Identifying mismatch repair-deficient colon cancer: near-perfect concordance between immunohistochemistry and microsatellite instability testing in a large, population-based series Aims: Establishing the mismatch repair (MMR) status of colorectal cancers is important to enable the detection of underlying Lynch syndrome and inform prognosis and therapy. Current testing typically involves either polymerase chain reaction (PCR)-based microsatellite instability (MSI) testing or MMR protein immunohistochemistry (IHC). The aim of this study was to compare these two approaches in a large, population-based cohort of stage 2 and 3 colon cancer cases in Northern Ireland. Methods and results: The study used the Promega pentaplex assay to determine MSI status and a four-antibody MMR IHC panel. IHC was applied to tumour tissue microarrays with triplicate tumour sampling, and assessed manually. Of 593 cases with available MSI and MMR IHC results, 136 (22.9%) were MSI-high (MSI-H) and 135 (22.8%) showed abnormal MMR IHC. Concordance was extremely high, with 97.1% of MSI-H cases showing abnormal MMR IHC, and 97.8% of cases with abnormal IHC showing MSI-H status. Under-representation of tumour epithelial cells in samples from heavily inflamed tumours resulted in misclassification of several cases with abnormal MMR IHC as microsatellite-stable. MMR IHC revealed rare cases with unusual patterns of MMR protein expression, unusual combinations of expression loss, or secondary clonal loss of expression, as further illustrated by repeat immunostaining on whole tissue sections. Conclusions: MSI PCR testing and MMR IHC can be considered to be equally proficient tests for establishing MMR/MSI status, when there is awareness of the potential pitfalls of either method. The choice of methodology may depend on available services and expertise.

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Radioresistance remains a significant challenge in treating pancreatic ductal adenocarcinoma (PDAC), contributing to the poor survival rates of this cancer. MicroRNAs (miRs) are small non-coding RNA molecules that may play an essential role in regulating radioresistance by altering the levels of oxidative stress. In this study, we investigated the role and potential mechanisms linking miR-31 to PDAC radioresistance. A pCMV-miR vector containing a miR-31 mimic was stably expressed into a miR-31-deficient PDAC cell line, BxPC-3. Additionally, a pmiRZip lentivector suppressing miR-31 was stably expressed in a miR-31 abundant PDAC cell line, Panc-1. Clonogenic assays were conducted to explore the role of miR-31 manipulation on radiosensitivity. Fluorometric ROS assays were performed to quantify ROS levels. The expression of potential miR-31 targets was measured by Western blot analysis. It was found that the manipulation of miR-31 altered the radiosensitivity in PDAC cells by regulating oxidative stress. Using online bioinformatics tools, we identified the 3′UTR of GPx8 as a predicted target of miR-31. Our study demonstrates, for the first time, that manipulating miR-31 alters GPx8 expression, regulating ROS detoxification and promoting either a radioresistant or radiosensitive phenotype. MiR-31 may represent a promising therapeutic target for altering radiosensitivity in PDAC cells.

Chemotherapy upregulates immune checkpoint (IC) expression on the surface of tumour cells and IC-intrinsic signalling confers a survival advantage against chemotherapy in several cancer-types including oesophageal adenocarcinoma (OAC). However, the signalling pathways mediating chemotherapy-induced IC upregulation and the mechanisms employed by ICs to protect OAC cells against chemotherapy remain unknown. Longitudinal profiling revealed that FLOT-induced IC upregulation on OE33 OAC cells was sustained for up to 3 weeks post-treatment, returning to baseline upon complete tumour cell recovery. Pro-survival MEK signalling mediated FLOT-induced upregulation of PD-L1, TIM-3, LAG-3 and A2aR on OAC cells promoting a more immune-resistant phenotype. Single agent PD-1, PD-L1 and A2aR blockade decreased OAC cell viability, proliferation and mediated apoptosis. Mechanistic insights demonstrated that blockade of the PD-1 axis decreased stem-like marker ALDH and expression of DNA repair genes. Importantly, combining single agent PD-1, PD-L1 and A2aR blockade with FLOT enhanced cytotoxicity in OAC cells. These findings reveal novel mechanistic insights into the immune-independent functions of IC-intrinsic signalling in OAC cells with important clinical implications for boosting the efficacy of the first-line FLOT chemotherapy regimen in OAC in combination with ICB, to not only boost anti-tumour immunity but also to suppress IC-mediated promotion of key hallmarks of cancer that drive tumour progression.

Aim Use of immune checkpoint blockade to enhance T cell-mediated immunity within the hostile tumour microenvironment (TME) is an attractive approach in oesophageal adenocarcinoma (OAC). This study explored the effects of the hostile TME, including nutrient deprivation and hypoxia, on immune checkpoint (IC) expression and T cell phenotypes, and the potential use of nivolumab to enhance T cell function under such conditions. Methods and Results ICs were upregulated on stromal immune cells within the tumour including PD-L2, CTLA-4 and TIGIT. OAC patient-derived PBMCs co-cultured with OE33 OAC cells upregulated LAG-3 and downregulated the co-stimulatory marker CD27 on T cells, highlighting the direct immunosuppressive effects of tumour cells on T cells. Hypoxia and nutrient deprivation altered the secretome of OAC patient-derived PBMCs, which induced upregulation of PD-L1 and PD-L2 on OE33 OAC cells thus enhancing an immune-resistant phenotype. Importantly, culturing OAC patient-derived PBMCs under dual hypoxia and glucose deprivation, reflective of the conditions within the hostile TME, upregulated an array of ICs on the surface of T cells including PD-1, CTLA-4, A2aR, PD-L1 and PD-L2 and decreased expression of IFN-γ by T cells. Addition of nivolumab under these hostile conditions decreased the production of pro-tumorigenic cytokine IL-10. Conclusion Collectively, these findings highlight the immunosuppressive crosstalk between tumour cells and T cells within the OAC TME. The ability of nivolumab to suppress pro-tumorigenic T cell phenotypes within the hostile TME supports a rationale for the use of immune checkpoint blockade to promote anti-tumour immunity in OAC. Graphical abstract Study schematic: (A) IC expression profiles were assessed on CD45+ cells in peripheral whole blood and infiltrating tumour tissue from OAC patients in the treatment-naïve setting. (B) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then co-cultured for 48 h with OE33 cells. T cell phenotypes were then assessed by flow cytometry. (C) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then further cultured under conditions of nutrient deprivation or hypoxia for 48 h and T cell phenotypes were then assessed by flow cytometry. Key findings: (A) TIGIT, CTLA-4 and PD-L2 were upregulated on CD45+ immune cells and CTLA-4 expression on CD45+ cells correlated with a subsequent decreased response to neoadjuvant regimen. (B) Following a 48 h co-culture with OE33 cells, T cells upregulated LAG-3 and decreased CD27 co-stimulatory marker. (C) Nutrient deprivation and hypoxia upregulated a range of ICs on T cells and decreased IFN-γ production by T cells. Nivolumab decreased IL-10 production by T cells under nutrient deprivation-hypoxic conditions.