Mismatch Repair Protein Expression Research Articles

ARID1A deficiency associated with MMR deficiency and a high abundance of tumor-infiltrating lymphocytes predicts a good prognosis of endometrial carcinoma

BackgroundARID1A alterations have been detected in 40% of endometrial carcinomas (ECs) and are associated with loss of its expression. The role of ARID1A in tumorigenesis and development is complex, and the prognostic role in EC remains controversial. Hence, it is of great significance to confirm the role of ARID1A in EC. MethodsA total of 549 EC patients (cohort A) from TCGA were evaluated to explore the prognostic role of ARID1A. NGS was performed for 13 EC patients (cohort B), and expression of ARID1A, CD3, CD8 and mismatch repair (MMR) proteins in 52 patients (cohort C) from our center was determined by immunohistochemistry (IHC). The Kaplan–Meier method was used to perform survival analyses. ResultsARID1A alterations were detected in 32% of EC patients and correlated with good disease-free survival (DFS, P = 0.004) and overall survival (OS, P = 0.0353). ARID1A alterations were found to co-occur with MMR-related gene mutations and correlated with higher PD-L1 expression. Patients concomitantly harboring ARID1A alterations and MMR-related gene mutations had the best prognosis (DFS: P = 0.0488; OS: P = 0.0024). A cohort from our center showed that ARID1A deficiency was an independent prognostic factor and predicted longer recurrence-free survival (P = 0.0476). ARID1A loss was associated with a tendency toward MSI-H (P = 0.0060). ARID1A alterations and expression loss were associated with a higher abundance of CD3+ (P = 0.0406) and CD8+ (P = 0.0387) T cells. ConclusionARID1A alterations and expression loss are tightly associated with MMR deficiency and a high abundance of tumor-infiltrating lymphocytes, which might contribute to the good prognosis of EC.

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 expression of programmed death-ligand 1 and programmed death-ligand 2 in endometrial carcinosarcoma: Correlation with mismatch repair protein expression status, tumor-infiltrating lymphocyte infiltration, and clinical outcomes

BackgroundEndometrial carcinosarcomas have high malignant potential with a high recurrence rate and poor prognosis. Immunotherapy may be a promising treatment option. The aim of this study is to evaluate the expression of PD-L1/PD-L2 and its relationship to mismatch repair (MMR) protein status and tumor-infiltrating lymphocyte (TIL) density. MethodsWe performed immunohistochemical analyses of PD-L1 (clone 22C3), PD-L2 (clone TY25), MSH-2, MSH-6, PMS-2, and MLH-1 in 77 tumors. We count TILs using CD8 antibody. Clinicopathologic features were recorded and statistically correlated with immunohistochemical results. Kaplan-Meier analyses were used to analyze the prognosis. ResultsWhile PD-L1 positivity was seen more commonly in MMR protein deficient tumors (p = 0.010), PD-L2 positivity was seen more commonly in MMR protein proficient tumors (p = 0.003). PD-L1 positivity was also found to be more common in carcinosarcoma with high TIL infiltration. PD-L2 positivity was associated with decreased overall survival (OS) rates (p = 0.043, p = 0.043, respectively), whereas the PD-L1 positivity and TIL density were not significantly associated with OS rate. The OS rate of patients with MMR protein proficient tumors was significantly lower compared with those with MMR protein deficient tumors (p = 0.042). The lower TILs infiltration was associated with a shorter disease-free survival (DFS) rate. PD-L1 and PD-L2 positivity did not affect the DFS rate. ConclusionsPD-L1/PD-L2 might be a better target for immunotherapy in endometrial carcinosarcoma. PD-L2 positivity was also associated with a worse clinical outcome in patients with endometrial carcinosarcoma, suggesting that PD-L2 status can be used to predict clinical behavior. Further studies are needed to elucidate the relationship between PD-L1/PD-L2 expression and therapeutic response.

Mesonephric-like Adenocarcinoma of the Uterine Corpus: Comparison Between Mismatch Repair Protein Immunostaining and Microsatellite Instability Testing.

The utility of mismatch repair (MMR) immunohistochemistry (IHC) and microsatellite instability (MSI) testing in uterine mesonephric-like adenocarcinoma (MLA) has seldom been reported. This study aimed to compare MMR IHC and MSI testing in uterine MLA. We analyzed the MMR protein expression of 25 patients with MLA and compared the results with the MSI status. Of the cases, one (4.0%) was initially interpreted to have a loss of mutS homolog 2 (MSH2) and mutS homolog 6 (MSH6) expression, and eight (32.0%) to have a loss of MSH6 immunoreactivity. Upon re-evaluation of the slides and repeat IHC, all cases were revealed to demonstrate at least focal (5-20%) immunoreactivity for MSH2 or MSH6. MSI testing revealed all cases to be microsatellite stable (MSS). Uterine MLA is an MSS/MMR-proficient tumor. We observed complete concordance between MMR IHC and MSI testing. MMR IHC should be carefully interpreted, and discordant cases must be thoroughly reviewed. Repeat IHC and confirmatory MSI testing may be beneficial in resolving uncertain cases.

Subcellular Epithelial HMGB1 Expression Is Associated with Colorectal Neoplastic Progression, Male Sex, Mismatch Repair Protein Expression, Lymph Node Positivity, and an 'Immune Cold' Phenotype Associated with Poor Survival.

New treatment targets are needed for colorectal cancer (CRC). We define expression of High Mobility Group Box 1 (HMGB1) protein throughout colorectal neoplastic progression and examine the biological consequences of aberrant expression. HMGB1 is a ubiquitously expressed nuclear protein that shuttles to the cytoplasm under cellular stress. HMGB1 impacts cellular responses, acting as a cytokine when secreted. A total of 846 human tissue samples were retrieved; 6242 immunohistochemically stained sections were reviewed. Subcellular epithelial HMGB1 expression was assessed in a CRC Tissue Microarray (n = 650), normal colonic epithelium (n = 75), adenomatous polyps (n = 52), and CRC polyps (CaP, n = 69). Stromal lymphocyte phenotype was assessed in the CRC microarray and a subgroup of CaP. Normal colonic epithelium has strong nuclear and absent cytoplasmic HMGB1. With progression to CRC, there is an emergence of strong cytoplasmic HMGB1 (p < 0.001), pronounced at the leading cancer edge within CaP (p < 0.001), and reduction in nuclear HMGB1 (p < 0.001). In CRC, absent nuclear HMGB1 is associated with mismatch repair proteins (p = 0.001). Stronger cytoplasmic HMGB1 is associated with lymph node positivity (p < 0.001) and male sex (p = 0.009). Stronger nuclear (p = 0.011) and cytoplasmic (p = 0.002) HMGB1 is associated with greater CD4+ T-cell density, stronger nuclear HMGB1 is associated with greater FOXP3+ (p < 0.001) and ICOS+ (p = 0.018) lymphocyte density, and stronger nuclear HMGB1 is associated with reduced CD8+ T-cell density (p = 0.022). HMGB1 does not directly impact survival but is associated with an 'immune cold' tumour microenvironment which is associated with poor survival (p < 0.001). HMGB1 may represent a new treatment target for CRC.

Highly Sensitive Microsatellite Instability and Immunohistochemistry Assessment in Endometrial Aspirates as a Tool for Cancer Risk Individualization in Lynch Syndrome

Women with Lynch syndrome (LS) are at increased risk of endometrial cancer (EC), among other tumors, and are characterized by mismatch repair (MMR) deficiency and microsatellite instability (MSI). While risk-reducing gynecologic surgeries effectively decrease EC incidence, doubts arise regarding the appropriate timing of the surgery. We explored the usefulness of highly sensitive MSI (hs-MSI) assessment in endometrial aspirates for individualizing gynecologic surveillance in LS carriers. Ninety-three women with LS, 25 sporadic EC patients (9 MMR-proficient and 16 MMR-deficient), and 30 women with benign gynecologic disease were included in this study. hs-MSI was assessed in prospectively collected endometrial aspirates in 67 LS carriers, EC cases, and controls. MMR, PTEN, ARID1A, and PAX2 protein expression patterns were evaluated in the LS samples. Follow-up aspirates from 8 LS carriers were also analyzed. Elevated hs-MSI scores were detected in all aspirates from MMR-deficient EC cases (3 LS and 16 sporadic) and negative in aspirates from controls and MMR-proficient EC cases. Positive hs-MSI scores were also detected in all 4 LS aspirates reported as complex hyperplasia. High hs-MSI was also present in 10 of 49 aspirates (20%) from LS carriers presenting a morphologically normal endometrium, where MMR protein expression loss was detected in 69% of the samples. Interestingly, the hs-MSI score was positively correlated with MMR-deficient gland density and the presence of MMR-deficient clusters, colocalizing PTEN and ARID1A expression loss. High hs-MSI scores and clonality were evidenced in 2 samples collected up to 4 months before EC diagnosis; hs-MSI scores increased over time in 5 LS carriers, whereas they decreased in a patient with endometrial hyperplasia after progestin therapy. In LS carriers, elevated hs-MSI scores were detected in aspirates from premalignant and malignant lesions and normal endometrium, correlating with MMR protein loss. hs-MSI assessment and MMR immunohistochemistry may help individualize EC risk assessment in women with LS.

Neoadjuvant immunotherapy for colorectal cancer: Right regimens, right patients, right directions?

Neoadjuvant chemoradiotherapy (NACRT) or chemotherapy (NACT) followed by radical resection and then adjuvant therapy is considered the optimal treatment model for locally advanced colorectal cancer (LACRC). A recent total neoadjuvant therapy (TNT) strategy further improved the tumour regression rate preoperatively and reduced local-regional recurrence in locally advanced rectal cancer (LARC). However, distant metastasis was still high, and little overall survival benefit was obtained from these preoperative treatment models. According to mismatch repair protein expression, MSI-H/dMMR and non-MSI-H/pMMR statuses were defined in colorectal cancer (CRC) patients. Due to the special features of biologics in MSI-H/dMMR CRC patients, this subgroup of patients achieved little treatment efficacy from chemoradiotherapy but benefited from immune checkpoint inhibitors (ICIs). The KEYNOTE-177 trial observed favourable survival outcomes in metastatic CRC patients treated with one-line pembrolizumab with tolerable toxicity. Given the better systemic immune function, increased antigenic exposure, and improved long-term memory induction before surgery, neoadjuvant ICI (NAICI) treatment was proposed. The NICHE trial pioneered the use of NAICI treatment in LACRC, and recent reports from several phase II studies demonstrated satisfactory tumour downsizing in CRC. Preclinical rationales and preliminary early-phase human trials reveal the feasibility of NAICI therapy and the therapeutic efficacy provided by this treatment model. Better tumour regression before surgery also increases the possibility of organ preservation for low LARC. However, the optimal treatment strategy and effective biomarker identification for beneficiary selection remain unknown, and potential pitfalls exist, including tumour progression during neoadjuvant treatment due to drug resistance and surgery delay. Given these foundations and questions, further phase II or III trials with large samples need to be conducted to explore the right regimens for the right patients.

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.

Mismatch repair protein expression in gastric carcinoma patients

Gastric cancer is one of the most aggressive malignancies. Although there has been a slight increase in overall survival due to the inclusion of targeted medications in the standard chemotherapeutic treatment paradigm, the prognosis remains poor. New evidence suggests that certain molecular and histopathologic tumor characteristics, as well as staging, may also influence patients’ prognosis. Indeed, two detailed genomic classifications of gastric cancer have recently been developed by The Cancer Genome Atlas (TCGA) and the Asian Cancer Research Group (ACRG), showing that microsatellite instability (MSI) is a distinct subgroup related to gastric cancer patients’ prognosis. Interestingly, the MSI-H subgroup was identified as a separate entity of GC in both of these classifications that are relevant to the prognosis of gastric cancer. Microsatellite instability (MSI) is caused most frequently by deficiency in the mismatch repair proterin (MMR) (including MLH1, MSH2, PMS2, MSH6) and can be defined by immunohistochemistry (IHC). Microsatellite instability offers a good prognostic marker associated with different cancer types. Therefore, the identification of MMR in gastric cancer is very important to provide valuable prognostic information and personalized treatment Key words: gastric cancer, microsatellite instability, mismatch protein repair deficiency, immunohistochemistry, prognosis.

Impact of DNA mismatch repair proteins deficiency on number and ratio of lymph nodal metastases in colorectal adenocarcinoma.

Approximately 15 % of colorectal adenocarcinomas (CRCs) are characterized by an altered expression of DNA mismatch repair (MMR) proteins (i.e. MMR deficiency [MMRd]). Lymph node ratio (LNR) represents one of the most important prognostic markers in non-advanced CRCs. No significant data are available regarding LNR distribution depending on MMR status. The aim of the present work was to compare pathological and clinical characteristics of MMRd tumors versus MMR proficient (MMRp) cases. Particular attention was paid to how these molecular sub-groups relate to the LNR. A mono-Institutional series of 1037 consecutive surgically treated stage I-IV CRCs were retrospectively selected and data were obtained from pathological reports. Cases were characterized for MMR/MSI status by means of immunohistochemistry or for microsatellite instability (MSI) analysis. MMRd/MSI tumors (n=194; 18.7 %) showed significant differences in comparison to MMRp lesions for sex (female prevalence 50.5 % vs 40.7 %; p=0.013), age (74.2 vs 69.2; p<0.001), location (right side; p<0.001), diameter (larger than MMRp; p<0.001), growth pattern (expansive pattern of growth; p<0.001), peri- (p=0.0002) and intra-neoplastic (p=0.0018) inflammatory infiltrate, presence of perineural invasion (p<0.001), stage (lower stage at presentation; p<0.001), grade (higher prevalence of high-grade tumors; p<0.001), and LNR (lower; p<0.001). MMRd/MSI tumors are a distinct molecular CRC subtype characterized by a significantly lower LNR in comparison to MMRp lesions. These data further support the prognostic impact of MMRd/MSI status in early-stage CRCs.

Clinicopathological features of patients with RET fusion-positive non-small cell lung cancer

Objective: To investigate the clinicopathological features, treatment and prognosis of patients with RET fusion positive non-small cell lung cancer (NSCLC). Methods: A total of 1 089 NSCLCs were retrieved at Affiliated Hospital of Jiangnan University from August 2018 to April 2020. In all cases, multiple gene fusion detection kits (fluorescent PCR method) were used to detect the gene status of RET, EGFR, ALK, ROS1, KRAS, BRAF and HER2; and immunohistochemical method was used to detect the expression of PD-L1 and mismatch repair related proteins. The correlation between RET-fusion and patients' age, gender, smoking history, tumor stage, grade, pathologic type, and PD-L1, mismatch repair related protein expression was analyzed. Results: There were 22 cases (2.02%) detected with RET fusion-positive in 1 089 NSCLC patients, in which 11 males and 11 females; and the median age was 63.5 years. There were 20 adenocarcinomas, including 11 acinar predominant adenocarcinoma (APA), five solid predominant adenocarcinoma (SPA) and four lepidic predominant adenocarcinoma (LPA); There were one case each of squamous cell carcinoma (non-keratinizing type) and sarcomatoid carcinoma (pleomorphic carcinoma). There were 6 and 16 patients with RET fusion-positive who were in stage Ⅰ-Ⅱ and Ⅲ-Ⅳ respectively, and 16 cases with lymph node metastasis, 11 cases with distant metastasis. Among RET fusion-positive cases, one was detected with HER2 co-mutation. The tumor proportion score of PD-L1≥1% in patients with RET fusion positive lung cancer was 54.5% (12/22). Defects in mismatch repair protein expression were not found in patients with RET fusion positive NSCLC. Four patients with RET fusions positive (two cases of APA and two cases of SPA) received pratinib-targeted therapy, and two showed benefits from this targeted therapy. Conclusions: The histological subtypes of RET fusions positive NSCLC are more likely to be APA or SPA. RET fusion-positive NSCLC patients are associated with advanced clinical stage, lymph node metastases, and they may benefit from targeted therapy with RET-specific inhibitors.

Impact of mismatch repair deficiency on tumour regression grade after neoadjuvant chemotherapy in localized gastroesophageal adenocarcinoma.

The use of neoadjuvant chemotherapy (NAC) in patients with mismatch repair (MMR) deficient (dMMR) localized gastric and oeso-gastric junction (OGJ) adenocarcinoma is subject of debate. Histological response assessment might help to better evaluate the impact of dMMR on response to NAC. Patients with localized gastric/OGJ adenocarcinoma resected after NAC were retrospectively identified. MMR protein expression status was assessed by immunohistochemistry. The primary objective was the frequency of histological responders to NAC defined by tumour regression grade (TRG) using Mandard's (TRG1-2) and Becker's (TRG1) classifications, according to the MMR status. In total, 247 patients with 43 dMMR and 204 pMMR gastric/OGJ adenocarcinoma were identified. Among dMMR tumours, 18 (42%) arose from the OGJ. Histological response (Becker TRG1-2) was observed for 28% and 35% of dMMR and pMMR tumours, respectively (p=0.35). Similar results were observed with Mandard classification. With a median follow-up of 37.5 months, median disease-free and overall survival were not reached for the dMMR group. Histological response after NAC in patients with localized dMMR gastric/OGJ adenocarcinoma is not statistically different to those with pMMR tumours. This study provides additional data for the discussion about avoiding NAC in patients with dMMR gastric/OGJ adenocarcinomas.

Prognostic value of mismatch repair deficiency in patients receiving first-line fluoropyrimidine plus platinum chemotherapy for metastatic, recurrent, or locally advanced unresectable gastric cancer.

461 Background: In localized gastric cancer (GC), deficient mismatch repair (dMMR) tumors reportedly have a favorable prognosis. However, a potentially detrimental effect of the dMMR status was suggested in patients treated with adjuvant chemotherapy. It remains unclear whether the mismatch repair (MMR) status can impact clinical outcomes in patients with metastatic GC (mGC) receiving palliative chemotherapy. We examined the impact of the MMR status on survival outcomes of patients with HER2-negative mGC receiving first-line fluoropyrimidine plus platinum (FP) chemotherapy. Methods: We reviewed patients with histologically confirmed metastatic, recurrent, or locally advanced unresectable adenocarcinoma of the stomach or gastroesophageal junction receiving first-line FP chemotherapy between January 2015 and August 2018 at Asan Medical Center, Korea. MMR status was correlated with clinical characteristics and survival outcomes for patients with available MMR immunohistochemistry results. Results: Of 895 patients, we analyzed 543 with available MMR protein expression results, and dMMR was detected in 4.4% (n=24). Patients with dMMR exhibited a significantly higher median age than those with proficient MMR (pMMR) (64 vs. 58 years, p=0.044). No signet ring cell carcinoma (SRCC) was detected among dMMR tumors, whereas SRCC was found in 17.5% of pMMR tumors. Based on our prognostic model (Koo DH et al, 2011), only 4.2% of patients with dMMR were classified as the poor-risk group (vs. 16.8% of patients with pMMR, p=0.097). No significant difference in progression-free survival (PFS) was noted between patients with dMMR and pMMR tumors (median, 5.6 vs. 5.8 months, p=0.266). Patients with dMMR tumors tended to have better overall survival than those with pMMR tumors; this difference was not statistically significant (median, 17.9 vs. 12.2 months, p=0.183). In the good-risk subgroup, patients with dMMR tumors had a significantly worse PFS than those with pMMR tumors (median, 5.6 vs. 9.2 months, p=0.009); multivariate analysis revealed that MMR status was an independent factor for poor PFS in this subgroup. Conclusions: Survival outcomes of dMMR mGC did not differ from pMMR in patients who received first-line FP chemotherapy. However, in the good-risk subgroup, patients with dMMR tumors had a significantly shorter PFS than those with pMMR, suggesting a potentially reduced efficacy of cytotoxic chemotherapy in these patients.

Unique Patterns of Heterogeneous Mismatch Repair Protein Expression in Colorectal Cancer Unveil Different Degrees of Tumor Mutational Burden and Distinct Tumor Microenvironment Features

Mismatch repair (MMR) protein expression in colorectal cancer (CRC) cells is usually homogeneously retained or lost. Rare lesions may show a heterogeneous pattern of MMR protein expression. We evaluated MMR protein expression (MLH1, MSH2, MSH6, and PMS2) in 200 CRCs, identifying 3 groups with proficient MMR protein expression (MMRp), deficient MMR protein expression (MMRd), and heterogeneous MMR protein expression (MMRh). MMRh tumors were microdissected on the basis of the expression of the heterogeneous marker. DNA was extracted and subjected to targeted sequencing. RNA was purified from bulk tumors of all MMRh cases and in a control series of 15 MMRp and 10 MMRd CRCs and analyzed using the PanCancer IO 360 Panel (NanoString Technologies). Twenty-nine of the 200 cases (14.5%) were MMRd. Nine cases (4.5%) showed a heterogeneous pattern of MMR expression, with 6 tumors harboring concomitant loss of one of the other MMR proteins, thus featuring areas with double loss at immunohistochemistry (IHC) testing (MMRh double-loss cases). Four of the 6 MMRh double-loss cases were suitable for a separate sequence variant analysis of IHC double-negative and IHC single-negative components of the tumor. In all lesions, both components exhibited a high tumor mutation burden (TMB). Nevertheless, a significant increase in TMB in the double-negative components was observed (mean TMB: negative, 70 mut/Mb vs positive, 59 mut/Mb) because of a higher number of subclonal variants compared with the other component. Comparative gene expression analyses among MMRd, MMRp, and MMRh CRCs highlighted differential gene expression patterns and an increased number of tumor-infiltrating lymphocytes in MMRh lesions, which is also characterized by a substantial population of exhausted CD8+ lymphocytes. We describe a unique subgroup of CRCs showing heterogeneous expression of MMR proteins in a background of concomitant loss of one of the other markers.

Extensive molecular profiling of squamous cell anal carcinoma in a phase 2 trial population: Translational analyses of the “CARACAS” study

BackgroundMolecular characteristics of squamous cell anal carcinoma (SCAC) are poorly explored. Immune checkpoint inhibitors showed limited activity in phase I/II trials, but predictive and prognostic biomarkers are lacking. Patients and methodsIn the phase II randomised trial CARACAS (NCT03944252), avelumab alone (Arm A) or with cetuximab (Arm B) was tested in pre-treated advanced SCAC , with overall response rate being the primary end-point. On pre-treatment tumour tissue samples, we assessed Human papillomavirus status, programmed-death ligand 1 (PD-L1) expression, mismatch repair proteins expression, tumour mutational burden (TMB) and comprehensive genomic profiling by FoundationOne CDx. Tumour-infiltrating lymphocytes were characterised on haematoxylin-eosine-stained samples. Primary objective was to describe response to immunotherapy in the CARACAS trial population according to molecular and histological characteristics. Secondary objectives were to assess progression-free survival (PFS) and overall survival (OS) according to molecular biomarkers. ResultsHigh PD-L1 (>40 with combined positive score) was significantly more frequent in patients with disease control (p = 0.0109). High TMB (>10 mutations per megabase) was related to better OS (hazard ratio (HR) = 0.09; 95%confidence interval (CI) 0.01–0.68; p = 0.019) and PFS (HR = 0.44; 95%CI = 0.15–1.27; p = 0.129). High expression of PD-L1 conferred longer OS (HR = 0.46; 95%CI = 0.19–1.08; p = 0.075) and PFS (HR = 0.42; 95%CI = 0.20–0.92; p = 0.03). Neither OS (HR = 1.30; 95%CI = 0.72–2.36; p = 0.39) or PFS (HR = 1.31; 95%CI = 0.74–2.31; p = 0.357) was affected by high (>1.2) Tumour-infiltrating lymphocytes count. High TMB and PD-L1identified patients were with significantly better OS (HR = 0.33; 95%CI = 0.13–0.81; p = 0.015) and PFS (HR = 0.48; 95%CI = 0.23–1.00; p = 0.015). ConclusionsTo our knowledge, TranslaCARACAS is the first study to document prognostic role of TMB and PD-L1 in advanced SCAC patients treated with immune checkpoint inhibitors.

Mis-Match Repair (MMR) Protein Expression and Microsatellite Instability (MSI) Analysis in Sporadic Adenocarcinoma Diagnosed on Colonoscopic Biopsy

Background and aim Colorectal adenocarcinoma is a major cause of morbidity and mortality worldwide. Mismatch repair MMR protein expression plays a crucial role in the development and progression of colorectal adenocarcinomas. Immunohistochemistry IHC is a widely used technique to assess MMR protein expression. However there is limited data on the prospective evaluation of MMR protein expression by IHC on endoscopic biopsies of colorectal adenocarcinomas. In this study we aimed to evaluate the MMR protein expression by IHC on endoscopic biopsies of colorectal adenocarcinomas at a tertiary care center.Methods Forty-Two colonoscopic endoscopic biopsies pathologically diagnosed as colorectal adenocarcinoma were analyzed for IHC expression of MMR proteins MLH1 PMS2 MSH2 and MSH6.Results Out of the 42 cases 22 52.3 were mismatch repair proficient pMMR while the remaining 20 47.6 were mismatch repair deficient dMMR. MLH1PMS2- was the most common pattern accounting for 20 of dMMR cases and MLH1-PMS2-MSH2-MSH6- or the ldquoNull Patternrdquo was the least common with 10 of dMMR cases.Conclusion Evaluation of MMR proteins using immunohistochemistry is relatively easy in routine testing and is a useful tool in detecting MSI in CRC.nbspnbsp

Histopathologic Features for Overall Survival in Merkel Cell Carcinoma: A Case Series with Intact Mismatch Repair Protein Expression.

In a study of Merkel cell carcinoma (MCC), a fusion transcript between MLH1 and SPATA4 was identified. This fusion has the potential to generate the inactive or dominant-negative form of the protein. Therefore, we aimed to investigate whether mismatch repair protein deficiency occurr in MCC cases or not, in addition to the overall survival association with histopathologic features. A retrospective review of 15 patients diagnosed with a biopsy-proven Merkel Cell Carcinoma between 2012 and 2019 was performed. Mismatch repair (MMR) protein expressions were evaluated by immunohistochemistry. The median follow-up time was 36 months (mean 41, range 2-103 months). Six (40%) patients died during follow-up. The overall survival (OS) at 1 year, 2 years, 3 years, and 5 years were 87%, 80%, 62%, and 53%, respectively. The patients diagnosed at < 60 years had an improved OS compared to those ≥60 years of age (p=0.016). Patients in clinical stage I had better OS than patients in clinical stage IV (p=0.011). Cases with pathological tumor stage (pT) 1 had better OS than pT3 and pT4 (p=0.045). Adjuvant radiotherapy or adjuvant radiotherapy+chemotherapy treatment improved OS compared to adjuvant chemotherapy (p=0.003). MMR protein nuclear expression was intact in 12 cases available for immunohistochemical study. To the best of our knowledge, this is the second study that preferentially investigated the mismatch repair protein status of Merkel Cell Carcinoma. No mismatch repair protein deficiency of MCC cases was identified in the current study.

Expression of PD-L1 in Microsatellite Instability High Tumours: A Retrospective Study

Introduction: Microsatellite Instability (MSI) is the hallmark of Lynch syndrome/Constitutional Mismatch Repair Deficiency (CMMRD) and is also found in many sporadic cancers like colorectal cancer, endometrial, gastric, small intestine, urothelial, central nervous system and sebaceous gland neoplasms. MSI is a predictive biomarker for immunotherapy and Immunohistochemistry (IHC) antibodies against four Mismatch Repair (MMR) proteins: MutL Homolog 1 (MLH1), MutS Homolog 2 (MSH2), MutS Homolog 6 (MSH6) and Postmeiotic Segregation increased 2 (PMS2) can identify the MSI status of the tumour. In addition to MSI, immune checkpoint programmed cell death protein 1 (PD-1) expression and its ligand PD-L1 are biomarkers that can predict response to immunotherapy. Considering this increasing interest to identify deficient MMR (dMMR) status in different cancers, authors have assessed the expression of PDL1 and status of MSI in various cancer types. Aim: To evaluate the expression of PD-L1 in MSI-high status tumours. Materials and Methods: This retrospective cross-sectional study was done in the Department of Pathology, Panimalar Medical College Hospital and Research Institute for a period of six months from July 2022 to December 2022. A total of 151 cases were identified for the period of three years from January 2020 to December 2022. The slides and blocks were retrieved from the archives. Tumour sections from the paraffin embedded tissues were deparaffinised and antigen retrieval was done. IHC using four antibodies (MLH1, MH2, MSH6 and PMS2) was performed on these slides to assess the MSI status. The slides were reviewed and were further subjected to PD-L1 IHC. PD-L1 expression on tumour cells was compared with the MSI status of different cancer types. The p-value was calculated using t-test and p&lt;0.5 was considered statistically significant. Statistical analysis was done using International Business Machines (IBM) Statistical Package for the Social Sciences (SPSS) software version 21.0. Results: A total of 151 cases were included in the present study. A positive nuclear stain for the four MMR proteins denote expression of wild type MMR proteins, hence MSI low status. A loss of nuclear expression denotes mutation of MMR proteins and hence MSI high (MSI-H) status. The MSI was high in nine out of 48 cases (18.75%) of colon cancer, three out of 15 cases (20%) of endometrial cancers, three out of 20 cases (15%) of gastric cancers. Rest were one out of sixteen cases of ovarian cancer, one out of two malignant melanoma and one out of three glioblastoma. Out of these, PD-L1 was positive in seven of the 18 MSI-H cases (38.88% of MSI-H cases). The MSI was low/ stable in the remaining 133 cases. The p-value of significance was 0.03 (statistically significant). Conclusion: This study shows a significant association of MSI-H with PD-L1 expression in tumours. Further large scale studies can help in assessing the role of PD-L1 as an effective therapeutic biomarker in MSI-high status patients who can benefit from targeted therapy

Comprehensive Study of Microsatellite Instability Testing and Its Comparison With Immunohistochemistry in Gastric Cancers.

In this study, polymerase chain reaction (PCR)-based microsatellite instability (MSI) testing was comprehensively analyzed and compared with immunohistochemistry (IHC) for mismatch repair (MMR) protein expression in patients with gastric cancer (GC). In 5,676 GC cases, PCR-based MSI testing using five microsatellites (BAT-26, BAT-25, D5S346, D2S123, and D17S250) and IHC for MLH1 were performed. Re-evaluation of MSI testing/MLH1 IHC and additional IHC for MSH2, MSH6, and PMS2 were performed in discordant/indeterminate cases. Of the 5,676 cases, microsatellite stable (MSS)/MSI-low and intact MLH1 were observed in 5,082 cases (89.5%), whereas MSI-high (MSI-H) and loss of MLH1 expression were observed in 502 cases (8.8%). We re-evaluated the remaining 92 cases (1.6%) with a discordant/indeterminate status. Re-evaluation showed 1) 37 concordant cases (0.7%) (18 and 19 cases of MSI-H/MMR-deficient (dMMR) and MSS/MMR-proficient (pMMR), respectively), 2) 6 discordant cases (0.1%) (3 cases each of MSI-H/pMMR and MSS/dMMR), 3) 14 MSI indeterminate cases (0.2%) (1 case of dMMR and 13 cases of pMMR), and 4) 35 IHC indeterminate cases (0.6%) (22 and 13 cases of MSI-H and MSS, respectively). Finally, MSI-H or dMMR was observed in 549 cases (9.7%), of which 47 (0.8%) were additionally confirmed as MSI-H or dMMR by re-evaluation. Sensitivity was 99.3% for MSI testing and 95.4% for MMR IHC. Considering the low incidence of MSI-H or dMMR, discordant/indeterminate results were occasionally identified in GCs, in which case complementary testing is required. These findings could help improve the accuracy of MSI/MMR testing in daily practice.