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Abstract Background Since the FDA approved immune-enhancing therapies for patients with high microsatellite instability (MSI-H) and/or mismatch repair deficiency (dMMR), recognizing these biomarkers in solid tumors has gained clinical importance. Although MSI-H and dMMR are considered uncommon in breast cancer, previous studies on bilateral breast cancer (biBC) identified a surprisingly high frequency of MSI. Methods In this study, we aimed to describe the prevalence of dMMR and its association with clinicopathologic parameters in biBC. We performed immunohistochemistry with anti-MMR proteins on tissue microarrays (TMAs) with 58 bilateral breast cancer cases. The biomarkers used were MLH1, PMS2, MSH2, MSH6, ER, PR, HER2 and Ki67. SPSS was used for data analysis. Results Four (6.9%) cases showed dMMR on TMAs. Three (75%) of the dMMR cases were luminal and one (25%) was triple negative. Two biBC cases presented unilateral dMMR. No association between dMMR status and clinicopathologic parameters was found. Conclusions This work highlights a noticeable frequency of dMMR in bilateral breast cancer and builds upon previous research in this area, suggesting routine MMR protein testing as part of the immunohistochemical panel for biBC to identify candidates for immune-enhancing therapies.

Abstract Background Microsatellite instability (MSI) due to mismatch repair deficiency (dMMR) is common in colorectal cancer (CRC). These cancers are associated with somatic coding events, but the noncoding pathophysiological impact of this genomic instability is yet poorly understood. Here, we perform an analysis of coding and noncoding MSI events at the different steps of colorectal tumorigenesis using whole exome sequencing and search for associated splicing events via RNA sequencing at the bulk-tumor and single-cell levels. Results Our results demonstrate that MSI leads to hundreds of noncoding DNA mutations, notably at polypyrimidine U2AF RNA-binding sites which are endowed with cis-activity in splicing, while higher frequency of exon skipping events are observed in the mRNAs of MSI compared to non-MSI CRC. At the DNA level, these noncoding MSI mutations occur very early prior to cell transformation in the dMMR colonic crypt, accounting for only a fraction of the exon skipping in MSI CRC. At the RNA level, the aberrant exon skipping signature is likely to impair colonic cell differentiation in MSI CRC affecting the expression of alternative exons encoding protein isoforms governing cell fate, while also targeting constitutive exons, making dMMR cells immunogenic in early stage before the onset of coding mutations. This signature is characterized by its similarity to the oncogenic U2AF1-S34F splicing mutation observed in several other non-MSI cancer. Conclusions Overall, these findings provide evidence that a very early RNA splicing signature partly driven by MSI impairs cell differentiation and promotes MSI CRC initiation, far before coding mutations which accumulate later during MSI tumorigenesis.

Molecular tumor profiling has become an important diagnostic for prostate cancer, allowing for personalized treatment regimens based on somatic and germline genetic information. We report a 67-year-old patient with metastatic castrate-resistant prostate cancer which was intermittently responsive to androgen-deprivation therapy, docetaxel, abiraterone, radium-223, Sipuleucel-T, and radiotherapy who ultimately demonstrated a remarkable and durable response to pembrolizumab. Our case report underlines the significance of early tumor molecular profiling in aggressive or atypical prostate cancer patients and exhibits the potential for a remarkable clinical response with immunotherapy in candidates with the appropriate tumor profiles.

Context: DNA mismatch repair (MMR) deficiency (dMMR) testing is now recommended in endometrial cancer. Defect identification in the molecules participating in this pathway, or the presence of microsatellite instability, are commonly employed for this purpose. Novel methods are continuously evolving to report dMMR/microsatellite instability and to easily perform routine diagnoses. Objective: The main aim of this study was to compare the concordance of the Idylla microsatellite instability test for the identification of dMMR endometrial cancer samples defined by immunohistochemistry and MMR genomic status. Design: We applied the Idylla MSI test to 126 early-stage endometrial cancer cases with MMR testing by immunohistochemistry and genomic characterization (methylation in MLH1 and sequence alterations in MLH1, PMS2, MSH2 and MSH6). Individual markers and overall specific performance indicators were explored. Results: The Idylla platform achieved a higher global concordance rate with MMR genomic status than with immunohistochemistry (75 % and 66 %, respectively). Sensitivity and specificity are also higher (75 % vs 66 % and 96 % vs 90 %, respectively). Clustering analysis split the patients into 2 well-differentiated clusters, the pMMR and the dMMR group, represented by MLH1/PMS2 loss and the MLH1 methylated promoter. Overall, immunohistochemistry and MMR genomic status identified more dMMR cases than did the Idylla test, although correlations were improved with a modified Idylla test cut-off. Conclusions: Performance of the Idylla test was better correlated with MMR genomic status than MMR immunohistochemistry status, which improved with a modified test cut-off. Further studies are needed to confirm the cut-off accuracy.

Summary: Testing for DNA mismatch repair deficiency (MMRd) is recommended for all colorectal cancers (CRCs). Automating this would enable precision medicine, particularly if providing information on etiology not captured by deep learning (DL) methods. We present AIMMeR, an AI-based method for determination of mismatch repair (MMR) protein expression at a single-cell level in routine pathology samples. AIMMeR shows an area under the receiver-operator curve (AUROC) of 0.98, and specificity of ≥75% at 98% sensitivity against pathologist ground truth in stage II/III in two trial cohorts, with positive predictive value of ≥98% for the commonest pattern of somatic MMRd. Lower agreement with microsatellite instability (MSI) testing (AUROC 0.86) reflects discordance between MMR and MSI PCR rather than AIMMeR misclassification. Analysis of the SCOT trial confirms MMRd prognostic value in oxaliplatin-treated patients; while MMRd does not predict differential benefit of chemotherapy duration, it correlates with difference in relapse by regimen (PInteraction = 0.04). AIMMeR may help reduce pathologist workload and streamline diagnostics in CRC.

Abstract Background Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers. Methods We conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation. Results Similar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs. Conclusions These data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC. Graphical abstract

Functional analyses are the main method to classify mismatch repair (MMR) gene variants of uncertain significance (VUSs). However, the pathogenicity remains unclear for many variants because of conflicting results between clinical, molecular, and functional data. In this study, we evaluated whether whole exome sequencing (WES) could add another layer of evidence to elucidate the pathogenicity of MMR variants with conflicting interpretations. WES was performed on formalin-fixed paraffin-embedded tumor tissue of eight patients with a constitutional MMR VUS (seven families), including eight colorectal and two endometrial carcinomas and one ovarian carcinoma. Cell-free CIMRA assays were performed to assign Odds of Pathogenicity to these VUSs. In four families, seven tumors showed MMR deficiency-associated mutational signatures, supporting the pathogenicity of the VUS. Moreover, somatic (second) MMR hits identified in the WES data were found to explain MMR staining patterns when the MMR staining was discordant with the reported germline MMR gene variant. In conclusion, WES did not significantly reclassify VUS in these cases but clarified some phenotypic aspects such as age of onset and explanations in case of discordant MMR stainings.

Abstract Background The tumor-agnostic indication of immune checkpoint inhibitors to treat cancers with mismatch repair deficiency (dMMR)/microsatellite instability (MSI) increased the demand for such tests beyond Lynch syndrome. International guideline recommendations accept immunohistochemistry (IHC) for dMMR or molecular techniques (PCR or NGS) for MSI status determinations considering the two tests are equal, although there are scattered reports contradicting to this presumption. Materials and methods Here we have directly compared four protein MMR immunohistochemistry (IHC) to MSI Pentaplex PCR test in a large cancer patient cohort (n = 1306) of our diagnostic center where the two tests have been run parallel in 703 cases. Results In this study we have found a high discrepancy rate (19.3%) of the two tests which was independent of the tumor types. The MSI PCR sensitivity for MMR IHC status was found to be very low resulting in a relatively low positive and negative predicting values. As a consequence, the correlation of the two tests was low (kappa

Current clinical guidelines recommend mismatch repair (MMR) protein immunohistochemistry (IHC) or molecular microsatellite instability (MSI) tests as predictive markers of immunotherapies. Most of the pathological guidelines consider MMR protein IHC as the gold standard test to identify cancers with MMR deficiency and recommend molecular MSI tests only in special circumstances or to screen for Lynch syndrome. However, there are data in the literature which suggest that the two test types may not be equal. For example, molecular epidemiology studies reported different rates of deficient MMR (dMMR) and MSI in various cancer types. Additionally, direct comparisons of the two tests revealed relatively frequent discrepancies between MMR IHC and MSI tests, especially in non-colorectal and non-endometrial cancers and in cases with unusual dMMR phenotypes. There are also scattered clinical data showing that the efficacy of immune checkpoint inhibitors is different if the patient selection was based on dMMR versus MSI status of the cancers. All these observations question the current dogma that dMMR phenotype and genetic MSI status are equal predictive markers of the immunotherapies.

Background: Neoadjuvant radiotherapy and chemotherapy, followed by surgical resection, are standard treatments for locally advanced rectal cancer (LARC). Emerging evidence has shown the efficacy of anti-programmed cell death protein 1 (anti-PD-1) therapy for patients with mismatch repair-deficient (dMMR) colorectal cancer, particularly in managing metastatic disease. Several ongoing clinical trials evaluating the efficacy of anti-PD-1 therapy in patients with dMMR LARC have reported outstanding responses. Patients and methods: Here, we present the VOLTAGE-2 study (EPOC 2201), a non-randomized, single-arm, phase II trial that aims to investigate the efficacy and safety of nivolumab monotherapy for 1 year in patients with dMMR-resectable LARC. Patients with clinical complete response (cCR) or near-complete response (nCR) will be observed with non-operative management (NOM) using the Memorial Sloan Kettering Regression Schema.The primary endpoint will be investigator-determined 2-year cCR rate for nivolumab monotherapy. We will investigate the surrogacy of circulating tumor DNA assay as a cCR using whole-genome sequencing (WGS)-based molecular residual disease (MRD) assay and will evaluate the biomarkers of the response to anti-PD-1 antibody using whole-exome sequencing (WES) plus whole-transcriptome sequencing (WTS)-based tumor genomics and immune microenvironment evaluations. We plan to carry out spatiotemporal trans-omics analyses using artificial intelligence and deep learning-driven genomics, transcriptomics, radiomics, pathomics, colonoscopic imaging, quality of life, and clinical features.

Objective: To evaluate the association between mismatch repair (MMR) protein expression and clinico-pathologic outcomes in patients with endometrioid endometrial cancer (EC). Materials and methods: A retrospective review of the clinico-pathologic outcomes was performed on patients who were diagnosed with EC and had results of MMR protein immunohistochemistry. MMR-deficient (MMR-d) was defined as absence of expression in any of the 4 MMR proteins (MLH1, MSH2, MSH6, and PMS2). Demographics, pathologic variables, and survival outcomes were compared according to the MMR status. Results: A total of 193 EC patients with available MMR expression data were included, of whom 163 patients had endometrioid type EC. Overall, 44 patients (27.0%) were classified as MMR-d. Compared with MMR-proficient (MMR-p) group, MMR-d group was associated with more frequent lymphovascular space invasion (LVSI, p = 0.001). MMR-d was also related with higher risk of lymph node (LN) metastasis in endometrioid type EC (p = 0.008), especially para-aortic LN metastasis. During the median follow-up period of 19.1 months (1–44.5), MMR-d group, especially MLH1/PMS2 subgroup, showed a tendency of reduced PFS (p = 0.036 and p = 0.008, respectively). On Cox regression analysis, however, LN metastasis remained as the only independent risk factor for PFS (p = 0.004) in endometrioid EC, and MLH1/PMS2 loss showed a marginally significant association (p = 0.054). Conclusion: Our findings of the associations between MMR deficiency and poor prognostic factors, such as LVSI and LN metastasis, may suggest the prognostic value of MMR status in EC and need further prospective validation studies.

Purpose: To evaluate the effectiveness of machine learning model based on magnetic resonance imaging (MRI) in identifying microsatellite instability (MSI) status and PD-L1 expression in endometrial cancer (EC). Methods: This retrospective study included 82 EC patients from 2 independent centers. Radiomics features from the intratumoral and peritumoral regions, obtained from four conventional MRI sequences (T2-weighted images; contrast-enhanced T1-weighted images; diffusion-weighted images; apparent diffusion coefficient), were combined with clinicopathologic characteristics to develop machine learning model for predicting MSI status and PD-L1 expression. 60 patients from center 1 were used as the training set for model construction, while 22 patients from center 2 were used as an external validation set for model evaluation. Results: For predicting MSI status, the clinicopathologic model, radscore model, and combination model achieved area under the curves (AUCs) of 0.728, 0.833, and 0.889 in the training set, respectively, and 0.595, 0.790, and 0.848 in the validation set, respectively. For predicting PD-L1 expression, the clinicopathologic model, radscore model, and combination model achieved AUCs of 0.648, 0.814, and 0.834 in the training set, respectively, and 0.660, 0.708, and 0.764 in the validation set, respectively. Calibration curve analysis and decision curve analysis demonstrated good calibration and clinical utility of the combination model. Conclusion: The machine learning model incorporating MRI-based radiomics features and clinicopathologic characteristics could be a potential tool for predicting MSI status and PD-L1 expression in EC. This approach may contribute to precision medicine for EC patients.

Abstract Background In this study we aimed to evaluate the efficacy and safety of the PD-L1 inhibitor durvalumab across various mismatch repair deficient (dMMR) or microsatellite instability-high (MSI-H) tumours in the Drug Rediscovery Protocol (DRUP). This is a clinical study in which patients are treated with drugs outside their labeled indication, based on their tumour molecular profile. Patients and methods Patients with dMMR/MSI-H solid tumours who had exhausted all standard of care options were eligible. Patients were treated with durvalumab. The primary endpoints were clinical benefit ((CB): objective response (OR) or stable disease ≥16 weeks) and safety. Patients were enrolled using a Simon like 2-stage model, with 8 patients in stage 1, up to 24 patients in stage 2 if at least 1/8 patients had CB in stage 1. At baseline, fresh frozen biopsies were obtained for biomarker analyses. Results Twenty-six patients with 10 different cancer types were included. Two patients (2/26, 8%) were considered as non-evaluable for the primary endpoint. CB was observed in 13 patients (13/26, 50%) with an OR in 7 patients (7/26, 27%). The remaining 11 patients (11/26, 42%) had progressive disease. Median progression-free survival and median overall survival were 5 months (95% CI, 2-not reached) and 14 months (95% CI, 5-not reached), respectively. No unexpected toxicity was observed. We found a significantly higher structural variant (SV) burden in patients without CB. Additionally, we observed a significant enrichment of JAK1 frameshift mutations and a significantly lower IFN-γ expression in patients without CB. Conclusion Durvalumab was generally well-tolerated and provided durable responses in pre-treated patients with dMMR/MSI-H solid tumours. High SV burden, JAK1 frameshift mutations and low IFN-γ expression were associated with a lack of CB; this provides a rationale for larger studies to validate these findings. Trial registration Clinical trial registration: NCT02925234. First registration date: 05/10/2016.

Abstract Background Large-scale genomic alterations, especially CD274/PD-L1 gene amplification, have great impact on anti-PD-1 efficacy on cancers such as Hodgkin’s lymphoma. However, the prevalence of PD-L1 genetic alterations in colorectal cancer (CRC) and its correlation with the tumor immune microenvironment and clinical implications remain unknown. Materials and Methods PD-L1 genetic alterations were evaluated in 324 patients with newly diagnosed CRC including 160 mismatch repair-deficient (dMMR) patients and 164 mismatch repair-proficient (pMMR) patients using fluorescence in situ hybridization (FISH) method. The correlation between PD-L1 and the expression of the common immune markers was analyzed. Results Totally 33 (10.2%) patients were identified with aberrant PD-L1 genetic alternations including deletion (2.2%), polysomy (4.9%), and amplification (3.1%); They had more aggressive features such as advanced stage (P = 0.02), shorter overall survival (OS) (P

For colorectal cancer (CRC), surgical resection remains essential for achieving good prognoses. Unfortunately, numerous patients with locally advanced CRC and metastatic CRC failed to meet surgical indications or achieve pathological complete response after surgery. Perioperative therapy has been proven to effectively lower tumor staging and reduce recurrence and metastasis. Immune checkpoint inhibitors (ICIs) have shown unprecedented prolongation of survival time and satisfactory safety in patients with high microsatellite instability/deficient mismatch repair (MSI-H/dMMR), while the therapeutic effect obtained by patients with mismatch repair-proficient or microsatellite stable (pMMR/MSS) was considered minimal. However, recent studies found that certain CRC patients with dMMR/MSI-H presented intrinsic or acquired immune resistance, and pMMR/MSS CRC patients can also achieve better efficacy. Therefore, more predictors are required for screening patients with potential clinical benefits. Since the discovery of synergistic effects between immunotherapy, chemotherapy, and radiotherapy, different immunotherapy-based therapies have been applied to the perioperative therapy of CRC in an increasing number of research. This review comprehensively summarized the past and current progress of different combinations of immunotherapy in perioperative clinical trials for CRC, focusing on the efficacy and safety, and points out the direction for future development.

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

Abstract Colon cancers with mismatch repair deficiency (dMMR) have specific clinicopathologic characteristics compared with mismatch repair proficiency (pMMR); however, the effect of MMR status on the efficiency of neoadjuvant chemotherapy (NCT) remains unclear. In our study, 439 dMMR and 26 pMMR colon cancer patients with or without NCT from 2010 to 2017 were retrospectively collected. Clinicopathological features, treatment response, and survival were compared between different groups. In the dMMR group, patients with NCT were likely to have higher CEA (abnormal CEA: 51.6% vs. 17.4%, p

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.

Abstract Background Lynch Syndrome (LS) is an inherited cancer predisposition syndrome defined by pathogenic variants in the mismatch repair (MMR) or EPCAM genes. In the United Kingdom, people with LS are advised to undergo biennial colonoscopy from as early as 25 until 75 years of age to mitigate a high lifetime colorectal cancer (CRC) risk, though the consideration of additional surveillance intervention(s) through the application of non-invasive diagnostic devices has yet to be longitudinally observed in LS patients. In this study, we will examine the role of annual faecal immunochemical testing (FIT) alongside biennial colonoscopy for CRC surveillance in people with LS. Methods/design In this single-arm, prospective, non-randomised study, 400 LS patients will be recruited across 11 National Health Service (NHS) Trusts throughout the United Kingdom. Study inclusion requires a LS diagnosis, between 25 and 73 years old, and a routine surveillance colonoscopy scheduled during the recruitment period. Eligible patients will receive a baseline OC-Sensor™ FIT kit ahead of their colonoscopy, and annually for 3 years thereafter. A pre-paid envelope addressed to the central lab will be included within all patient mailings for the return of FIT kits and relevant study documents. A questionnaire assessing attitudes and perception of FIT will also be included at baseline. All study samples received by the central lab will be assayed on an OC-Sensor™ PLEDIA Analyser. Patients with FIT results of ≥6 μg of Haemoglobin per gram of faeces (f-Hb) at Years 1 and/or 3 will be referred for colonoscopy via an urgent colonoscopy triage pathway. 16S rRNA gene V4 amplicon sequencing will be carried out on residual faecal DNA of eligible archived FIT samples to characterise the faecal microbiome. Discussion FIT may have clinical utility alongside colonoscopic surveillance in people with LS. We have designed a longitudinal study to examine the efficacy of FIT as a non-invasive modality. Potential limitations of this method will be assessed, including false negative or false positive FIT results related to specific morphological features of LS neoplasia or the presence of post-resection anastomotic inflammation. The potential for additional colonoscopies in a subset of participants may also impact on colonoscopic resources and patient acceptability. Trial registration Trial Registration: ISRCTN, ISRCTN15740250 . Registered 13 July 2021.

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 PD-L1 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

Mismatch repair deficiency (d-MMR)/microsatellite instability (MSI), KRAS, and BRAF mutational status are crucial for treating advanced colorectal cancer patients. Traditional methods like immunohistochemistry or polymerase chain reaction (PCR) can be challenged by artificial intelligence (AI) based on whole slide images (WSI) to predict tumor status. In this systematic review, we evaluated the role of AI in predicting MSI status, KRAS, and BRAF mutations in colorectal cancer. Studies published in PubMed up to June 2023 were included (n = 17), and we reported the risk of bias and the performance for each study. Some studies were impacted by the reduced number of slides included in the data set and the lack of external validation cohorts. Deep learning models for the d-MMR/MSI status showed a good performance in training cohorts (mean AUC = 0.89, [0.74–0.97]) but slightly less than expected in the validation cohort when available (mean AUC = 0.82, [0.63–0.98]). Contrary to the MSI status, the prediction of KRAS and BRAF mutations was less explored with a less robust methodology. The performance was lower, with a maximum of 0.77 in the training cohort, 0.58 in the validation cohort for KRAS, and 0.82 AUC in the training cohort for BRAF.

Mismatch repair deficiency (MMRd) is caused by the biallelic inactivation of an MMR gene, which can be attributed either to an inherited or an acquired pathway. MMRd is characterized by the inability of cells to repair spontaneous mutations in microsatellites that occur during replication. Microsatellites are repetitive nucleotide sequences composed of one to six base pairs. Mutations in microsatellites lead to deletions or insertions of sequence units that are designated as microsatellite instability (MSI). MMRd is diagnosed by immunochemistry and is characterized by loss of nuclear immunostaining for at least one of the four MMR proteins that are routinely examined, i.e., MSH2, MSH6, MLH1 and PMS2. Available tests for MSI are PCR and next generation sequencing. MMRd and MSI predispose to tumor initiation and progression, increase tumor mutational burden as well as tumor immunogenicity, facilitate the activation of the programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) immune checkpoint pathway and serve as prognostic and predictive biomarkers in solid tumors.

IntroductionLynch 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.MethodsNinety-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.ResultsAmong 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.ConclusionOur 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.

ObjectivesDNA mismatch repair deficiency (dMMR) status has served as a positive predictive biomarker for immunotherapy and long-term prognosis in gastric cancer (GC). The aim of the present study was to develop a computed tomography (CT)-based nomogram for preoperatively predicting mismatch repair (MMR) status in GC.MethodsData from a total of 159 GC patients between January 2020 and July 2021 with dMMR GC (n=53) and MMR-proficient (pMMR) GC (n=106) confirmed by postoperative immunohistochemistry (IHC) staining were retrospectively analyzed. All patients underwent abdominal contrast-enhanced CT. Significant clinical and CT imaging features associated with dMMR GC were extracted through univariate and multivariate analyses. Receiver operating characteristic (ROC) curve analysis, decision curve analysis (DCA) and internal validation of the cohort data were performed.ResultsThe nomogram contained four potential predictors of dMMR GC, including gender (odds ratio [OR] 9.83, 95% confidence interval [CI] 3.78-28.20, P < 0.001), age (OR 3.32, 95% CI 1.36-8.50, P = 0.010), tumor size (OR 5.66, 95% CI 2.12-16.27, P < 0.001) and normalized tumor enhancement ratio (NTER) (OR 0.15, 95% CI 0.06-0.38, P < 0.001). Using an optimal cutoff value of 6.6 points, the nomogram provided an area under the curve (AUC) of 0.895 and an accuracy of 82.39% in predicting dMMR GC. The calibration curve demonstrated a strong consistency between the predicted risk and observed dMMR GC. The DCA justified the relatively good performance of the nomogram model.ConclusionThe CT-based nomogram holds promise as a noninvasive, concise and accurate tool to predict MMR status in GC patients, which can assist in clinical decision-making.

Abstract Mismatch repair deficiency (dMMR) is a hallmark of Lynch syndrome (LS), but its prevalence in early‐onset (diagnosed under the age of 50 years) duodenal, ampullary, and pancreatic carcinomas (DC, AC, and PC, respectively) is largely unknown. We explored the prevalence of dMMR and the underlying molecular mechanisms in a retrospectively collected cohort of 90 early‐onset carcinomas of duodenal, ampullary, and pancreatic origin. dMMR was most prevalent in early‐onset DCs (47.8%); more than half of those were associated with hereditary cancer syndromes (LS or constitutional mismatch repair deficiency syndrome). All dMMR AC and PC were due to LS. Concordance of dMMR with underlying hereditary condition warrants ubiquitous dMMR testing in all early‐onset DC, AC, and PC.

Approximately 20–30% of endometrial carcinomas (EC) are characterized by mismatch repair (MMR) deficiency (dMMR) or microsatellite instability (MSI), and their testing has become part of the routine diagnosis. The aim of this study was to establish and compare the MMR status using various approaches. Immunohistochemistry (IHC), PCR-based MSI, and the detection of defects in the four key MMR genes (MLH1, PMS2, MSH2, and MSH6) via methylation-specific multiplex ligation-dependent probe amplification (MLPA) and targeted next-generation sequencing (NGS) were performed. MSH3 expression was also evaluated. A set of 126 early-stage EC samples were analyzed, 53.2% of which were dMMR and 46.8% of which were proficient MMR (pMMR) as determined using IHC, whereas 69.3% were classified as microsatellite stable, while 8.8% and 21.9% were classified MSI-low (MSI-L) and MSI-high (MSI-H), respectively. In total, 44.3% of the samples showed genetic or epigenetic alterations in one or more genes; MLH1 promoter methylation was the most common event. Although acceptable concordance was observed, there were overall discrepancies between the three testing approaches, mainly associated with the dMMR group. IHC had a better correlation with MMR genomic status than the MSI status determined using PCR. Further studies are needed to establish solid conclusions regarding the best MMR assessment technique for EC.

Background: About 12%–15% of sporadic colorectal cancers (CRCs) display a defect in the DNA mismatch repair (MMR) system resulting in microsatellite instability (MSI). Many authors have described certain clinicopathological predictors of MSI and confirmed with ancillary studies. The purpose of this study was to determine the clinicopathological features and their association with MMR deficiency (dMMR) among CRC patients. Materials and Methods: A cross-sectional study was designed among patients presented with CRC. A predesigned proforma was used to document the particulars of the patient along with histological parameters to be assessed. Cases were analyzed for dMMR using MLH1 and MSH2 immunostains and categorized into dMMR and MMR-proficient. The association of clinicopathological features with MMR status was statistically analyzed. Results: Sixty-four CRC cases were analyzed in the study. Thirteen out of 64 cases showed dMMR. Most of the dMMR tumors were located in the right-sided colon (P < 0.001). Three patients with a family history of CRC exclusively had dMMR (P = 0.01). Mucinous (P = 0.04), signet ring cell differentiation (P = 0.04), and lack of dirty necrosis (P < 0.001) showed a significant difference between deficient and proficient MMR categories. Gender, mean tumor-infiltrating lymphocytes per hpf, Crohn's-like reaction, and tumor stage did not show any significant difference between the two categories. Conclusions: Clinicopathological features such as family history, tumor location, tumor size, histologic type, tumor differentiation, mucinous, signet ring cell component, and dirty necrosis are associated with MMR status in CRC.

Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is a common genetic predisposition to cancer due to germline mutations in genes affecting DNA mismatch repair. Due to mismatch repair deficiency, developing tumors are characterized by microsatellite instability (MSI-H), high frequency of expressed neoantigens and good clinical response to immune checkpoint inhibitors. Granzyme B (GrB) is the most abundant serine protease in the granules of cytotoxic T-cells and natural killer cells, mediating anti-tumor immunity. However, recent results confirm a diverse range of physiological functions of GrB including that in extracellular matrix remodelling, inflammation and fibrosis. In the present study, our aim was to investigate whether a frequent genetic variation of GZMB, the gene encoding GrB, constituted by three missense single nucleotide polymorphisms (rs2236338, rs11539752 and rs8192917) has any association with cancer risk in individuals with LS. In silico analysis and genotype calls from whole exome sequencing data in the Hungarian population confirmed that these SNPs are closely linked. Genotyping results of rs8192917 on a cohort of 145 individuals with LS demonstrated an association of the CC genotype with lower cancer risk. In silico prediction proposed likely GrB cleavage sites in a high proportion of shared neontigens in MSI-H tumors. Our results propose the CC genotype of rs8192917 as a potential disease-modifying genetic factor in LS.

Genetic mutations that render mismatch repair defective may result in microsatellite instability, which is common in colorectal carcinomas and gastric cancers as well as Lynch syndrome. Mismatch repair deficiency/high microsatellite instability (dMMR/MSI-H) predicts the tumor response to immune checkpoint inhibitors. However, few studies have evaluated the efficacy of immune checkpoint inhibitors in non-small cell lung cancer (NSCLC) patients with dMMR/MSI-H. In this work, we present a patient with advanced squamous lung cancer with dMMR/MSI-H and a high tumor mutational burden (TMB-H) who obtained a long-term benefit from immunotherapy. NSCLC patients with dMMR/MSI-H/TMB-H may thus benefit from immune checkpoint inhibitors.

Colon tumors with deficient DNA mismatch repair (dMMR) are generally infiltrated by T cells more densely than tumors with proficient mismatch repair (pMMR). However, high numbers of tumor-infiltrating lymphocytes (TILs) are found in select pMMR tumors, and low numbers of TILs are seen in select dMMR tumors. In this study, we compared T-cell repertoires in 20 pMMR and 27 dMMR colon tumors with high and low TIL counts. We found that T cells in dMMR tumors are more clonal and their repertoire is less rich compared with T cells in pMMR tumors. In the dMMR group, T cells in TIL-high tumors were more clonal and their repertoire was less rich compared with T cells in TIL-low tumors, but in the pMMR group, T-cell diversity in TIL-high tumors was comparable to T-cell diversity in TIL-low tumors. These findings suggest that T cells clonally expand in dMMR tumors, possibly in response to MMR deficiency-induced tumor neoantigens.

Objective: DNA damage response (DDR) genes have low mutation rates, which may restrict their clinical applications in predicting the outcomes of immune checkpoint inhibitor (ICI) treatment. Thus, a systemic analysis of multiple DDR genes is needed to identify potential biomarkers of ICI efficacy. Methods: A total of 39,631 patients with mutation data were selected from the cBioPortal database. A total of 155 patients with mutation data were obtained from the Fudan University Shanghai Cancer Center (FUSCC). A total of 1,660 patients from the MSK-IMPACT cohort who underwent ICI treatment were selected for survival analysis. A total of 249 patients who underwent ICI treatment from the Dana-Farber Cancer Institute (DFCI) cohort were obtained from a published dataset. The Cancer Genome Atlas (TCGA) level 3 RNA-Seq version 2 RSEM data for gastric cancer were downloaded from cBioPortal. Results: Six MMR and 30 DDR genes were included in this study. Six MMR and 20 DDR gene mutations were found to predict the therapeutic efficacy of ICI, and most of them predicted the therapeutic efficacy of ICI, in a manner dependent on TMB, except for 4 combined DDR gene mutations, which were associated with the therapeutic efficacy of ICI independently of the TMB. Single MMR/DDR genes showed low mutation rates; however, the mutation rate of all the MMR/DDR genes associated with the therapeutic efficacy of ICI was relatively high, reaching 10%–30% in several cancer types. Conclusions: Coanalysis of multiple MMR/DDR mutations aids in selecting patients who are potential candidates for immunotherapy.

Immunotherapy is an effective new approach in the treatment of many malignancies. However, pancreatic ductal adenocarcinoma (PDAC) does not usually respond to immunotherapy. We discuss the case of a patient with metastatic microsatellite instability-high PDAC who had a prolonged response to single-agent pembrolizumab for almost 3 years.

Microsatellite instability (MSI) is a molecular signature of mismatch repair deficiency (dMMR), a predictive marker of immune checkpoint inhibitor therapy response. Despite its recognized pan-cancer value, most methods only support detection of this signature in colorectal cancer. In addition to the tissue-specific differences that impact the sensitivity of MSI detection in other tissues, the performance of most methods is also affected by patient ethnicity, tumor content, and other sample-specific properties. These limitations are particularly important when only tumor samples are available and restrict the performance and adoption of MSI testing. Here we introduce MSIdetect, a novel solution for NGS-based MSI detection. MSIdetect models the impact of indel burden and tumor content on read coverage at a set of homopolymer regions that we found are minimally impacted by sample-specific factors. We validated MSIdetect in 139 Formalin-Fixed Paraffin-Embedded (FFPE) clinical samples from colorectal and endometrial cancer as well as other more challenging tumor types, such as glioma or sebaceous adenoma or carcinoma. Based on analysis of these samples, MSIdetect displays 100% specificity and 96.3% sensitivity. Limit of detection analysis supports that MSIdetect is sensitive even in samples with relatively low tumor content and limited microsatellite instability. Finally, the results obtained using MSIdetect in tumor-only data correlate well (R=0.988) with what is obtained using tumor-normal matched pairs, demonstrating that the solution addresses the challenges posed by MSI detection from tumor-only data. The accuracy of MSI detection by MSIdetect in different cancer types coupled with the flexibility afforded by NGS-based testing will support the adoption of MSI testing in the clinical setting and increase the number of patients identified that are likely to benefit from immune checkpoint inhibitor therapy.

BackgroundPatients with Lynch syndrome are at an increased risk of developing simultaneous or metachronous tumors, while sarcomas have been occasionally reported. Sarcomas are generally not considered part of the common Lynch syndrome tumor spectrum. However, more and more studies and case reports suggested that sarcoma could be a rare clinical manifestation of Lynch syndrome, leading to new treatment strategies for sarcoma.Case summaryWe report the case of a 74-year-old male patient with Lynch syndrome who had rectal mucinous adenocarcinoma and prostate adenocarcinoma and then developed undifferentiated sarcoma of the left neck two years later. Mismatch repair deficiency (dMMR) was confirmed by immunohistochemical staining for the mismatch repair proteins MSH2, MSH6, MLH1 and PMS2. The result of polymerase chain reaction (PCR) microsatellite instability (MSI) testing of sarcoma showed high-level microsatellite instability (MSI-H). Additionally, a pathogenic germline mutation in MSH2 (c.2459-12A>G) was detected by next-generation sequencing (NGS). Taking into account HE morphology, immunohistochemical phenotype, MSI status, NGS result, medical history and germline MSH2 gene mutation, the pathological diagnosis of left neck biopsy tissue was Lynch syndrome related undifferentiated sarcoma with epithelioid morphology. The patient has been receiving immunotherapy (sintilimab) combined with chemotherapy (tegafur, gimeracil and oteracil potassium capsules) and currently has stable disease. We also reviewed the literature to understand the association between sarcoma and Lynch syndrome.ConclusionSarcoma may now be considered a rare clinical manifestation of Lynch syndrome. Attention and awareness about the association between Lynch syndrome and sarcoma need to be increased. Therefore, timely detection of MMR proteins and validation at the gene level for suspicious patients are the keys to avoiding missed or delayed diagnosis and to identifying patients suited for immunotherapy, which may also help to provide appropriate genetic counseling and follow-up management for patients.

Abstract Objective: To determine the frequency of mismatch repair deficiency in colorectal carcinoma determined by immunohistochemistry. Study Design: Cross-sectional study. Place and Duration of Study: Department of Histopathology, Armed Forces Institute of Pathology, Rawalpindi, from Aug 2018 to Jan 2019. Methodology: A total of 101 patients of adenocarcinoma of colorectum who underwent surgical resections and their characteristic and clinical data were recorded. Immunohistochemical stains were performed using antibodies MLH1, MSH2, PMS2 and MSH6. Results were interpreted and mismatch repair deficiency status of all patients was determined. Patients with loss of expression for MLH1, MSH2, PMS2 and MSH6 antibodies were observed and noted. Results: In this study, out of 101 patients with CRC, 71 (70.3%) were male and 30 (29.7%) female. The mean age was (54 years ± 15.9). Amongst the 101 cases loss of immunohistochemical staining for MMR proteins was noted in 19 patients (18.8%). The combined loss of all four antibodies was seen in one case, loss of MLH1 and PMS2 in 7, MSH2 and MSH6 in 5 and MLH1 only in 6 patients. However, no mismatch repair deficiency was detected in remaining 82 cases. According to statistical analysis no significant association between mismatch repair deficiency and variables was found. Conclusion: The frequency of mismatch repair deficiency in colorectal carcinoma patients was found to be 18.8% in our population.

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 < 2 mm thickness and low-level MLH1, three patients with primary cSCC > 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 < 2 mm, cSCC > 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.

Hai Luo,1,* Lingzhi Cui,2,* Kexin Shen,1 Ruiqi Li,1 Zeming Wang,1 Zhongshi Xie1 1Gastrointestinal Colorectal and Anal Surgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin, People’s Republic of China; 2General Surgery, The Second Affiliated Hospital of Baotou Medical College, Inner Mongolia University of Science and Technology, Baotou, Inner Mongolia, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhongshi XieChina-Japan Union Hospital of Jilin University, 126 Xian Tai Street, Changchun, Jilin Province, 130033, People’s Republic of ChinaTel +86 13944108332Email surgeonxzs@163.comPurpose: This study was designed to investigate the correlation between the expression of human epidermal growth factor receptor 2 (HER2), mismatch repair (MMR), and clinicopathological parameters and serum tumor markers in a total of 522 resection samples materials from colorectal cancer (CRC) patients. These data were also used to determine the links between HER2 and MMR expression and prognosis.Methods: We conducted a retrospective analysis of the clinical data from 522 CRC patients. Immunohistochemistry (IHC) was used to detect HER2 overexpression and MMR deficiency (dMMR) in tumor specimens which were then correlated with various clinicopathological parameters. Prognostic value for HER2 and MMR expression was then evaluated using the data from 105 CRC patients.Results: HER2 overexpression was identified in 35.63% of the samples evaluated in this study, while the total dMMR rate was 12.64%. Expression of HER2 and several, MMR proteins (MLH1, MSH-2, MSH-6, and PMS-2) were then correlated with tumor location. HER2 overexpression is significantly associated with increased depth of tumor invasion, lymph node metastasis, distant metastases, pTNM staging, vascular invasion, nerve infiltration, and serum carcinoembryonic antigen (CEA) levels. HER2 overexpression and dMMR increased with advancing clinical stage. In addition, deficiencies in MLH1 and PMS2 correlated with HER2 overexpression. Finally, the prognostic evaluations revealed that HER2 overexpression was closely associated with poorer clinical outcomes.Conclusion: HER2 overexpression is significantly correlated with multiple clinicopathological parameters resulting in a poorer prognosis. Moreover, the prognosis of patients with HER2 overexpression was worse, confirming its significance during disease assessment. In clinical practice, clinicians should pay close attention to the HER2 profile of patients as they may require more extensive clinical intervention. In addition, deficiencies in MLH1, MSH-2, MSH-6, or PMS-2 correlate with tumor location, and MLH1 and PMS2 expression is associated with lymph node metastasis and pTNM stage, suggesting that these may be additional markers in CRC risk assessments.Keywords: human epidermal growth factor receptor 2, mismatch repair deficiency, colorectal cancers, prognosis

Patients with locally advanced colorectal cancer (LACRC) have a high risk of recurrence and metastasis, although neoadjuvant therapy may provide some benefit. However, patients with high microsatellite instability/deficient mismatch repair (MSI-H/dMMR) LACRC receive little benefit from neoadjuvant chemoradiotherapy (nCRT) or neoadjuvant chemotherapy (nCT). The 2015 KEYNOTE-016 trial identified MSI-H/dMMR as a biomarker indicative of immunotherapy efficacy, and pointed to the potential use of immune checkpoint inhibitors (ICIs). In 2017, the FDA approved two ICIs (pembrolizumab and nivolumab) for treatment of MSI-H/dMMR metastatic CRC (mCRC). In 2018, the CheckMate-142 trial demonstrated successful treatment of mCRC based on “double immunity” provided by nivolumab with ipilimumab, a regimen that may become a standard first-line treatment for MSI-H mCRC. In 2018, the FDA approved nivolumab alone or with ipilimumab for patients who progressed to MSI-H/dMMR mCRC after standard chemotherapy. The FDA then approved pembrolizumab alone as a first-line treatment for patients with MSI-H/dMMR CRC that was unresectable or metastatic. There is now interest in using these drugs in neoadjuvant immunotherapy (nIT) for patients with MSI-H/dMMR non-mCRC. In 2020, the NICHE trial marked the start of using nIT for CRC. This novel treatment of MSI-H/dMMR LACRC may change the approaches used for neoadjuvant therapy of other cancers. Our review of immunotherapy for CRC covers diagnosis and treatment, clinical prognostic characteristics, the mechanism of nIT, analysis of completed prospective and retrospective studies, and ongoing clinical trials, and the clinical practice of using nIT for MSI-H/dMMR LACRC. Our team also proposes a new organ-preservation strategy for patients with MSI-H/dMMR low LARC.

Abstract Background Individuals who have colorectal or endometrial cancers displaying loss of immunohistochemical staining of one or more mismatch repair proteins without an identifiable causative germline pathogenic variant have unexplained mismatch repair deficiency (UMMRD). Comprehensive germline genetic testing for Lynch syndrome (LS) includes sequencing and deletion/duplication analysis of MLH1, MSH2, MSH6, and PMS2, deletion analysis of EPCAM, and MSH2 inversion analysis. Updated genetic testing to include elements of comprehensive LS testing not previously completed could further clarify LS status in individuals with UMMRD, allowing for tailored screening guidelines for affected individuals and their family members. However, patient understanding of the potential impact of updated genetic testing for LS is unclear. This study aimed to evaluate the interest in and perceived impact of updated genetic testing among individuals with UMMRD at a tertiary academic center. Methods A survey evaluating interest in and perceived impact of updated genetic testing was mailed to 98 potential participants. Electronic health record review was completed for all individuals meeting eligibility criteria. Thirty-one individuals responded to the survey. Results Results indicate this population is highly interested in updated genetic testing with the perceived impact being primarily for family members to have appropriate genetic testing and screening. Electronic health record review indicates that clinicians have an evolving understanding of causes of UMMRD, representing a potential change in assessment of cancer risk. Conclusions Updated risk assessment and genetic counseling with a discussion of the benefits and limitations of germline and somatic genetic testing, is essential as the understanding of UMMRD and genetic testing recommendations for this population evolve.

Abstract Background Immunohistochemistry (IHQ) is commonly used for the detection of mismatch repair proteins deficiency (MMRD). One very infrequent abnormal pattern of MMR protein expression is the loss of PMS2 and MSH6, with intact expression of MLH1 and MSH2. Case presentation We review the frequency of this MMRD IHC pattern among 108 colorectal (CRCs) and 35 endometrial cancers in our files with loss of expression of at least one protein, and present two CRCs showing loss of PMS2 and MSH6 protein expression (1.9% of CRCs). NGS analysis of these tumours identified PMS2 mutations (R134* germline mutation in one tumour and M1R and c.1239delA somatic mutation in the other) as the primary event and somatic MSH6 mutation (c.3261dupC) as the secondary event in both tumours. Conclusions This study suggests that Next Generation Sequencing (NGS) tumour analysis should be considered in the algorithm of Lynch syndrome screening to detect MMR gen somatic mutation in inconclusive cases.

Abstract Background Glioblastoma (GBM) is a highly malignant brain neoplasm with poor survival. Despite its aggressive nature, metastatic spread of GBM is identified only rarely. While the molecular alterations associated with GBM and its subtypes are well-described, there remains a gap in understanding which alterations may predispose towards metastasis. In this report, we present a case of GBM with multi-organ metastases and discuss its genomic alterations. Case presentation A 74-year-old woman was diagnosed with left occipital glioblastoma (IDH-wildtype, MGMT-unmethylated), for which she underwent resection, standard chemoradiation, and then stereotactic radiosurgery (SRS) for local recurrence. One month after SRS, work-up for a pathologic hip fracture revealed a left breast mass, lytic lesions involving pelvic bones, and multiple pulmonary and hepatic lesions. Biopsies of the breast and bone lesions both demonstrated metastatic IDH-wildtype GBM. For worsening neurologic symptoms, the patient underwent debulking of a large right temporal lobe recurrence and expired shortly thereafter. Autopsy confirmed metastatic GBM in multiple systemic sites, including bilateral lungs, heart, liver, thyroid, left breast, small bowel, omentum, peritoneal surfaces, visceral surfaces, left pelvic bone, and hilar lymph nodes. Targeted sequencing was performed on tissue samples obtained pre- and postmortem, as well as on cell cultures and an orthotopic mouse xenograft derived from premortem surgical specimens. A BRCA1 mutation (p.I571T) was the only variant found in common among the primary, recurrence, and metastatic specimens, suggesting its likely status as an early driver mutation. Multiple subclonal ARID1A mutations, which promote genomic instability through impairment of DNA mismatch repair, were identified only in the recurrence. Mutational spectrum analysis demonstrated a high percentage of C:G to T:A transitions in the post-treatment samples but not in the primary tumor. Conclusion This case report examines a rare case of widely metastatic IDH-wildtype GBM with a clonal somatic mutation in BRCA1. Post-treatment recurrent tumor in the brain and in multiple systemic organs exhibited evidence of acquired DNA mismatch repair deficiency, which may be explained by functional loss of ARID1A. We identify a potential role for immune checkpoint and PARP inhibitors in the treatment of metastatic GBM.

Introduction: Invasive micropapillary carcinoma (IMPC) of the breast is a rare and aggressive subtype of invasive ductal carcinomas, associated with poor prognosis and without a well-established treatment. Programmed death-ligand 1 (PD-L1) expression, high tumor-infiltrating lymphocytes (TILs), and microsatellite instability have recently been linked to susceptibility to immunotherapies against PD-1/PD-L1 axis. No exhaustive data is available on the status of these predictive markers in IMPCs of the breast. The aim of our study is to analyze PD-L1 expression, stromal TIL (sTIL), and mismatch repair (MMR) gene status in IMPCs of the breast, to extend the therapeutic possibilities of these rare aggressive tumors. Materials and Methods: Thirty-seven cases of IMPCs diagnosed in two European institutions between 2003 and 2017 with detailed clinical and pathologic data were analyzed. sTILs were assessed in hematoxylin and eosin-stained sections. MMR deficiency was tested by either immunohistochemistry (IHC) for MMR proteins (MLH1, MSH2, MSH6, and PMS2) or capillary electrophoresis for microsatellite instability using a standardized panel of five loci (Bat25, Bat26, D2S123, D5S346, and D17S250). For PD-L1, expression in both tumor cells (TCs) and immune cells (ICs) was determined using the antibody clone SP263. Results: The median sTILs was 3% (mean: 6%, range: 0–40). Thirty-one cases (84%) showed ≤10% of sTILs and only one case had 40% of sTILs. Higher median TILs were more frequently observed in lymph node metastases. PD-L1 expression (≥1%) was observed in 4 (11%) and 14 (38%) cases in TCs and ICs, respectively. None of the tumors showed PD-L1 expression in >1% of TCs. Only three cases showed expression in >10% of ICs. All cases were microsatellite stable by either IHC or polymerase chain reaction analyses. Conclusions: IMPCs of the breast are microsatellite-stable and immune desert tumors with low PD-L1 expression, thus arguing against the use of immune-checkpoint inhibitors in these patients. Active immunotherapy strategies attempting to stimulate self-immune system to attack tumor are needed.

Abstract Immunotherapies have led to substantial changes in cancer treatment and have been a persistently popular topic in cancer research because they tremendously improve the efficacy of treatment and survival of individuals with various cancer types. However, only a small proportion of patients are sensitive to immunotherapy, and specific biomarkers are urgently needed to separate responders from nonresponders. Mismatch repair pathways play a vital role in identifying and repairing mismatched bases during DNA replication and genetic recombination in normal and cancer cells. Defects in DNA mismatch repair proteins and subsequent microsatellite instability-high lead to the accumulation of mutation loads in cancer-related genes and the generation of neoantigens, which stimulate the anti-tumor immune response of the host. Mismatch repair deficiency/microsatellite instability-high represents a good prognosis in early colorectal cancer settings without adjuvant treatment and a poor prognosis in patients with metastasis. Several clinical trials have demonstrated that mismatch repair deficiency or microsatellite instability-high is significantly associated with long-term immunotherapy-related responses and better prognosis in colorectal and noncolorectal malignancies treated with immune checkpoint inhibitors. To date, the anti-programmed cell death-1 inhibitor pembrolizumab has been approved for mismatch repair deficiency/microsatellite instability-high refractory or metastatic solid tumors, and nivolumab has been approved for colorectal cancer patients with mismatch repair deficiency/microsatellite instability-high. This is the first time in the history of cancer therapy that the same biomarker has been used to guide immune therapy regardless of tumor type. This review summarizes the features of mismatch repair deficiency/microsatellite instability-high, its relationship with programmed death-ligand 1/programmed cell death-1, and the recent advances in predicting immunotherapy efficacy.

Abstract Background Mismatch repair (MMR)-deficiency increases the risk of colorectal tumorigenesis. To determine whether the tumors develop on a normal or disturbed epigenetic background and how radiation affects this, we quantified genome-wide histone H3 methylation profiles in macroscopic normal intestinal tissue of young radiated and untreated MMR-deficient VCMsh2 LoxP/LoxP (Msh2 −/− ) mice months before tumor onset. Results Histone H3 methylation increases in Msh2 −/− compared to control Msh2 +/+ mice. Activating H3K4me3 and H3K36me3 histone marks frequently accumulate at genes that are H3K27me3 or H3K4me3 modified in Msh2 +/+ mice, respectively. The genes recruiting H3K36me3 enrich in gene sets associated with DNA repair, RNA processing, and ribosome biogenesis that become transcriptionally upregulated in the developing tumors. A similar epigenetic effect is present in Msh2 +/+ mice 4 weeks after a single-radiation hit, whereas radiation of Msh2 −/− mice left their histone methylation profiles almost unchanged. Conclusions MMR deficiency results in genome-wide changes in histone H3 methylation profiles preceding tumor development. Similar changes constitute a persistent epigenetic signature of radiation-induced DNA damage.

Abstract Background Lynch syndrome is the most common hereditary colorectal cancer syndrome, and adenoma is one of the important premalignant lesions to colorectal cancer in Lynch syndrome. The first objective of this study was to calculate the detection rate of Lynch syndrome in colorectal polyps by using mismatch repair immunohistochemistry as the initial screening strategy. The second objective of this study was to optimize screening strategies for adenoma associated with Lynch syndrome by integrating polyp and/or patient characteristics such as polyp size, location, dysplasia, age of onset and/or family history of cancer. Methods From June 2014 to May 2016, immunohistochemistry was performed for mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2) using endoscopically resected specimens obtained from newly diagnosed colorectal adenomas. Gene analysis was performed in patients with missing expression of mismatched repair protein. Results Five hundred and ten patients underwent colorectal polyp resection, with a total of 718 polyps. Five hundred and eight resected adenomas underwent mismatch repair protein immunohistochemical testing. Loss of mismatch repair protein expression was observed in six adenomas, accounting for 1.18% of all adenomas. Five patients then underwent genetic tests to identify two pathogenic mutations from different individuals, while another patient was suspected to have a pathogenic mutation. Three patients were younger than 50 years old. Two patients had advanced histology (high-grade dysplasia and malignant components) and one patient had a family history of cancer. Conclusion Immunohistochemical detection of colorectal polyp mismatch repair protein as Lynch syndrome screening efficiency is low. Effective screening strategies may be improved by optimizing patient/polyp selection, such as focusing on young adenoma patients with a family history of cancer, or patients who present with high-risk features (large size, villous, high-grade dysplasia and malignant components).

ObjectiveTo explore the computed tomography (CT) features of gastric cancer (GC) patients with DNA mismatch repair deficiency (dMMR).Materials and MethodsThis study reviewed the clinical and CT features of GC patients with dMMR, confirmed by the postoperative results, between September 2017 and December 2019. The expression pattern of MMR major proteins (MLH1, MSH2, MSH6, and PMS2) in immunohistochemistry was used to confirm the MMR status in GC tissues. The correlation between pre-treatment CT features and MMR status was statistically analyzed.ResultsA total of 28 patients with GC were diagnosed as dMMR in our study, and 49 patients were MMR-proficient (pMMR). The tumor locations were significantly different between the dMMR and pMMR groups (p = 0.006). The CT tumor thickness, CT long and short diameters of the largest lymph node, and the number of lymph nodes on CT of the dMMR group were significantly different from the pMMR group.ConclusionThe dMMR GC exhibited a lower stomach location, smaller tumor thickness and lymph node diameter, and fewer lymph nodes on CT imaging.

ObjectiveAlthough Polymerase-epsilon (POLE)-mutated and mismatch repair (MMR)-deficient endometrial cancers (ECs) are considered as promising candidates for anti-PD-1/PD-L1 therapy, selecting only these patients may exclude other patients who could potentially respond to this treatment strategy, highlighting the need of additional biomarkers for better patient selection. This study aims to evaluate potential predictive biomarkers for anti-PD-1/PD-L1 therapy in addition to POLE mutation (POLEm) and MMR deficiency (MMRd).MethodsWe performed next generation sequencing for POLE from 202 ECs, and immunohistochemistry for MLH1, MSH2, MSH6, PMS2, CD3, CD8, PD-1 and PD-L1 on full-section slides from these ECs. We assessed the association of POLEm and MMRd with clinicopathologic features, expression of check point proteins, and density of tumor-infiltrating lymphocytes (TILs). Prognostic impact of these immune markers was also evaluated.ResultsPOLEm, MMRd and high-grade tumors exhibited elevated level of TILs. Increased expression of PD-1 and PD-L1 was observed in MMRd and high-grade ECs. A subgroup of MMR proficient ECs also harbored increased density of TILs, and positive expression of PD-1 and PD-L1. In addition, negative expression of checkpoint proteins and high density of TILs in combination was associated with good prognosis.ConclusionsCandidates for PD-1 blockade may extend beyond POLEm and MMRd ECs, additional factors such as tumor grade, and combination of TILs levels and expression of checkpoint proteins may need to be considered for better patient selection.

Mismatch repair-deficient (dMMR) prostate cancer is rare and has not been well studied. We aimed to evaluate the clinical characterization of dMMR metastatic castration-resistant prostate cancer (mCRPC) patients. The MMR genes include MLH1, MLH3, MSH2, MSH6, PMS1, PMS2, and EPCAM, and were analyzed by targeted sequencing of plasma cell-free DNA samples. A total of 109 mCRPC patients were identified, including 50 patients with MMR alterations (pathogenic alterations, n = 7; alterations of unknown significance, n = 43) and 59 patients with wild-type MMR. For the seven patients with pathogenic MMR alterations, the median age at diagnosis was 63.5 years, and 42.9% had a Gleason score ≥8. The median time from androgen deprivation therapy (ADT) initiation to CRPC was 24 months. Compared with the wild-type MMR subgroup, patients with MMR alterations, pathogenic MMR alterations, or MMR alterations of unknown significance showed higher rates of hotspot missense mutations or copy number amplifications in the AR gene (24/50 vs. 10/59, P = 7.8 × 10–4; 7/7 vs. 10/59, P = 2.5 × 10–5; 17/43 vs. 10/59, P = 0.013). The presence of any MMR alterations was associated with an inferior response to abiraterone [median progression-free survival (PFS): 5.0 vs. 10.9 months, P = 0.022]. Shorter PFS times were observed in both the pathogenic MMR alteration subgroup (median PFS: 5 months) and the MMR alterations of unknown significance subgroup (median PFS: 5.3 months), compared with the PFS of those with wild-type MMR genes (median PFS: 10.9 months, P = 0.052). There was no statistically significant difference in response to docetaxel chemotherapy between the MMR alterations of unknown significance and the wild-type MMR subgroups (median PFS: 8.2 vs. 8.1 months, P = 0.23). Our results demonstrate that dMMR mCRPC patients have an equivalent response to standard ADT and taxane-based chemotherapy treatments compared with wild-type MMR patients. Patients with both pathogenic and unknown significance alterations of MMR genes had poorer responses to abiraterone therapy.

Abstract Background MMR-D pancreatic cancer have been reported to respond to checkpoint inhibitor therapy. Here, we report the first case of acquired resistance to immunotherapy in MMR-D pancreatic cancer. Case presentation A 45-year-old woman with unresectable MMR-D pancreatic cancer was initially treated with FOLFIRINOX, FOLFIRI, and stereotactic body radiation with stable disease burden. After 3 months, imaging showed progression of disease with an increase in CA19-9. She was subsequently enrolled in a clinical trial of an anti-PD-L1 antibody in combination with an IDO1 inhibitor. She demonstrated a partial response to therapy by RECIST 1.1 criteria with declining tumor markers. Twenty-two months after beginning immunotherapy, imaging revealed an increasing left ovarian cystic mass. There were no other sites of progressive disease. The patient underwent a total hysterectomy and bilateral salpingo-oophorectomy, appendectomy, omentectomy and pelvic lymphadenopathy. Pathology was consistent with a metastasis from the pancreas involving the endometrium and left ovary. Thereafter, the patient continued with PD-1 blockade therapy off protocol with no further progressive disease. Immune profiling showed high levels of CD8+ T cells and PD-1 positive immune cells infiltrating the tumor, with a moderate level of PD-L1 expression in both the immune cells and the tumor cells. Next generation sequencing found only the KRAS G12D and RNF43 G659Vfs*41 mutations were retained from the pre-treatment tumor in the treatment-resistant tumor. Conclusions This is the first report describing acquired resistance to immunotherapy in MMR-D pancreatic cancer with accompanying genomic and immune profiling. This case of oligoprogression in the setting of immunotherapy demonstrates the feasibility of localized treatment followed by continuation of immunotherapy to sustain ongoing response.

Endometrial cancer (EC) can easily be cured when diagnosed at an early stage. However, advanced and metastatic EC is a common disease, affecting more than 15,000 patients per year in the United Sates. Only limited treatment options were available until recently, with a taxane–platinum combination as the gold standard in first-line setting and no efficient second-line chemotherapy or hormone therapy. EC can be split into four molecular subtypes, including hypermutated cases with POLE mutations and 25–30% harboring a microsatellite instability (MSI) phenotype with mismatch repair deficiency (dMMR). These tumors display a high load of frameshift mutations, leading to increased expression of neoantigens that can be targeted by the immune system, including (but not limited) to T-cell response. Recent data have demonstrated this impact of programmed death 1 and programmed death ligand 1 (PD-1/PD-L1) inhibitors on chemo-resistant metastatic EC. The uncontrolled KEYNOTE-158 and GARNET trials have shown high response rates with pembrolizumab and dostarlimab in chemoresistant MSI-high tumors. Most responders experiment long responses that last more than one year. Similar, encouraging results were obtained for MMR proficient (MMRp) cases treated with a combination of pembrolizumab and the angiogenesis inhibitor lenvatinib. Approvals have, thus, been obtained or are underway for EC with immune checkpoint inhibitors (ICI) used as monotherapy, and in combination with antiangiogenic agents. Combinations with other targeted therapies are under evaluation and randomized studies are ongoing to explore the impact of ICI-chemotherapy triplets in first-line setting. We summarize in this review the current knowledge of the immune environment of EC, both for MMRd and MMRp tumors. We also detail the main clinical data regarding PD-1/PD-L1 inhibitors and discuss the next steps of development for immunotherapy, including various ICI-based combinations planned to limit resistance to immunotherapy.