Differences in the immunosurveillance pattern associated with DNA mismatch repair status between right-sided and left-sided colorectal cancer.

Tumor location and immunity play important roles in the progression of colorectal cancer (CRC). This study aimed to investigate the differences in the immunosurveillance pattern between right- and left-sided CRC and analyze their association with clinicopathologic features, including mismatch repair (MMR) status. We included surgically resected stage II/III CRC cases and evaluated the immunohistochemical findings of HLA class I, HLA class II, programmed cell death-ligand 1 (PD-L1), PD-1, CTLA-4, CD3, CD4, CD8, TIA-1, T-bet, GATA3, RORγT, Foxp3, and CD163. A total of 117 patients were included in the analyses; of these, 30 and 87 had right- and left-sided cancer, respectively. Tumor immunity varied according to the tumor location in the overall cohort. Analysis of the tumors excluding those with DNA mismatch repair (MMR) deficiency also revealed that tumor immunity differed according to the tumor location. In right-sided colon cancer (CC), high expression of Foxp3 (P = .0055) and TIA-1 (P = .0396) were associated with significantly better disease-free survival (DFS). High CD8 (P = .0808) and CD3 (P = .0863) expression tended to have better DFS. Furthermore, in left-sided CRC, only high PD-L1 expression in the stroma (P = .0426) was associated with better DFS. In multivariate analysis, high Foxp3 expression in right-sided CC was an independent prognostic factor for DFS (hazard ratio, 7.6445; 95% confidence interval, 1.2091-150.35; P = .0284). In conclusion, the immunosurveillance pattern differs between right- and left-sided CRC, even after adjusting for MMR deficiency.

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

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

Genomics and emerging biomarkers for immunotherapy of colorectal cancer.

Colorectal cancer (CRC) is a common and lethal disease with a high therapeutic need. For most patients with metastatic CRC, chemotherapy is the only viable option. Currently, immunotherapy is restricted to the particular genetic subgroup of mismatch-repair deficient (MMRd)/microsatellite instable (MSI) CRC. Anti-PD1 therapy was recently FDA-approved as a second-line treatment in this subgroup. However, in a metastatic setting, these MMRd/MSI tumors are vastly outnumbered by mismatch-repair proficient (MMRp)/microsatellite stable (MSS) tumors. These MMRp/MSS tumors do not meaningfully respond to any traditional immunotherapy approach including checkpoint blockade, adoptive cell transfer and vaccination. This resistance to immunotherapy is due to a complex tumor microenvironment that counteracts antitumor immunity through a combination of poorly antigenic tumor cells and an immunosuppressive tumor microenvironment. To find ways of overcoming immunotherapy resistance in the majority of CRC patients, it is necessary to analyze the immunological makeup in an in-depth and personalized way and in the context of their tumor genetic makeup. Flexible, biomarker-guided early-phase immunotherapy trials are needed to optimize this workflow. In this review, we detail key mechanisms for immune evasion and emerging immune biomarkers for personalized immunotherapy in CRC. Also, we present a template for biomarker-guided clinical trials that are needed to move new immunotherapy approaches closer to clinical application.

Mismatch Repair Status of Gastric Cancer and Its Association with the Local and Systemic Immune Response.

BACKGROUND: Microsatellite instability (MSI)-high (MSI-H) colorectal cancer is known to be associated with increased tumor-infiltrating lymphocytes (TILs), elevated host systemic immune response, and a favorable prognosis. In gastric cancer, however, MSI status has rarely been evaluated in the context of TILs and systemic immune response. MATERIALS AND METHODS: We evaluated data for 345 patients with gastric cancer who underwent gastrectomy with MSI typing. The numbers of TILs were counted after immunohistochemical staining with anti-CD3, CD4, CD8, forkhead box P3 (Foxp3), and granzyme B to quantify the subsets of TILs. To evaluate the systemic immune response, the differential white blood cell count and prognostic nutritional index (PNI) were obtained. RESULTS: Of the 345 patients, 57 demonstrated MSI-H tumors and 288 demonstrated non-MSI-H tumors. MSI-H tumors carried significantly higher densities of CD8+ T cells, Foxp3+ T cells, and granzyme B+ T cells and a higher ratio of Foxp3/CD4 and granzyme B/CD8. The prognostic impact of TILs differed between patients with MSI-H tumors and those with non-MSI-H tumors. The TIL subsets were not found to be significant prognostic factors for recurrence-free survival (RFS) or overall survival (OS) in the MSI-H tumor group. In the non-MSI-H tumor group, multivariate analysis showed that stage, PNI, and CD4+ T cells were independent prognostic factors for RFS, and stage, PNI, and the Foxp3/CD4 ratio were independent prognostic factors for OS. CONCLUSIONS: The association between systemic/local immune response and prognosis differed according to MSI status. Different tumor characteristics and prognoses according to MSI status could be associated with the immunogenicity caused by microsatellite instability and subsequent host immune response. IMPLICATIONS FOR PRACTICE: This study demonstrates that the density of each subset of tumor-infiltrating lymphocytes (TILs) differed between microsatellite instability (MSI)-high and non-MSI-high tumors. Moreover, the prognostic effect of the preoperative systemic immune response status and TILs differed between the MSI-high (MSI-H) and non-MSI-H tumor groups. The present study may help to identify the mechanisms of cancer progression and develop treatment strategies for MSI-high gastric cancer.

Comparison of immunological characteristics between paired mismatch repair-proficient and -deficient colorectal cancer patients.

BACKGROUND: Currently, mismatch repair-deficient (dMMR) status is a promising candidate for targeted immune checkpoint inhibition therapy in colorectal cancer (CRC) patients, however, the potential immunological mechanism has not yet been well clarified and some other predictors need to be excavated as well. METHODS: We collected 330 CRC patients by the match of mismatch repair-proficient (167) and dMMR (163), explored the relationship between MMR status and some important immune molecules including MHC class I, CD3, CD4, CD8, CD56, programmed death-1 and programmed death ligand-1, and investigated the risk factors for dMMR status as well as low MHC class I expression. The Pearson Chi square test was used for analyzing the associations between clinicopathological and immune characteristics and MMR status, and two categories logistic regression model was used for univariate and multivariate analysis to predict the odds ratio of risk factors for dMMR status and low MHC class I expression. RESULTS: Multivariate logistic regression analysis showed that low MHC class I and CD4 expression and high CD8 expression were significant risk factors for dMMR status [odds ratio (OR) = 24.66, 2.94 and 2.97, respectively; all p < 0.05] and dMMR status was the only risk factor for low MHC class I expression (OR = 15.34; p < 0.001). CONCLUSIONS: High CD8 and low MHC class I expression suggests the contradiction and complexity of immune microenvironment in dMMR CRC patients. Some other immunocytes such as CD56+ cells might also participate in the process of immune checkpoint inhibition, whereas needs further investigations.

Nivolumab in the treatment of microsatellite instability high metastatic colorectal cancer.

Nivolumab is a PD-1 inhibitor approved for the use in treatment of multiple tumor types (such as melanoma, non-small-cell lung cancer, renal cell carcinoma, urothelial cancer and Hodgkin's lymphoma). In July 2017, the US FDA granted accelerated approval of this agent for the treatment of metastatic colorectal cancer patients whose tumor harbors deficient mismatch repair, or microsatellite-instability high and have progressed on conventional chemotherapy. In this review, we will discuss the use, efficacy, and safety of this agent in microsatellite-instability high metastatic colorectal cancer.

Non-canonical uracil processing in DNA gives rise to double-strand breaks and deletions: relevance to class switch recombination.

During class switch recombination (CSR), antigen-stimulated B-cells rearrange their immunoglobulin constant heavy chain (CH) loci to generate antibodies with different effector functions. CSR is initiated by activation-induced deaminase (AID), which converts cytosines in switch (S) regions, repetitive sequences flanking the CH loci, to uracils. Although U/G mispairs arising in this way are generally efficiently repaired to C/Gs by uracil DNA glycosylase (UNG)-initiated base excision repair (BER), uracil processing in S-regions of activated B-cells occasionally gives rise to double strand breaks (DSBs), which trigger CSR. Surprisingly, genetic experiments revealed that CSR is dependent not only on AID and UNG, but also on mismatch repair (MMR). To elucidate the role of MMR in CSR, we studied the processing of uracil-containing DNA substrates in extracts of MMR-proficient and -deficient human cells, as well as in a system reconstituted from recombinant BER and MMR proteins. Here, we show that the interplay of these repair systems gives rise to DSBs in vitro and to genomic deletions and mutations in vivo, particularly in an S-region sequence. Our findings further suggest that MMR affects pathway choice in DSB repair. Given its amenability to manipulation, our system represents a powerful tool for the molecular dissection of CSR.

Interleukin 6 alters localization of hMSH3, leading to DNA mismatch repair defects in colorectal cancer cells.

BACKGROUND & AIMS: Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is the most common DNA mismatch repair defect in colorectal cancers, observed in approximately 60% of specimens. This acquired genotype correlates with metastasis and poor outcomes for patients, and is associated with intra-epithelial inflammation and heterogeneous nuclear levels of the mismatch repair protein hMSH3. Inflammation and accompanying oxidative stress can cause hMSH3 to change its intracellular location, but little is known about the source of oxidative stress in cancer cells. We investigated whether cytokines mediate this process. METHODS: We analyzed levels of interleukin 6 (IL6) and its receptor (IL6R) in human colon and lung cancer cell lines by flow cytometry and enzyme-linked immunosorbent assay; proteins were localized by immunofluorescence and immunoblot analyses. IL6 signaling was blocked with antibodies against IL6, soluble glycoprotein 130 Fc fragments, and the signal transducers and activators of transcription 3 inhibitor NSC74859; a constitutively active form of STAT3 was expressed in colon and lung cancer cell lines to replicate IL6R signaling. EMAST was detected by DNA fragment analysis. Immunohistochemistry was used to examine levels of IL6 in 20 colorectal tumor and adjacent nontumor tissues. RESULTS: Incubation of colon and lung cancer cell lines with IL6, but not other cytokines, caused hMSH3, but no other mismatch repair proteins, to move from the nucleus to the cytosol after generation of oxidative stress; inhibition of IL6 signaling prevented this shift. Expression of constitutively active STAT3 also caused hMSH3 to translocate from the nucleus to the cytoplasm in cancer cell lines. Incubation of cells with IL6 led to tetranucleotide frameshifts, the signature for EMAST. EMAST-positive colorectal tumors had significantly higher levels of IL6 than EMAST-negative tumors. CONCLUSIONS: IL6 signaling disrupts the nuclear localization of hMSH3 and DNA repair, leading to EMAST in cancer cell lines. Inflammatory cytokines might therefore promote genetic alterations in human cancer cells.

Mismatch repair proteins and AID activity are required for the dominant negative function of C-terminally deleted AID in class switching.

Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. The AID C terminus is required for CSR, but not for S-region DNA double-strand breaks (DSBs) during CSR, and it is not required for SHM. AID lacking the C terminus (ΔAID) is a dominant negative (DN) mutant, because human patients heterozygous for this mutant fail to undergo CSR. In agreement, we show that ΔAID is a DN mutant when expressed in AID-sufficient mouse splenic B cells. To have DN function, ΔAID must have deaminase activity, suggesting that its ability to induce DSBs is important for the DN function. Supporting this hypothesis, Msh2-Msh6 have been shown to contribute to DSB formation in S regions, and we find in this study that Msh2 is required for the DN activity, because ΔAID is not a DN mutant in msh2(-/-) cells. Our results suggest that the DNA DSBs induced by ΔAID are unable to participate in CSR and might interfere with the ability of full-length AID to participate in CSR. We propose that ΔAID is impaired in its ability to recruit nonhomologous end joining repair factors, resulting in accumulation of DSBs that undergo aberrant resection. Supporting this hypothesis, we find that the S-S junctions induced by ΔAID have longer microhomologies than do those induced by full-length AID. In addition, our data suggest that AID binds Sµ regions in vivo as a monomer.

Separation of mutational and transcriptional enhancers in Ig genes.

Secondary Ig gene diversification relies on activation-induced cytidine deaminase (AID) to create U:G mismatches that are subsequently fixed by mutagenic repair pathways. AID activity is focused to Ig loci by cis-regulatory DNA sequences named targeting elements. In this study, we show that in contrast to prevailing thought in the field, the targeting elements in the chicken IGL locus are distinct from classical transcriptional enhancers. These mutational enhancer elements (MEEs) are required over and above transcription to recruit AID-mediated mutagenesis to Ig loci. We identified a small 222-bp fragment in the chicken IGL locus that enhances mutagenesis without boosting transcription, and this sequence represents a key component of an MEE. Lastly, MEEs are evolutionarily conserved among birds, both in sequence and function, and contain several highly conserved sequence modules that are likely involved in recruiting trans-acting targeting factors. We propose that MEEs represent a novel class of cis-regulatory elements for which the function is to control genomic integrity.

Characterization of the immunological microenvironment of tumour buds and its impact on prognosis in mismatch repair-proficient and -deficient colorectal cancers.

AIMS: Tumour budding in colorectal cancer is established as a poor prognostic factor. The inverse correlation of tumour buds with peritumoural lymphocytic inflammation suggests an interaction with specific immune responses. The aims of this study were to characterize the immunological microenvironment of tumour buds and its impact on prognosis in mismatch repair (MMR)-proficient and -deficient colorectal cancers. METHODS AND RESULTS: A total of 297 colorectal cancers were double-immunostained for CK22 plus one of the following: CD138, CD16, CD20, CD21, CD56, CD68, CD8, forkhead box P2 (FoxP3), granzyme B, mast cell tryptase, CD3 or T cell intracellular antigen-1 (TIA)-1. Tumour buds and immune cells within the region of densest budding were evaluated [×40 high-power field (HPF)] simultaneously. In both MMR-proficient and -deficient cancers, CD8(+), FoxP3(+) and CD68(+) cells were observed most frequently (>40 cells/HPF) and were independent prognostic factors. A combined prognostic score of tumour budding and CD8(+), FoxP3(+) and CD68(+) distinctly identified patients with low-, moderate- or high-risk colorectal cancers with 5-year survival rates of 75.2% [confidence interval 95% (CI): 66-83], 56.3% (95% CI: 43-68) and 25.2% (95% CI: 14-38), respectively, in MMR-proficient and -deficient cancers. CONCLUSION: The combined assessment of tumour budding with CD8, FoxP3 and CD68 lymphocytes could represent a basis for a prognostic score similar to the Bloom Richardson grade (BRE) and Gleason scores for breast and prostatic cancers.

Anti-tumor immunity in mismatch repair-deficient colorectal cancers requires type I IFN-driven CCL5 and CXCL10.

Colorectal cancers (CRCs) deficient in DNA mismatch repair (dMMR) contain abundant CD8+ tumor-infiltrating lymphocytes (TILs) responding to the abundant neoantigens from their unstable genomes. Priming of such tumor-targeted TILs first requires recruitment of CD8+ T cells into the tumors, implying that this is an essential prerequisite of successful dMMR anti-tumor immunity. We have discovered that selective recruitment and activation of systemic CD8+ T cells into dMMR CRCs strictly depend on overexpression of CCL5 and CXCL10 due to endogenous activation of cGAS/STING and type I IFN signaling by damaged DNA. TIL infiltration into orthotopic dMMR CRCs is neoantigen-independent and followed by induction of a resident memory-like phenotype key to the anti-tumor response. CCL5 and CXCL10 could be up-regulated by common chemotherapies in all CRCs, indicating that facilitating CD8+ T cell recruitment underlies their efficacy. Induction of CCL5 and CXCL10 thus represents a tractable therapeutic strategy to induce TIL recruitment into CRCs, where local priming can be maximized even in neoantigen-poor CRCs.

Phase II Single-arm Study of Durvalumab and Tremelimumab with Concurrent Radiotherapy in Patients with Mismatch Repair-proficient Metastatic Colorectal Cancer.

PURPOSE: Immune checkpoint inhibition (ICI) alone is not active in mismatch repair-proficient (MMR-P) metastatic colorectal cancer (mCRC), nor does radiotherapy alone result in objective systemic benefit. However, combined radiotherapy plus ICI can induce systemic antitumor immunity in preclinical and clinical models. PATIENTS AND METHODS: In this single-center, phase II study, patients with chemotherapy-refractory MMR-P mCRC received durvalumab 1,500 mg plus tremelimumab 75 mg every 4 weeks plus radiotherapy. The primary endpoint was objective response rate (ORR) in nonirradiated lesions. Treatment and efficacy were correlated with peripheral immune cell profiles. RESULTS: We enrolled 24 patients, and report outcomes after a median follow-up of 21.8 (range: 15.9-26.3) months. The ORR was 8.3% (2 patients) [95% confidence interval (CI), 1.0-27.0]. The median progression-free survival was 1.8 (95% CI, 1.7-1.9) months, median overall survival was 11.4 (95% CI, 10.1-17.4) months. Twenty five percent of patients (n = 6) had treatment-related grade 3-4 adverse events. We observed increased circulating CD8+ T lymphocyte activation, differentiation, and proliferation in patients with objective response. CONCLUSIONS: This combination of radiotherapy plus ICI study did not meet the prespecified endpoint criteria to be considered worthwhile for further study. However, rare instances of systemic immune augmentation and regression in nonirradiated lesions were observed (an abscopal response). Combination durvalumab and tremelimumab plus radiotherapy is feasible in MMR-P mCRC with a manageable safety profile. Further studies of novel immunotherapy combinations, and identification of biomarkers predictive of abscopal response are warranted.

Proteogenomic identification of an immunogenic HLA class I neoantigen in mismatch repair-deficient colorectal cancer tissue.

Although CD8+ T cells recognize neoantigens that arise from somatic mutations in cancer, only a small fraction of nonsynonymous mutations give rise to clinically relevant neoantigens. In this study, HLA class I ligandomes of a panel of human colorectal cancer (CRC) and matched normal tissues were analyzed using mass spectrometry-based proteogenomic analysis. Neoantigen presentation was rare; however, the analysis detected a single neoantigen in a mismatch repair-deficient CRC (dMMR-CRC) tissue sample carrying 3967 nonsynonymous mutations, where abundant tumor-infiltrating lymphocytes (TILs) and inflamed gene expression status were observed in the tumor microenvironment (TME). Using the HLA class I ligandome data and gene expression profiles, a set of nonmutated tumor-associated antigen (TAA) candidates was concomitantly identified. Interestingly, CD8+ TILs predominantly recognized the detected neoantigen over the array of TAA candidates. Neoantigen-reactive CD8+ TILs showed PD-1 positivity and exhibited functional and specific responses. Moreover, T cell receptor (TCR) profiling identified the sequence of the neoantigen-reactive TCR clonotype and showed its expansion in the TME. Transduction of the sequenced TCR conferred neoantigen specificity and cytotoxicity to peripheral blood lymphocytes. The proteogenomic approach revealed the antigenic and reactive T cell landscape in dMMR-CRC, demonstrating the presence of an immunogenic neoantigen and its potential therapeutic applications.

Analysis of Immunological Characteristics and Genomic Alterations in HPV-Positive Oropharyngeal Squamous Cell Carcinoma Based on PD-L1 Expression.

Programmed death-ligand 1 (PD-L1) expression has been approved as an immune checkpoint inhibitor (ICI) response predictive biomarker; however, the clinicopathological and molecular features of HPV-positive oropharyngeal squamous cell carcinoma [HPV(+)OPSCC] based on PD-L1 expression are not well studied. We aimed to characterize clinicopathological, tumor immune microenvironmental, and molecular features of HPV(+)OPSCC with different PD-L1 expression scored by combined positive score (CPS). A total of 112 cases were collected from 2008-2021 and received PD-L1 and CD8 immunohistochemistry (IHC) staining. 71 samples received DNA sequencing out of which 32 samples received RNA sequencing for immune-related gene alterations or expression analysis. The 32 samples were also subjected to analysis of CD20, CD4, CD8, CD68, Foxp3 and P16 by multiplex immunofluorescence (mIF) staining, and the immune markers were evaluated in the tumor body (TB), tumor margin (TM) and normal stroma (NS) regions separately. Our results showed that of 112 HPV(+)OPSCC tumors, high(CPS≥20), intermediate(1≤CPS<20), and low(CPS<1) PD-L1 expression was seen in 29.5%, 43.8% and 26.8% cases respectively. Non-smoking patients and patients with tumors occurring at the tonsils or having rich lymphocytes infiltration had significantly higher PD-L1 expression. Patients with CPS≥20 had significantly higher tumor mutation burden (TMB, p=0.0058), and PD-L1 expression correlated significantly with CD8+ T cells infiltration, which were ample in tumor regions than in NS in mIF. CD20+, CD4+, CD68+, Foxp3+CD4+ cells were demonstrated to infiltrate higher in TM while CD20+ and CD68+ cells were also enriched in NS and TB regions respectively. However, none of them showed correlations with PD-L1 expression. ARID1A, STK11 alterations were enriched in the low PD-L1 group significantly, while anti-viral immune associated APOBEC mutation signature and immune-related genes expression such as XCL1 and IL11 were positively associated with PD-L1 expression (p<0.05). This is a comprehensive investigation revealing immune and molecular features of HPV(+)OPSCC based on PD-L1 expression. Our study suggested that 73.2% of HPV(+)OPSCC patients may benefit from immunotherapy, and high PD-L1 expression reflects immune-active status of HPV(+)OPSCC accompanied by higher immune effect factors such as TMB, CD8+ cytotoxic T cells and immune-related genomic alterations. Our study offers valuable information for understanding the immune features of HPV(+)OPSCC.

Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.

Based on its substrate specificity, activation-induced cytidine deaminase can directly induce C:G mutations in Ig genes. However the origin of A:T mutations, which occur in a similar proportion in germinal center (GC) B cells, is unclear. Genetic evidence suggests that the induction of A:T mutations requires the components of the mismatch repair system and DNA polymerase eta (POLH). We found that fibroblasts and GC B cells expressed similar levels of the mismatch repair components, but nonetheless the fibroblasts failed to generate a significant proportion of A:T mutations in a GFP reporter gene even after POLH overexpression. To investigate whether the ability to generate A:T mutations is dependent on the cellular environment (i.e., GC B cell or fibroblast) or the target gene (i.e., Ig or GFP), we developed a mutation detection system in a human GC-like cell line. We introduced a GFP gene with a premature stop codon into Ramos cells and compared the activation-induced cytidine deaminase-induced mutations in the endogenous V(H) and the transgenic GFP genes. Remarkably, a high proportion of A:T mutations was induced in both genes. Ectopic expression of POLH did not further increase the proportion of A:T mutations but diminished the strand bias of these mutations that is normally observed in V(H) genes. Intriguingly, the total mutation frequency in the GFP gene was consistently one-fifth of that in the V(H) gene. These results demonstrate that the ability to generate A:T mutations is dependent on the GC B cell environment but independent of the mutation frequency and target gene location.

Could Mismatch Repair Status Serve as a Biomarker for Immunotherapy in Endometrial Carcinoma?

AIM: To study whether mismatch repair (MMR) status is related to the expression of programmed cell death-ligand 1 (PD-L1) and CD8 counts in a series of grade 3 endometrial carcinomas. MATERIALS AND METHODS: The expression of MMR protein PD-L1 and CD8+ cell count were evaluated by immunohistochemistry and related to several clinicopathological parameters. RESULTS: Among 105 endometrial carcinomas, 40% were of endometrioid and 60% of non-endometrioid histology. MMR deficiency was observed in 28.6% of cases and was related to endometrioid histology (p<0.001), positive PD-L1 expression (p=0.047) and high CD8+ cell count (p=0.022). When examined by histotype, endometrioid MMR-deficient tumors were related only to PD-L1 expression (p=0.032) but not to high CD8+ cell count (p=0.231), whereas non-endometrioid MMR-deficient carcinomas were not related to either of these markers. MMR deficiency was associated with PD-L1+/CD8high status (p=0.006), whilst MMR proficiency was associated with PD-L1-/CD8low status. In MMR-proficient tumors, high CD8+ cell infiltration alone and combined with PD-L1- status was associated with better progression-free survival (p=0.013 and p=0.04, respectively). CONCLUSION: MMR-deficient high-grade endometrioid tumors might be more likely to benefit from immunotherapy compared to other grade 3 endometrial carcinomas.

A PoleP286R mouse model of endometrial cancer recapitulates high mutational burden and immunotherapy response.

Cancer is instigated by mutator phenotypes, including deficient mismatch repair and p53-associated chromosomal instability. More recently, a distinct class of cancers was identified with unusually high mutational loads due to heterozygous amino acid substitutions (most commonly P286R) in the proofreading domain of DNA polymerase ε, the leading strand replicase encoded by POLE. Immunotherapy has revolutionized cancer treatment, but new model systems are needed to recapitulate high mutational burdens characterizing human cancers and permit study of mechanisms underlying clinical responses. Here, we show that activation of a conditional LSL-PoleP286R allele in endometrium is sufficient to elicit in all animals endometrial cancers closely resembling their human counterparts, including very high mutational burden. Diverse investigations uncovered potentially novel aspects of Pole-driven tumorigenesis, including secondary p53 mutations associated with tetraploidy, and cooperation with defective mismatch repair through inactivation of Msh2. Most significantly, there were robust antitumor immune responses with increased T cell infiltrates, accelerated tumor growth following T cell depletion, and unfailing clinical regression following immune checkpoint therapy. This model predicts that human POLE-driven cancers will prove consistently responsive to immune checkpoint blockade. Furthermore, this is a robust and efficient approach to recapitulate in mice the high mutational burdens and immune responses characterizing human cancers.

Inflammation-Associated Microsatellite Alterations Caused by MSH3 Dysfunction Are Prevalent in Ulcerative Colitis and Increase With Neoplastic Advancement.

OBJECTIVES: Inflammation-associated microsatellite alterations (also known as elevated microsatellite alterations at selected tetranucleotide repeats [EMAST]) result from IL-6-induced nuclear-to-cytosolic displacement of the DNA mismatch repair (MMR) protein MSH3, allowing frameshifts of dinucleotide or longer microsatellites within DNA. MSH3 also engages homologous recombination to repair double-strand breaks (DSBs), making MSH3 deficiency contributory to both EMAST and DSBs. EMAST is observed in cancers, but given its genesis by cytokines, it may be present in non-neoplastic inflammatory conditions. We examined ulcerative colitis (UC), a preneoplastic condition from prolonged inflammatory duration. METHODS: We assessed 70 UC colons without neoplasia, 5 UC specimens with dysplasia, 14 UC-derived colorectal cancers (CRCs), and 19 early-stage sporadic CRCs for microsatellite instability (MSI) via multiplexed polymerase chain reaction capable of simultaneous detection of MSI-H, MSI-L, and EMAST. We evaluated UC specimens for MSH3 expression via immunohistochemistry. RESULTS: UC, UC with dysplasia, and UC-derived CRCs demonstrated dinucleotide or longer microsatellite frameshifts, with UC showing coincident reduction of nuclear MSH3 expression. No UC specimen, with or without neoplasia, demonstrated mononucleotide frameshifts. EMAST frequency was higher in UC-derived CRCs than UC (71.4% vs 31.4%, P = 0.0045) and higher than early-stage sporadic CRCs (66.7% vs 26.3%, P = 0.0426). EMAST frequency was higher with UC duration >8 years compared with ≤8 years (40% vs 16%, P = 0.0459). DISCUSSION: Inflammation-associated microsatellite alterations/EMAST are prevalent in UC and signify genomic mutations in the absence of neoplasia. Duration of disease and advancement to neoplasia increases frequency of EMAST. MSH3 dysfunction is a potential contributory pathway toward neoplasia in UC that could be targeted by therapeutic intervention.

Distinct Immunological Landscapes Characterize Inherited and Sporadic Mismatch Repair Deficient Endometrial Cancer.

Around 30% of endometrial cancers (EC) are mismatch repair (MMR) deficient, mostly as a consequence of mutations acquired during tumorigenesis, but a significant minority is caused by Lynch syndrome (LS). This inherited cancer predisposition syndrome primes an anti-cancer immune response, even in healthy carriers. We sought to explore the intra-tumoral immunological differences between genetically confirmed LS-associated MMR-deficient (MMRd), sporadic MMR-deficient, and MMR-proficient (MMRp) EC. Endometrial tumors from women with known LS were identified (n = 25). Comparator tumors were recruited prospectively and underwent microsatellite instability (MSI) testing, immunohistochemistry (IHC) for MMR expression and MLH1 methylation testing. Those found to have MLH1 hypermethylation formed the sporadic MMR-deficient group (n = 33). Those found to be mismatch repair proficient and microsatellite stable formed the MMR-proficient group (n = 35). A fully automated monoplex IHC panel was performed on sequential formalin-fixed paraffin-embedded tumor sections to identify CD3+, CD8+, CD45RO+, FoxP3+, and PD-1+ immune cells, and PD-L1 expression by tumor/immune cells. Two independent observers quantified immune marker expression at the tumor center and invasive margin. Mean and overall compartmental T-cell counts generated standard (binary: Low/High) and higher resolution (quaternary: 0-25, 25-50, 50-75, 75-100%) immune scores, which were used as explanatory features in neural network, support vector machine, and discriminant predictive modeling. Overall T-cell counts were significantly different between the three cohorts: CD3+ (p = <0.0001), CD8+ (p = <0.0001), CD45RO+ (<0.0001), FoxP3+ (p = <0.0001), and PD1+ (p = <0.0001), with LS-associated MMR-deficient tumors having highest infiltrations. There were significant differences in CD8+ (p = 0.02), CD45RO+ (p = 0.007), and PD-1+ (p = 0.005) T-cell counts at the invasive margin between LS-associated and sporadic MMR-deficient tumors, but not between sporadic MMR-deficient and MMR-proficient tumors. Predictive modeling could accurately determine MMR status based on CD8+ T-cell counts within the tumor center alone. This study shows that LS-associated and sporadic MMR-deficient EC are distinct immunological entities, which has important implications for treatment and prognosis.