan probe-based methylation-specific polymerase chain reaction (MethyLight). Two of the four patients were heterozygous for MSH6 germline pathogenic variation, and the other two were heterozygous for MSH2 germline pathogenic variation. In all cases, IHC detection of protein expression of the MMR gene with germline variation was negative in all primary and metastatic tumors; non-classical phenotypes of dMMR and MSI were present between primary and metastatic tumors at different sites. dMMR in Lynch colorectal cancer demonstrated high MSI, whereas MSI in primary and metastatic tumors outside the intestine mostly exhibited MSS or low MSI. The non-classical dMMR and MSI phenotype are mostly observed in Lynch syndrome, even in the context of MMR protein expression loss. Extraintestinal tumors infrequently present with a high degree of MSI and often exhibit a stable or low degree of MSI.

DNA Mismatch Repair Proteins and BRAF V600E Detection by Immunohistochemistry in Colorectal Cancer Demonstrates Concordance with Next Generation Sequencing

Background and Aims: Multiple laboratory methods are used to screen patients with colorectal cancer (CRC) for mismatch repair (MMR) protein deficiency to identify possible Lynch syndrome patients. The goal of this study was to compare the agreement between ready-to-use immunohistochemistry (IHC) assays for MLH-1, PMS-2, MSH-2, MSH-6, and mutated BRAF at V600E and molecular methods in CRC cases. The inclusion of the BRAF V600E mutation testing is important for the identification of patients with sporadic CRC, as the BRAF V600E mutation is very rarely observed in patients with Lynch syndrome tumors. Methods: CRC cases were analyzed by ColoSeqTM tumor sequencing assay and VENTANA MMR IHC Panel that included anti-MLH1, anti-PMS2, anti-MSH2, anti-MSH6, and anti-BRAF V600E antibodies. Additionally, CRC cases with MLH1 IHC loss were evaluated for MLH1 promoter hypermethylation. Results: One hundred and eighteen cases were analyzed. The overall percent agreement (OPA) for each evaluated marker status compared to next-generation sequencing (NGS) exceeded 96%. Twenty-three cases were positive for the BRAF V600E mutation by IHC and NGS, and twenty cases showed loss of MLH1 protein and were positive for MLH1 hypermethylation. Samples with loss of MMR protein expression by IHC demonstrated genetic and/or epigenetic alterations that were consistent with the observed protein expression patterns. Conclusions: The results of this study indicate that ready-to-use IHC assays can correctly identify the loss of MMR proteins and the presence of mutated BRAF V600E protein, supporting the utility of the VENTANA MMR IHC Panel as an aid to stratify patients with sporadic CRC vs. potential Lynch syndrome.

Clinicopathological characteristics and loss of mismatch repair protein expression in Chinese upper tract urothelial carcinomas.

Expression of DNA mismatch repair (MMR) protein (MLH1, PMS2, MSH2, and MSH6) in upper tract urothelial carcinoma (UTUC) has been explored in Western cohorts, but it is rarely reported in Eastern cohorts. We aimed to assess the loss of MMR protein expression among Chinese UTUC patients and study its clinicopathological implications. We enrolled 175 UTUC patients at our center and tested the expression of MMR proteins by immunohistochemistry. Then, we explored these patients' clinicopathological characteristics. We found loss of MMR proteins in 19 (10.9%) of 175 patients in our cohort (6 MSH2 and MSH6, 2 MSH6 alone, 6 MSH2 alone, 3 MLH1 and PMS2, and 2 PMS2 alone). Loss of MMR proteins was not a significant prognostic factor of relapse-free survival for these patients. In addition, patients with lower T stage or with bladder cancer history were more likely to have loss of MMR protein expression. At last, two metastatic patients (MSH2 and MSH6 loss; MSH2 loss) with loss of MMR protein experienced tumor recession after several cycles of anti-PD-1 immunotherapy. In conclusion, this is the largest Chinese UTUC cohort study to date that explores the loss of MMR protein expression. The rate of MMR loss observed was comparable to that in the Western UTUC cohort, supporting universal UTUC screening in China. Furthermore, a subset of advanced UTUCs with MMR protein loss are probably immunogenic, for whom single or combined immunotherapy may be potential therapeutic options in the future.

Low prevalence of biliary tract cancer with defective mismatch repair genes in a Japanese hospital-based population.

Recent studies have reported that immune checkpoint inhibitors are effective against various defective mismatch repair (dMMR)/microsatellite instability-high (MSI-H) cancers. A limited number of reports are available on the frequency of dMMR/MSI-H carcinoma in biliary tract cancer (BTC), describing its clinicopathological characteristics and prognosis. The latter carcinoma is also associated with Lynch syndrome (LS). The present study was performed to investigate the frequency of patients with dMMR/MSI-H in BTC and the clinical characteristics of BTC with dMMR/MSI-H in a single institution in Japan. A total of 116 patients with BTC who underwent curative surgical resection at Kagawa University Hospital between January 2008 and December 2017 were included. The protein expression levels of the mismatch repair (MMR) genes [mutL homolog 1 (MLH1), mismatch repair endonuclease PMS2 (PMS2), MutS homolog (MSH)2 and MSH6] were assessed by immunohistochemistry (IHC) using formalin-fixed paraffin-embedded tissue specimens. Subsequently, MSI testing was performed on patients who exhibited loss of MMR protein expression. Loss of expression of one or more proteins was detected in five cases (4.3%). Loss of MLH1/PMS2 expression was observed in one case of intrahepatic cholangiocarcinoma, whereas loss of PMS2 expression was noted in one case of perihilar cholangiocarcinoma. Loss of MSH2/MSH6 and MSH6 expression was noted in two cases of distal cholangiocarcinoma and loss of PMS2 expression in one case of ampullary carcinoma. Out of the five patients, two demonstrated MSI-H. Microsatellite stability was observed in two cases and for one case, no data were available. Two MSI-H cases were patients with loss of expression of MLH1/PMS2 and MSH2/MSH6. None of the five patients exhibited a past medical history or family history of suspected LS. The frequency of dMMR in BTC was ~5%, which was similar to that reported by similar studies performed in other countries. In the present study, IHC appeared to be more useful than MSI testing for detecting MMR abnormalities with regards to the detection rate. Furthermore, there may only be a limited number of patients with BTCs who are likely to benefit from the therapeutic effects of treatment with immune checkpoint inhibitors.

Identification of Lynch syndrome-associated DNA mismatch repair-deficient bladder cancer in a Japanese hospital-based population.

The prevalence of Lynch syndrome (LS)-associated DNA mismatch repair (MMR)-deficient bladder cancer (BC) has scarcely been investigated. Immunohistochemistry for four MMR proteins (MLH1, MSH2, MSH6, and PMS2) was performed in formalin-fixed paraffin-embedded (FFPE) sections prepared from the resected specimens of 618 consecutive newly diagnosed BC cases. Genetic/epigenetic analyses were performed in patients displaying the loss of any MMR proteins in the tumor. Of the 618 patients, 9 (1.5%) showed the loss of MMR protein expression via immunohistochemistry; specifically, 3, 3, 2, and 1 patients displayed the loss of MLH1/PMS2, PMS2, MSH6, and MSH2/MSH6, respectively. All nine patients were male with a median age of 68years (63-79years). One had been previously diagnosed as having LS with an MSH2 variant. Genetic testing demonstrated the presence of a pathogenic PMS2 variant (n = 1), a variant of uncertain significance in MSH2 (n = 1), and no pathogenic germline variants of the MMR genes (n = 1). One patient with MSH6-deficient BC did not complete the genetic testing because of severe degradation of DNA extracted from the FFPE specimen, but the patient was strongly suspected to have LS because of their history of colon cancer and MSH6-deficient upper urinary tract cancer. There remained a possibility that the remaining four patients who refused genetic testing had LS. The prevalence of LS-associated MMR-deficient BC was estimated to be 0.6-1.1% among unselected BC cases.

Implication of expression of MMR proteins and clinicopathological characteristics in gastric cancer

Introduction Microsatellite instability (MSI), referred to as variations at microsatellite loci, at mismatch repair (MMR) genes leads to the formation of an aberrant MMR system that fails to rectify errors occurring during DNA replication. MMR deficiency can be assessed by immunohistochemical analysis of the expression of mismatch repair proteins in the target tissues. Methods We investigated the expression of four key MMR proteins (MLH1, MSH2, MSH6, and PMS2) in formalin-fixed paraffin-embedded (FFPE) tumor and normal tissues obtained from thirty gastric cancer (GC) patients. The association of clinicopathological features with MMR status was also analyzed. Results A total of 12 (40%) GC patients exhibited loss of expression of MMR proteins, including loss of MLH1 and PMS2 in 3 cases and loss of MSH2 and MSH6 in 4 cases. Univariate analysis showed an association of loss of MMR protein expression with moderately differentiated GC. However, there was no statistically significant association between loss of MMR protein expression with gender, tumor location, depth of invasion, lymph node metastasis, WHO classification, lympho-vascular invasion, and infection with H. pylori. Conclusion Our results implicate the role of mismatch repair proteins in gastric tumorigenesis. The MMR protein status is an important aspect of tumorigenesis and can be prescribed for the screening of GC.

Mismatch-Repair Protein Expression in High-Grade Gliomas: A Large Retrospective Multicenter Study.

Background: DNA mismatch repair (MMR) is a system for repairing errors in DNA replication. Cancer cells with MMR deficiency can have immunohistochemical loss of MMR protein expression leading to a hypermutable phenotype that may correlate with anti-PD1 efficacy. Scant data exist about immunohistochemical loss of MMR protein expression in high-grade gliomas (HGG). Materials and Methods: We performed a large multicenter retrospective study to investigate the frequency and the prognostic role of immunohistochemical loss of MMR protein expression in HGG patients; we nevertheless evaluated the association between this status and clinical or molecular characteristics. Immunohistochemical loss of MMR protein expression was recorded as partial or complete loss of at least 1 MMR protein. Results: We analyzed the expression of MMR proteins in tumor tissue of 355 consecutive patients. Partial and complete immunohistochemical loss of MMR proteins was found in 43/355 samples (12.1%) and among these, 15 cases (4.2%) showed a complete loss of at the least one MMR protein. Alteration of MSH2 expression was found in 55.8%, MSH6 in 46.5%, PMS2 in 34.9%, and MLH1 in 30.2%. Alteration of MMR protein expression was statistically more frequent in anaplastic gliomas, in recurrent disease, in patients treated with temozolomide, and in IDH-mut gliomas. Immunohistochemical loss of MMR proteins was not associated with survival, adjusting for clinically relevant confounders. Conclusions: MMR protein expression status did not affect survival in HGG patients. We identified clinical and molecular characteristics correlating with immunohistochemical loss of MMR proteins expression. A large study should be performed to analyze its predictive role of immune checkpoint inhibitor efficacy in these subgroups of patients.

Pembrolizumab Activity in Recurrent High-Grade Gliomas with Partial or Complete Loss of Mismatch Repair Protein Expression: A Monocentric, Observational and Prospective Pilot Study.

Introduction: Pembrolizumab demonstrated promising results in hypermutated tumors of diverse origin. Immunohistochemical loss of mismatch repair (MMR) proteins has been suggested as a surrogate of hypermutation in high-grade gliomas (HGG). We evaluated the efficacy and safety of pembrolizumab in relapsing HGGs with immunohistochemical loss of at least 1 MMR protein. Molecular biomarkers of pembrolizumab activity were also analyzed. Methods: Consecutive patients with recurrent HGG and partial or complete loss of MMR protein expression were prospectively enrolled; they received pembrolizumab 200 mg once every 3 weeks until disease progression. The primary endpoint was disease control rate (DCR). Post hoc exploratory analyses included next-generation sequencing to assess tumor mutational burden (TMB), and immunostaining for CD8+ T-cells and CD68+ macrophages. Results: Among 310 HGG patients screened, 13 cases with MMR loss were enrolled: eight glioblastoma, four anaplastic astrocytoma, and one anaplastic oligodendroglioma. Median age was 43 years. DCR was 31%: four patients had stable disease and no patient had complete or partial response. TMB ranged between 6.8 and 23.4 mutations/megabase. Neither TMB nor gene mutations, nor CD8+ T-cell and CD68+ macrophage content, were associated with pembrolizumab activity. Conclusions: pembrolizumab showed no apparent benefit in these patients. No molecular biomarker was found to be associated with pembrolizumab activity.

The utility of immunohistochemical testing for mismatch repair proteins in fine needle aspiration specimens of pancreatic adenocarcinoma

IntroductionMicrosatellite instability (MSI) testing is recommended for all colonic and endometrial carcinomas to screen for Lynch syndrome. The role of MSI testing in pancreatic adenocarcinoma has not been well-established. Screening can be done via immunohistochemical (IHC) staining for mismatch repair (MMR) proteins (MLH1, MSH2, MSH6, PMS2). We report our experience and the clinical utility of MMR IHC on pancreatic adenocarcinomas in fine-needle aspiration (FNA) specimens. Materials and methodsWe performed a retrospective review to identify all patients diagnosed with pancreatic adenocarcinoma by FNA at our institution between December 2017 and September 2019. For cases with sufficient tumor cells for testing, the MMR results and morphology were summarized, as well as corresponding clinical information, including age, clinical stage, treatment, and concurrent other cancers. ResultsFrom December 2017 to September 2019, there were a total of 184 pancreatic FNAs with a diagnosis of adenocarcinoma. Of these 184 FNAs, 65 (35%) contained sufficient material in the cell block to perform IHC for MMR. The cell block material was collected in either RPMI or CytoLyt. Poor technical quality precluded interpretation of PMS2 in 4 cases and MSH6 in 2 cases. All other cases showed intact expression of all four proteins. ConclusionsIHC for MMR proteins can be done on specimens collected in RPMI or CytoLyt, but RPMI appears to be more reliable. None of the pancreatic adenocarcinomas in this study showed loss of MMR protein expression. Routine testing of MMR loss may not be indicated in pancreatic adenocarcinomas in the general patient population.

Abstract A08: Tumor microbiome in subtypes of mismatch repair-deficient colorectal cancer

Abstract Background: Colorectal cancer (CRC) comprises different molecular subtypes, including those tumors that develop defective DNA mismatch repair (MMR) evidenced by microsatellite instability (MSI) and/or loss of MMR protein expression (MMR-deficiency). Tumor MMR-deficiency can result from inherited causes, namely germline mutations in the MMR genes (Lynch syndrome) or from somatic inactivation resulting from hypermethylation of the MLH1 gene promoter or from biallelic somatic mutations. Little is known about the etiologies of these cancers or the avenues for risk amelioration. We hypothesized that dysbiosis of the gut microbiome is a risk factor for the development of MMR-deficient subtypes of CRC and that the microbiome composition differs between inherited and somatic MMR-deficient CRC. Materials and Methods: The microbiome composition was determined by 16S rRNA sequencing of the V3-V4 regions in DNA extracted from 94 FFPE-derived tumor specimens comprised of 14 MMR-proficient CRCs and 80 MMR-deficient CRCs from the Australasian Colorectal Cancer Family Registry. The MMR-deficient CRCs comprised three subtypes: 35 people with germline MMR gene mutations (Lynch syndrome), 13 people with sporadic MLH1 hypermethylated CRCs, and 32 people with suspected Lynch syndrome (no germline or MLH1 methylation). LEfse analyses were used to identify differential abundance in taxa between groups. Results: In all tumor samples, Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Fusobacteria were most abundant. Of these, the Firmicute population was significantly higher in the MMR-proficient than MMR-deficient CRCs (0.33 v 0.19, P < 0.05). When compared with the Lynch syndrome-related CRCs, we found 22 bacterial species, including Corynebacteriaceae, that were significantly more abundant in MLH1 hypermethylated tumors (P < 0.05), whereas we found 4 species significantly more abundant, including Enterobacter, in suspected Lynch syndrome group. Although the microbial diversity was notably smaller compared with the sporadic groups, several species of the family Comamanadaceae including Acidovorax were abundant in Lynch syndrome tumors (P < 0.05). Fusobacterium nucleatum was detected in 11/35 (31.4%) Lynch syndrome-related CRCs, 5/13 (38.5%) MLH1 hypermethylated CRCs, 13/32 (40.6%) suspected Lynch syndrome CRCs, and 4/14 (28.6%) sporadic MMR-proficient CRCs. Conclusions: Inherited and somatic subtypes of MMR-deficient CRC show significant differences in their tumor microbiome composition where Lynch syndrome-related MMR-deficient CRC showed a reduced microbiome diversity compared with somatic MMR-deficient CRC. Further validation of these findings will improve our understanding of MMR-deficient CRC tumorigenesis and aid in CRC prevention through the identification of modifiable microbial risk factors. Citation Format: Jihoon E. Joo, Mark Clendenning, Khalid Mahmood, Christophe Rosty, Ingrid M. Winship, Mark A. Jenkins, Daniel D. Buchanan. Tumor microbiome in subtypes of mismatch repair-deficient colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A08.

Frequent CTNNB1 or PIK3CA Mutations Occurred in Endometrial Endometrioid Adenocarcinoma With High Levels of Microsatellite Instability and Loss of MSH2/MSH6 Expression.

DNA mismatch repair (MMR) proteins form 2 heterodimers-MutSα formed by MSH2 and MSH6, and MutLα by MLH1 and PMS2. In endometrial endometrioid adenocarcinomas, cases with MMR protein defect also usually harbor other recurrent genetic mutations of the neoplasm. However, it remains unknown whether defects of the 2 functionally different heterodimers are linked to mutations in different genes. We aimed to study the MMR protein expression, microsatellite instability (MSI), and other common genetic mutations of endometrial endometrioid adenocarcinoma. We investigated the MSI status of 107 endometrial endometrioid adenocarcinoma patients. MMR protein expression, and mutation of KRAS, CTNNB1, and PIK3CA were also evaluated by immunohistochemistry and sequencing. An overall 34.6% (37/107) of endometrial endometrioid adenocarcinomas were MSI-H. All MSI-H tumors exhibited loss of MMR protein expression (loss of MLH1, PMS2, MSH6, and MSH2 was noted in 22, 25, 12, and 7 cases, respectively). CTNNB1, PIK3CA, and KRAS mutation were present in 9, 7, and 7 MSI-H tumors. Compared with patients with loss of PMS2 and/or MLH1 expression, patients with loss of MSH6 and/or MSH2 expression were associated with higher frequencies of CTNNB1 mutation (P=0.036) and PIK3CA mutation (P=0.025). In MSI-H endometrial endometrioid adenocarcinomas, different types of MMR protein deficiency indicate different molecular genetic alterations.

Prevalence of Mismatch Repair-Deficient Colorectal Adenoma/Polyp in Early-Onset, Advanced Cases: a Cross-Sectional Study Based on Iranian Hereditary Colorectal Cancer Registry.

Lynch syndrome (LS) increases the risk of many types of cancer, mainly colorectal cancer (CRC). The purpose of this study was to assess the prevalence of mismatch repair (MMR) deficiency in patients under the age of 50 with advanced adenomatous polyps, aiming at an early diagnosis of LS. This retrospective, cross-sectional study included eligible patients with advanced adenomas diagnosed ≤ 50years of age registered between April 2014 and February 2017 at three pathology centers in Mashhad. Pathological records were reviewed, and colon tissue specimens were analyzed by immunohistochemistry (IHC) staining to identify proteins which serve as markers for LS as they are related to loss of MMR gene (MLH1, MSH2, MSH6, and PMS2) expression. Of 862 consecutive patients, a total of 50 adenomas (54% males, 46% females of mean age 41.24 ± 6.5) met the eligibility criteria. Of the adenomas examined, 20 (40%) had a tubulovillous component, 34 (68%) had high-grade dysplasia, and 30 (60%) had were larger than 10mm protrusions. None of the patients had loss of MMR protein expression. No individual with MMR genetic disorder was identified by IHC screening of early-onset advanced colorectal adenomas. This strategy is therefore not an effective strategy for detecting MMR mutation carriers.

Loss of Mismatch-repair Protein Expression and Microsatellite Instability in Upper Tract Urothelial Carcinoma and Clinicopathologic Implications.

Upper tract urothelial carcinoma (UTUC) may arise in the setting of hereditary non-polyposis colorectal cancer (Lynch syndrome [LS]) or sporadically. Variable frequencies of microsatellite instability (MSI) were found in UTUC. For advanced solid MSI tumors, targeted therapy with programmed death-ligand 1 inhibitors is available. Therefore, we aimed to determine the prevalence of mismatch repair (MMR) protein loss and MSI in UTUC using a tissue microarray approach and further molecular and correlation analysis. We studied the immunohistochemical expression of MLH1, MSH2, MSH6, and PMS2 on tissue microarrays containing formalin-fixed, paraffin-embedded samples of 128 patients with UTUC. MSI analysis was performed in 79 cases with deficient MMR protein expression, and/or in patients aged 60 years and below, and/or other tumors possibly related to LS. Loss of MMR protein expression was seen in 24 (18.8%) of 128 cases. MSI analysis revealed MSI-high in 29, MSI-low in 7 cases. The Fisher exact test demonstrated significant differences between MSI and loss of MMR protein expression, clinically possible LS, tumor growth pattern, inverted growth pattern, and death (P< .001, P< .001, P=.002, P= .003, and P= .033, respectively). MSI does not appear to influence survival (overall and progression-free), but there was a significant shorter progression-free survival in MSI-high versus MSS patients who had received chemotherapy. The frequency of MSI in UTUC was 36 (28.1%) of 128 patients with a good accuracy of immunohistochemistry. In daily practice, MSI screening especially is recommended in patients with advanced UTUC and inverted papillary tumor growth pattern with the aim of screening patients for possible targeted therapy.