Loss Of Mismatch Repair Research Articles

Endometrial Carcinomas With Subclonal Loss of Mismatch Repair Proteins: A Clinicopathologic and Genomic Study.

Subclonal loss of mismatch repair (MMR) proteins has been described in a small subset of endometrial carcinomas (ECs), but the genomic basis for this phenomenon has received limited attention. Herein, we retrospectively evaluated all ECs with MMR immunohistochemistry (n=285) for subclonal loss, and in those (n=6), performed a detailed clinicopathologic and genomic comparison of the MMR-deficient and MMR-proficient components. Three tumors were FIGO stage IA, and one each stage IB, II, and IIIC2. Patterns of subclonal loss were as follows: (1) 3 FIGO grade 1 endometrioid carcinomas with subclonal MLH1/PMS2, MLH1 promoter hypermethylation, and no MMR gene mutations; (2) POLE -mutated FIGO grade 3 endometrioid carcinoma with subclonal PMS2, and PMS2 and MSH6 mutations limited to the MMR-deficient component; (3) dedifferentiated carcinoma with subclonal MSH2/MSH6, as well as complete loss of MLH1/PMS2, MLH1 promoter hypermethylation, and PMS2 and MSH6 mutations in both components; (4) dedifferentiated carcinoma with subclonal MSH6, and somatic and germline MSH6 mutations in both components, but with a higher allele frequency in MMR-deficient foci. Recurrences occurred in 2 patients, one consisted of the MMR-proficient component from a FIGO 1 endometrioid carcinoma, while the other was from the MSH6 -mutated dedifferentiated endometrioid carcinoma. At the last follow-up (median: 44mo), 4 patients were alive and disease-free and 2 were alive with disease. In summary, subclonal MMR loss reflects subclonal and often complex genomic and epigenetic alterations, which may have therapeutic implications and therefore must be reported when present. In addition, subclonal loss can occur in both POLE -mutated and Lynch syndrome-associated ECs.

Loss of mismatch repair promotes a direct selective advantage in human stem cells.

Lynch syndrome (LS) is the most common hereditary form of colon cancer, resulting from a germline mutation in a DNA mismatch repair (MMR) gene. Loss of MMR in cells establishes a mutator phenotype, which may underlie its link to cancer. Acquired downstream mutations that provide the cell a selective advantage would contribute to tumorigenesis. It is unclear, however, whether loss of MMR has other consequences that would directly result in a selective advantage. We found that knockout of the MMR gene MSH2 results in an immediate survival advantage in human stem cells grown under standard cell culture conditions. This advantage results, in part, from an MMR-dependent response to oxidative stress. We also found that loss of MMR gives rise to enhanced formation and growth of human colonic organoids. These results suggest that loss of MMR may affect cells in ways beyond just increasing mutation frequency that could influence tumorigenesis.

EXTH-95. UNCOVERING THERAPEUTIC VULNERABILITIES IN MISMATCH REPAIR-DEFICIENT GLIOMAS

Abstract Approximately 25% of recurrent gliomas exhibit hypermutation, which is most often acquired in chemotherapy sensitive gliomas post-treatment with standard of care alkylating agent temozolomide, Intriguingly, nearly all of these recurrent hypermutant tumors harbor deficiencies in mismatch repair (MMR) genes. Unlike other MMR-deficient cancers, hypermutant gliomas do not exhibit detectable microsatellite instability (MSI) at the population level, and do not share similar dependencies. Thus, strategies to therapeutically target MMR-deficient and hypermutant gliomas are urgently needed, and likely to impact many patients. To evaluate vulnerabilities associated with MMR-deficient gliomas, we have generated isogenic MMR-deficient models by systematic ablation of core MMR genes MSH2, MSH6, MLH1, and PMS2, in multiple patient-derived glioma cell lines using a robust all-in-one CRISPR-Cas9 system. We characterized these isogenic MMR-deficient glioma lines in comparison to respective MMR-proficient control line by performing differential gene expression analysis and subsequent gene set enrichment analyses. Our analyses reveal in an unbiased fashion an enrichment of several hallmark gene sets including cell cycle control and DNA repair in the MMR-deficient cell lines. We performed a CRISPR-Cas9 knockout screen in these MMR deficient and hypermutant models and identified candidate genes involved in DNA repair, cell cycle, RNA metabolism and other pathways as preferential dependencies in the MMR-deficient glioma cells. We also screened multiple MMR-deficient isogenic models against the high-throughput drug repurposing (REPO) chemical library and our preliminary results reveal a number of compounds from a variety of drug classes that selectively kill the MMR-deficient glioma cells, indicating that the loss of MMR confers differential dependencies to these small molecule inhibitors. The candidate drugs and genes identified from these high-throughput assays are currently being subjected to further validation, and they possibly will help us identify novel therapeutic strategies or improve existing therapeutic strategies to target recurrent MMR-deficient gliomas.

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

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

Significance of molecular classification in fertility-sparing treatment of endometrial carcinoma and atypical endometrial hyperplasia

Objective: To investigate the molecular classification of endometrial carcinoma (EC) and atypical endometrial hyperplasia (AEH) treated with fertility-sparing therapy, and to analyze its relationship with clinicopathological factors and treatment efficacy. Methods: A total of 46 EC and AEH patients who received fertility-sparing therapy and molecular classification tested by next generation sequencing in Peking University People's Hospital from June 2020 to December 2021, were retrospectively collected. The relationships between molecular classification and clinicopathological factors and treatment outcomes were analyzed. Results: (1) Of the 46 patients, including 40 EC and 6 AEH patients, 32 cases (71%, 32/45) had complete response (CR) after treatment, with median CR time of 8 months, 6 cases (13%, 6/45) had partial response, and 8 cases (25%, 8/32) had recurrence. (2) The cases were distributed as no specific molecular profile (NSMP) 34 cases (74%, 34/46) subtype mainly, high microsatellite instability (MSI-H) 7 cases (15%, 7/46), POLE ultra-mutated 3 cases (7%, 3/46), and copy number high (CNH) 2 cases (4%, 2/46). Patients with CNH had the hightest serum cancer antigen 125 (CA125) level [(34.3±35.2) kU/L]. MSI-H subtype had more family history of tumors (6/7), more with loss of mismatch repair (MMR) protein expression by immunohistochemical (7/7), and higher nuclear antigen associated with cell proliferation (Ki-67) expression level (3/3). (3) Patients in MSI-H subgroup had the lowest CR rate at 6 months (0/6; P=0.019), and survival analysis showed that they were less likely to achieve CR than those with NSMP subtype (P=0.022). Subgroup analysis of patients with NSMP showed that age ≥30 years related with longer treatment time to CR (P=0.010). In addition, CR was obtained after treatment in 2/3 POLE ultra-mutated cases and 2/2 CNH, respectively. Conclusions: Molecular classification relates with the treatment response in patients with EC and AEH treated with fertility-sparing therapy. Patients with MSI-H subtype have poor treatment efficacy, and patients with NSMP need to be further studied and predict treatment benefit. However, there are few cases in POLE ultra-mutated and CNH subtypes, which need further clinical research.

Systems approach to enhance Lynch syndrome diagnosis through tumour testing

BackgroundGuidelines recommend universal mismatch repair (MMR) tumour testing of colorectal adenocarcinomas (CRCs) to screen for Lynch syndrome (LS). However, its implementation remains disjointed and referral for genetic testing dismal, particularly...

BAT26 Only Microsatellite Instability with High Tumor Mutation Burden-A Rare Entity Associated with PTEN Protein Loss and High PD-L1 Expression.

Detecting microsatellite instability (MSI) in advanced cancers is crucial for clinical decision-making, as it helps in identifying patients with differential treatment responses and prognoses. BAT26 is a highly sensitive MSI marker that defines the mismatch repair (MMR) status with high sensitivity and specificity. However, isolated BAT26-only instability is rare and has not been previously reported. Of the 6476 cases tested using pentaplex MSI polymerase chain reaction, we identified two BAT26-only instability cases (0.03%) in this study. The case #1 patient was diagnosed with endometrial adenocarcinoma without MMR germline mutations. The endometrial tumor showed BAT26-only instability, partial loss of MLH1/PMS2 protein expression, and a high programmed cell death ligand 1 (PD-L1) combined positive score (CPS = 8). The tumor exhibited a somatic phosphatase and tensin homolog (PTEN) R303P missense mutation and loss of the PTEN protein. On a comprehensive cancer panel sequencing with ≥500 genes, the tumor showed an MSI score of 11.38% and high tumor mutation burden (TMB) (19.5 mt/mb). The case #2 patient was diagnosed with colorectal carcinoma with proficient MMR and PTEN protein loss without PTEN alteration, as well as a high PD-L1 CPS (CPS = 10). A pathogenic KRAS A146T mutation was detected with an MSI score of 3.36% and high TMB (13 mt/mb). In conclusion, BAT26-only instability is very rare and associated with PTEN protein loss, high TMB, and a high PD-L1 score. Our results suggest that patients with BAT26-only instability may show good responses to immunotherapy.

Molecular classification of endometrial cancers using an integrative DNA sequencing panel (271)

<b>Objectives:</b> The Cancer Genome Atlas (TCGA) has identified four molecular subgroups of endometrial cancers (EC) with distinct differences in prognosis and response to adjuvant treatment. Stratification of patients according to molecular profiles is critical for individualized therapy at diagnosis, but adoption of molecular classification into clinical practice remains challenging due to the complexity and costs associated with sequencing. We aimed to develop a simple and affordable molecular technique to classify EC into four TCGA subgroups using our high-depth targeted sequencing panel. <b>Methods:</b> Women with newly diagnosed EC were prospectively recruited from three cancer centers in Ontario, Canada, between 2015 and 2018. Tumors were reflexively assessed for mismatch repair (MMR) protein expression by immunohistochemistry (IHC). Using our custom-designed panel, 181 formalin-fixed paraffin-embedded (FFPE) EC samples were sequenced. Our panel was able to identify somatic mutations, copy number variants, insertions and deletions, copy neutral loss of heterozygosity, structural rearrangements, and promoter methylation from a single aliquot of DNA. Variants were analyzed for pathogenicity, and clinical-pathologic information was collected through medical records. <b>Results:</b> Of 181, 86 (48%) were classified into microsatellite instability-high/hypermutated cohort (MSI-h or MMRd), of which 62 (72%) harbored <i>MLH1</i> promoter methylation, and 24 (27%) had pathogenic variants in MMR genes. Concordance with clinical IHC and methylation testing results was high at 99%. Of cases with a single classifier, three (1.6%; <i>n</i>=181) had pathogenic <i>POLE</i> exonuclease domain mutation (<i>POLE</i>mut), 15 (8%) had a pathogenic p53 variant (p53mut), and 61 (34%) had no specific molecular profile subtype (NSMP). Sixteen (9%; <i>n</i>=181) had more than one molecular classifying feature, with eight (4%) having MMRd-p53mut, six (3%) with MMRd-<i>POLE</i>mut, one (0.5%) with MMRd-<i>POLE</i>mut-p53mut, and one (0.5%) with <i>POLE</i>mut-p53mut. Overall, there were 11 (6%) with <i>POLE</i> mutations (three single and nine multiple classifiers). Survival outcomes of MMRd, <i>POLEmut,</i> p53mut, and NSMP groups stratified similarly to TCGA data, but when the MMRd group was further subclassified according to the mechanism of MMR loss, the MLH1 promoter methylated group did worse than those with somatic MMR pathogenic variants (5-year OS: 0.72 [0.57-0.92] vs 1.00 [1.00-1.00]; p=0.013 and RFS: 0.70 [0.58-0.85] vs 0.92 [0.82-1.00]; p=0.032). In contrast, those with somatic MMR pathogenic variants had similar outcomes to the <i>POLE</i>mut subtype (p=0.396) (Figure 1). <b>Conclusions:</b> Our NGS panel can classify EC into four TCGA subgroups through a simplified process and can be implemented reflexively in clinical practice. The higher rate of multiple classifiers (9%) in this study warrants further investigation. The difference in survival between MLH1 methylated group within MMRd cohort suggests that further subclassification may be required for accurate prognostication.

Rad5 and Its Human Homologs, HLTF and SHPRH, Are Novel Interactors of Mismatch Repair.

DNA mismatch repair (MMR) repairs replication errors, and MMR defects play a role in both inherited cancer predisposition syndromes and in sporadic cancers. MMR also recognizes mispairs caused by environmental and chemotherapeutic agents; however, in these cases mispair recognition leads to apoptosis and not repair. Although mutation avoidance by MMR is fairly well understood, MMR-associated proteins are still being identified. We performed a bioinformatic analysis that implicated Saccharomyces cerevisiae Rad5 as a candidate for interacting with the MMR proteins Msh2 and Mlh1. Rad5 is a DNA helicase and E3 ubiquitin ligase involved in post-replicative repair and damage tolerance. We confirmed both interactions and found that the Mlh1 interaction is mediated by a conserved Mlh1-interacting motif (MIP box). Despite this, we did not find a clear role for Rad5 in the canonical MMR mutation avoidance pathway. The interaction of Rad5 with Msh2 and Mlh1 is conserved in humans, although each of the Rad5 human homologs, HLTF and SHPRH, shared only one of the interactions: HLTF interacts with MSH2, and SHPRH interacts with MLH1. Moreover, depletion of SHPRH, but not HLTF, results in a mild increase in resistance to alkylating agents although not as strong as loss of MMR, suggesting gene duplication led to specialization of the MMR-protein associated roles of the human Rad5 homologs. These results provide insights into how MMR accessory factors involved in the MMR-dependent apoptotic response interact with the core MMR machinery and have important health implications into how human cells respond to environmental toxins, tumor development, and treatment choices of tumors with defects in Rad5 homologs.

Abstract 1516: The role of protein tyrosine kinases in DNA mismatch repair

Abstract The DNA mismatch repair (MMR) pathway and its regulation are necessary for genomic stability. Cells deficient in MMR exhibit increased expansion and contraction of short repeat sequences in the genome, termed microsatellite instability (MSI). Loss of MMR also results in resistance to certain classes of DNA damaging chemotherapeutics. The standard treatment option for most patients includes chemotherapy, which has toxic side effects. Immunotherapy has recently been utilized as a treatment for various cancers. MSI-high cancers respond better to immunotherapy treatment compared to microsatellite stable (MSS) cancers. This increased response is believed to be due to the higher mutation frequencies associated with the MSI-high tumors, which lead to greater neoantigen production. The primary proteins required for MMR are known, however regulation of the pathway is not well understood. The MLH1 protein is critical for MMR and is required for the excision licensing of the daughter DNA strand. Loss or mutation of MLH1 leads to defective MMR, increased mutation frequency, and MSI. Recent studies by our laboratory and others have shown that treatment with a tyrosine kinase inhibitor, Imatinib, leads to decreased MLH1 protein expression, and resistance to MMR-dependent apoptosis. These observations suggest a link between Imatinib-sensitive protein tyrosine kinases (PTKs) and the regulation of MMR. We show that MLH1 protein reduction is not occurring at the mRNA level and is thought to be post-transcriptional. We have identified the Imatinib sensitive target most likely responsible for the MLH1 reduction phenotype. We show that the target protein and MLH1 have a physical interaction that likely prevents degradation targeting of MLH1 by the Hsp70 chaperone. A detailed understanding of MMR regulation by PTKs would significantly advance the knowledge in the field of MMR and have important implications for immunotherapy strategies. Citation Format: Hannah Daniels, Breanna Knicely, Eva M. Goellner. The role of protein tyrosine kinases in DNA mismatch repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1516.

Relationship between tumor infiltrating lymphocytes and clinicopathological features in Peruvian gastric cancer.

e16080 Background: Role of tumor-infiltrating lymphocytes (TIL) over clinicopathological features including Mismatch Repair (MMR), HER2 status, Epstein Barr Virus (EBV) and Helicobacter pylori (HP) infection is not well understood in gastric cancer (GC). Methods: TIL level and HP infection was also evaluated in H&amp;E from 503 Peruvian GC patients who underwent surgery. Density of CD3+ T cells, CD8+ cytotoxic T cells, CD163 M2 macrophages, as well as MMR and HER2 status were determined by immunohistochemistry. HP and EBV infection was also detected by qPCR in a 234 cases subset. Results: Median age was 62 years and 50.1% were female, most tumors were grade 3 (59.1%), intestinal subtype (44.8%) and stage III (59.8%). MMR loss was found in 29.4% and HER2+ in 4.7%. HP+ was detected by H&amp;E in 55.7% and by qPCR in 63.7%. EBV+ by qPCR was found in 20.5% and co-infection along with HP in 9.4% (22/234). Stage (p &lt; 0.001), intestinal histology (IH) (p = 0.019), grade (p = 0.006) and lymphovascular invasion (p &lt; 0.001) were associated with longer survival. Median TIL was higher in invasive border (IB) and stromal (ST) than intratumoral (IT) compartment but there were significantly correlated (p &lt; 0.001). High IT TIL was associated with absence of HP H&amp;E (p = 0.009). High ST TIL was associated with older age (p &lt; 0.001), IH (p &lt; 0.001), grade 1 (p = 0.009), lower stage (p = 002), MMR loss (p = 0.019) and EBV+ (p = 0.001). Density of CD3 and CD8 were significantly correlated in IT and ST compartments (p &lt; 0.001). High CD3 density in IT compartment was associated with grade 3 (p = 0.001) and HP- (p = 0.02), and in ST was associated with IH (p = 0.005), grade 1- 2 (p = 0.002) and MMR loss (p = 0.04). High IT CD8 was associated with HER2- (p = 0.016), HP- (p = 0.014) and EBV+ (p = 0.012). High ST CD8/CD3 ratio was associated with diffuse histology (p = 0.034), grade 3 (p = 0.013), more advanced stage (p = 0.022) and recurrence (p = 0.014). High ratio of IT CD8/CD3 was associated with MMR loss (p = 0.007). High ST TIL was associated with longer DFS (p = 0.007). Lower ratio of CD8/CD3 was associated with longer OS (p = 0.024) and DFS (p = 0.02). Conclusions: TIL levels and infiltrating immune subpopulations differs by clinicopathological features in GC including MMR loss, HER2 expression EBV and HP infection. ST CD8/CD3 was associated with recurrence and shorter DFS.

Prevalence of mismatch repair mutations in colorectal carcinoma patients in Mangalore

Colorectal cancer (CRC) is highly prevalent throughout the world and represents the 3rd most common cancer in men and the 2nd in women worldwide. Microsatellite instability (MSI) is a term used to denote a hypermutable phenotype caused by the loss of DNA mismatch repair (MMR) activity, and is a phenomenon now linked to the pathways of colorectal carcinogenesis. Compounding its importance is its integral association with Lynch syndrome, the most common cause for CRCs in young individuals. In the present study, we aimed to analyse the proportion of patients with risk of microsatellite instability by checking for loss of immunostaining for mismatch repair (MMR) proteins.From January 2016 to December 2016 and May 2017 to October 2017, 40 consecutive newly diagnosed cases of colorectal cancer were included in the study. The expression of MMR proteins in the tumour tissue using IHC for MSH2, MSH6, MLH1 and PMS2 was studied.Among the 40 cases, 3 (7.5%) demonstrated loss of MMR proteins and 37 (92.5%) cases had intact nuclear expression. Out of the three cases with MMR loss, one showed concurrent loss of MLH1 and PMS2, the second showed concurrent loss of MSH2 and MSH6 and the third showed an isolated loss of MSH6.Colorectal carcinomas showing MMR mutations are seen in the Mangalorean population. However, the incidence in our study was relatively low compared to most other studies, probably due to a variation in ethnicity.

Novel Imidazotetrazine Evades Known Resistance Mechanisms and Is Effective against Temozolomide-Resistant Brain Cancer in Cell Culture

Glioblastoma (GBM) is the most lethal primary brain tumor. Currently, frontline treatment for primary GBM includes the DNA-methylating drug temozolomide (TMZ, of the imidazotetrazine class), while the optimal treatment for recurrent GBM remains under investigation. Despite its widespread use, a majority of GBM patients do not respond to TMZ therapy; expression of the O6-methylguanine DNA methyltransferase (MGMT) enzyme and loss of mismatch repair (MMR) function as the principal clinical modes of resistance to TMZ. Here, we describe a novel imidazotetrazine designed to evade resistance by MGMT while retaining suitable hydrolytic stability, allowing for effective prodrug activation and biodistribution. This dual-substituted compound, called CPZ, exhibits activity against cancer cells irrespective of MGMT expression and MMR status. CPZ has greater blood-brain barrier penetrance and comparable hematological toxicity relative to TMZ, while also matching its maximum tolerated dose in mice when dosed once-per-day over five days. The activity of CPZ is independent of the two principal mechanisms suppressing the effectiveness of TMZ, making it a promising new candidate for the treatment of GBM, especially those that are TMZ-resistant.

HER2 Overexpression and Amplification in Uterine Carcinosarcomas With Serous Morphology.

Uterine carcinosarcoma (UCS) is an aggressive malignancy with few treatment options. A recent clinical trial has shown an increase in progression-free survival in patients with human epidermal growth factor receptor 2 (HER2)-positive serous endometrial carcinomas treated with anti-HER2-targeted therapies. Few studies have evaluated HER2 expression/amplification in UCS. Similar to serous endometrial carcinoma, the majority of UCS have TP53 mutations and a serous epithelial component, suggesting that UCS may show similar rates of HER2 positivity and therapeutic response. Therefore, we evaluated HER2 expression/amplification in a cohort of UCS over a 5-year period. HER2 immunohistochemistry (IHC) and chromogenic in situ hybridization were performed on tissue microarray and whole tissue sections and scored according to the most recent clinical trial recommendations. Three of 48 UCS (6%) had strong (3+) HER2 IHC expression, and 3 cases (6%) were equivocal (2+). Seven cases (15%) had HER2 amplification by chromogenic in situ hybridization, including all 3 with overexpression and 2 that were equivocal by IHC. Mismatch repair (MMR) protein, p53, and programmed cell death-ligand 1 (PD-L1) expression status was obtained from prior whole section analyses. All HER2-positive cases had a serous morphology and aberrant p53 expression. Only minimal PD-L1 expression was seen in the HER2-positive cases, and none had MMR loss. A subset of UCS with serous morphology have overexpression and/or amplification of HER2, which may predict response to HER2-targeted therapies. HER2-positive UCS may be less susceptible to immune checkpoint inhibition as they uncommonly show MMR deficiency and/or strong PD-L1 expression. Thus, HER2-targeted therapies could be of clinical utility in a subset of UCS without other adjuvant treatment options.

Loss of MMR and TGFBR2 Increases the Susceptibility to Microbiota-Dependent Inflammation-Associated Colon Cancer.

Background and AimsMutations in DNA mismatch repair (MMR) genes are causative in Lynch syndrome and a significant proportion of sporadic colorectal cancers (CRCs). MMR-deficient (dMMR) CRCs display increased mutation rates, with mutations frequently accumulating at short repetitive DNA sequences throughout the genome (microsatellite instability). The TGFBR2 gene is one of the most frequently mutated genes in dMMR CRCs. Therefore, we generated an animal model to study how the loss of both TGFBR2 signaling impacts dMMR-driven intestinal tumorigenesis in vivo and explore the impact of the gut microbiota.MethodsWe generated VCMsh2/Tgfbr2 mice in which Msh2loxP and Tgfbr2loxP alleles are inactivated by Villin-Cre recombinase in the intestinal epithelium. VCMsh2/Tgfbr2 mice were analyzed for their rate of intestinal cancer development and for the mutational spectra and gene expression profiles of tumors. In addition, we assessed the impact of chemically induced chronic inflammation and gut microbiota composition on colorectal tumorigenesis.ResultsVCMsh2/Tgfbr2 mice developed small intestinal adenocarcinomas and CRCs with histopathological features highly similar to CRCs in Lynch syndrome patients. The CRCs in VCMsh2/Tgfbr2 mice were associated with the presence of colitis and displayed genetic and histological features that resembled inflammation-associated CRCs in human patients. The development of CRCs in VCMsh2/Tgfbr2 mice was strongly modulated by the gut microbiota composition, which in turn was impacted by the TGFBR2 status of the tumors.ConclusionsOur results demonstrate a synergistic interaction between MMR and TGFBR2 inactivation in inflammation-associated colon tumorigenesis and highlight the crucial impact of the gut microbiota on modulating the incidence of inflammation-associated CRCs.

Identification of Lynch Syndrome Carriers among Patients with Small Bowel Adenocarcinoma

Simple SummarySmall bowel adenocarcinoma (SBA) is associated with Lynch syndrome (LS). This is the first study to evaluate the identification of LS patients based on mismatch repair deficiency (MMRd) tumor among SBA. The authors found a 21.3% prevalence of MMRd tumors and a 10.1% prevalence of LS. A germline mutation was identified in 60% of patients with a MMRd tumor. This data suggests that universal tumor MMR testing among SBA patients should be implemented for the identification of LS.Background: Small bowel adenocarcinoma (SBA) is a rare disease which can be associated with Lynch syndrome (LS). LS tumors are characterized by the presence of microsatellite instability (MSI) and/or the loss of mismatch repair (MMR) protein expression. In SBA, the frequency of MMR deficient (MMRd) tumors varies from 5% to 35%. This study aims to describe the prevalence of LS carriers among patients with MMRd small bowel adenocarcinomas. Methods: A multicenter retrospective study with identification and MMR testing of all consecutive SBA between 2004 and 2020 in a multicenter Spanish study. Demographical data, tumor characteristics, follow-up and survival information were collected. Germline testing was driven by identification of MMRd tumors. Results: A total of 94 individuals diagnosed with SBA were recruited. We observed 20 (21.3%) MMRd tumors. In 9/15 (60%) patients with MMRd tumors, a pathogenic variant was identified (three MLH1, four MSH2, one MSH6 and one PMS2). Accordingly, the prevalence of LS among all SBA cases was 10.1%. Conclusions: More than one-fifth of SBA display MMRd and in more than a half is due to LS. Our data supports the implementation of universal MMR tumor testing among SBA for the identification of LS families.

Microsatellite Instability Assessment by Immunohistochemistry in Acute Myeloid Leukemia: A Reappraisal and Review of the Literature

Microsatellite instability (MSI) is caused by defects in DNA mismatch repair (MMR) components. Inactivation of any MMR gene(s), including hMLH1, hMSH2, hMSH6, and hPMS2, can result in MSI. Immunohistochemistry (IHC) is a sensitive and specific screening tool for MSI that can detect loss of expression of one or more MMR components. Of the four MMR markers, hMLH1 and hMSH2 are considered most informative of MSI status. There has been renewed interest in MSI status in view of its favorable association with response to immune checkpoint inhibitors in some cancers. MMR expression patterns in acute myeloid leukemia (AML) have not been evaluated systematically. We used clinically-validated IHC assays to assess the expression of hMLH1, hMSH2, hMSH6, and/or hPMS2 in formalin-fixed paraffin-embedded tissue sections of bone marrow core biopsies from patients diagnosed with AML. Mutation profiling was performed using next-generation sequencing to assess for mutations in MMR genes. The study group included 236 patients with AML, including a cohort treated on a clinical trial of azacitidine and nivolumab (NCT02397720). In addition, hMSH6, and/or hPMS2 expression was assessed in 99 AML patients with diploid karyotype. All patients, except two, had retained expression of all MMR markers assessed: One patient from the azacytidine+nivolumab group had zonal patchy loss of staining of hMLH1 and, to a lesser extent, a similar staining pattern of hMSH2; and one patient from the AML with diploid karyotype group had loss of hMSH2 but retained expression of hMLH1, hMSH6 and hPMS2. In addition, a retrospective analysis on a separate cohort of 139 patients with primary AML, on which next generation sequencing profiling was performed, identified 14 cases with alterations in MMR genes. MMR loss is a rare event in AML, thus does not appear to underlie response patterns to anti-PD1 therapy.

PD-L1 expression, tumor-infiltrating lymphocytes, mismatch repair deficiency, EGFR alteration and HPV infection in sinonasal squamous cell carcinoma

The antitumor efficacies of immune checkpoint inhibitors (ICIs) and the usefulness of potential predictive markers such as programmed death-ligand 1 (PD-L1) expression, density of tumor-infiltrating lymphocytes (TILs) and microsatellite instability (MSI) in sinonasal squamous cell carcinoma (SNSCC) have not been fully elucidated. We retrospectively analyzed 131 SNSCCs with immunohistochemistry for PD-L1 expression, TIL subpopulations and loss of mismatch repair (MMR) proteins as a surrogate for MSI-high. We also comprehensively evaluated the mutual relationships among these immuno-markers, high-risk human papillomavirus (HPV) infection, epidermal growth factor receptor (EGFR) gene status, and KRAS mutation. PD-L1 expression (tumor proportion score ≥ 1%) was detected in 60 (45.8%) SNSCC cases and was significantly associated with worse overall survival (OS) (p = 0.0240). High density of cluster of differentiation 8 (CD8)-positive TILs was significantly associated with better progression-free survival (PFS) (p = 0.0368), and high density of forkhead box protein P3-positive TILs was significantly associated with better PFS and OS (p = 0.0007 and 0.0143, respectively). With respect to the combination of CD8 + TIL and PD-L1 expression, the high-CD8/PD-L1-negative group showed the most favorable prognosis, whereas the low-CD8/PD-L1-positive group showed the worst prognosis. MMR loss was detected in 3 (2.3%) of the 131 cases. HPV infection (6.1%), EGFR mutation (14.5%), EGFR copy number gain (26%), and MMR loss were essentially mutually exclusive; patients in these molecular groups showed significant differences in prognosis but not in the degree of PD-L1 expression or TILs. Among the nine ICI-treated patients, three (33.3%) were responders, and the EGFR-wild type cases (n = 7) showed better clinical responses to an ICI compared to the EGFR-mutant cases (n = 2). Among the patients with residual/recurrent EGFR-wild type tumors (n = 43), ICI treatment significantly improved OS (p = 0.0281). The results suggest that the evaluation of immuno-markers and molecular subclassification may be helpful for prognostic prediction and selecting an individualized therapeutic strategy for patients with SNSCC.

A biomarker study in Peruvian males with breast cancer.

BACKGROUNDBreast cancer (BC) frequency in males is extremely low and tumor features vary from its female counterpart. Breast cancer clinical and pathological features differ by race in women. Tumor infiltrating lymphocyte (TIL) levels, mismatch repair (MMR) protein loss, androgen receptor (AR) expression, and PIK3CA gene mutations are predictive biomarkers of response to biological therapy in female BC. There is limited information about clinical and pathological features as well as predictive biomarkers in males of non-Caucasian races with BC. AIMTo investigate clinicopathological features and biomarkers of BC tumors in males and their prognostic value in Peruvian population. METHODSThis study looked at a single-institution series of 54 Peruvian males with invasive BC who were diagnosed from Jan 2004 to June 2018. Standard pathological features, TIL levels, MMR proteins, AR immunohistochemistry staining, and PIK3CA gene mutations were prospectively evaluated in cases with available paraffin material. Percentage of AR and estrogen receptor (ER) positive cells was additionally calculated by software after slide scanning. Statistical analyses included association tests, intraclass correlation test and Kaplan Meier overall survival curves.RESULTSThe median age was 63 years and most cases were ER-positive (85.7%), HER2 negative (87.2%), Luminal-A phenotype (60%) and clinical stage II (41.5%) among our male breast tumors. Median TIL was 10% and higher levels tended to be associated with Luminal-B phenotype and higher grade. AR-positive was found in 85.3% and was correlated with ER (intraclass index of 0.835, P < 0.001). Loss of MMR proteins was found in 15.4% and PIK3CA mutation (H1047R) in 14.3% (belonged to the Luminal-A phenotype). Loss of MMR proteins was associated with AR-negative (P = 0.018) but not with ER (P = 0.43) or TIL (P = 0.84). Early stages (P < 0.001) and lower grade (P = 0.006) were associated with longer overall survival. ER status, phenotype, AR status, TIL level, MMR protein loss nor PIK3CA mutation was not associated with survival (P > 0.05).CONCLUSIONMale BC is usually ER and AR positive, and Luminal-A. MMR loss and PIK3CA mutations are infrequent. Stage and grade predicted overall survival in our South American country population.

Ethanol-induced formation of colorectal tumours and precursors in a mouse model of Lynch syndrome.

Lynch syndrome (LS) confers inherited cancer predisposition due to germline mutations in a DNA mismatch repair (MMR) gene, e.g. MSH2. MMR is a repair pathway for removal of base mismatches and insertion/deletion loops caused by endogenous and exogenous factors. Loss of MMR through somatic alteration of the wild‐type allele in LS results in defective MMR (dMMR). Lifestyle/environmental factors can modify colorectal cancer risk in sporadic and LS patients. Ethanol and its metabolite acetaldehyde are classified as group one carcinogens, and acetaldehyde causes a range of DNA lesions. However, DNA repair pathways responsible for correcting most of such DNA lesions remain uncharacterised. We hypothesised that MMR plays a role in protecting colorectal epithelium from ethanol/acetaldehyde‐induced DNA damage. Here, an LS mouse model (intestinal epithelial conditional‐knockout for Msh2) was used to determine if there is a gene–environment interaction between dMMR and ethanol/acetaldehyde that accelerates colorectal tumourigenesis in LS. Mice underwent either long‐term ethanol treatment or water treatment. Most ethanol‐treated mice demonstrated colonic hyperproliferation and adenoma formation (with some invasive adenocarcinomas) within 6 months (15/23, 65%), compared with one colonic tumour after 15 months in water‐treated mice (1/23, 4%) (p < 0.0001, Fisher's exact test). A significantly greater number of dMMR colonic crypt foci precursors were observed in ethanol‐treated compared with water‐treated mice (p = 0.0029, Student's t‐test). Moreover, increased plasma acetaldehyde levels were detected in ethanol‐treated compared with water‐treated mice (p = 0.0019, Mann–Whitney U‐test), along with significantly increased DNA damage response in the colonic epithelium. Long‐term ethanol treatment was associated with significantly increased colonic epithelial proliferation and markedly reduced apoptosis in dMMR adenomas, consistent with enhanced survival of aberrant dMMR relative to MMR‐proficient colonic epithelium. In conclusion, there is strong evidence for a gene–environment interaction between dMMR and acetaldehyde, causing acceleration of dMMR‐driven colonic tumour formation in this LS model, indicating that advice to limit alcohol consumption should be considered for LS patients. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.