BACKGROUND/OBJECTIVES:Mucinous adenocarcinoma (MAC) is a rare and clinically problematic subtype of rectal cancer, tending to present at an advanced stage and to respond poorly to neoadjuvant therapy. The consistently worse prognosis than that of not-otherwise-specified adenocarcinoma (NOS-AC) is not fully understood, potentially owing to intrinsically more aggressive biology or specific immune evasion mechanisms. We used the IMMUNOREACT multicentre cohort, with external validation in TCGA, to investigate the clinical and immunological features of rectal MAC in detail. METHODS:Two hundred patients with rectal adenocarcinoma (16 MAC, 184 NOS-AC) from the IMMUNOREACT 1 (NCT04915326) and IMMUNOREACT 2 (NCT04917263) prospective cohorts were included. To account for the imbalance in baseline characteristics, propensity score matching (PSM) was performed on age, sex, neoadjuvant treatment and TNM stage. The immune microenvironment was characterised using immunohistochemistry (CD3, CD4, CD8, CD8β, Tbet, FoxP3, PD-L1, MSH6, PMS2, CD80), flow cytometry and NanoString PanCancer IO 360™ transcriptomics of adjacent healthy mucosa. Findings were externally validated against TCGA rectal and colon adenocarcinoma datasets. RESULTS:MAC presented at significantly more advanced stage than NOS-AC across all TNM parameters: higher T stage (p = 0.006), N stage (p < 0.001), M stage (p = 0.039) and overall TNM stage (p < 0.001). In the unmatched cohort, MAC was associated with worse overall survival (HR 2.53; 95% CI 1.03-6.23; p = 0.043) and disease-free survival (HR 2.86; 95% CI 1.25-6.55; p = 0.013), but both differences became non-significant after PSM. MAC patients had higher haemoglobin after adjusting for confounders (mean difference [MD] 1.26 g/dL, 95% CI 0.30-2.31, p = 0.012), consistent with a hypothesis of reduced chronic rectal bleeding as a possible mechanism for late presentation. Transcriptomically, MAC showed suppression of HLA class II antigen presentation genes (HLA-DQA1, HLA-DQB1, HLA-DRB1) and myeloid activation genes (S100A8/A9/A12) in adjacent healthy mucosa. Loss of MMR proteins MSH6 and PMS2 in histologically normal mucosa was significantly more frequent in MAC. These findings were replicated in the TCGA cohort, which also showed lower tumour mutational burden and a distinct mucin-associated transcriptomic profile in MAC. CONCLUSIONS:The worse outcomes of rectal MAC appear to be driven largely by late-stage presentation, possibly owing to later diagnosis. MAC nonetheless carries a distinct immune phenotype, detectable even in histologically normal surrounding mucosa, that likely contributes to its treatment resistance. These observations provide a basis for developing histotype-specific approaches to both early detection and treatment in this uncommon but clinically challenging tumour subtype.

Introduction: Most rectal cancers are microsatellite-stable (MSS) and derive limited benefit from immune checkpoint inhibition. We assessed whether familial colorectal cancer aggregation defines a distinct biological context within MSS rectal cancer.Materials and Methods: We performed a prespecified analysis of two prospective multicentre cohorts conducted between 2018 and 2024. Patients with MSS rectal adenocarcinoma were classified as familial (FH⁺) or sporadic (FH⁻), with a predefined subgroup of patients with an affected first-degree relative (FDR⁺). Known hereditary colorectal cancer syndromes were excluded. Immune profiling, transcriptomic analysis and targeted sequencing were performed on tumour-adjacent histologically normal rectal mucosa and, when appropriate, tumour tissue. Analyses were stratified by neoadjuvant treatment status.Results: Among 374 patients, 92 were FH⁺ and 65 were FDR⁺. In NAT-naïve patients, FDR⁺ cases showed higher epithelial CD80⁺ cell density than FH⁻ cases. After neoadjuvant therapy, FH⁺ patients had increased activated CD8⁺CD28⁺ T cells and reduced epithelial HLA-ABC expression, whereas FDR⁺ cases showed lower CD3⁺ T-cell density. Transcriptomic analysis indicated a quiescent mucosal phenotype in FH⁺ patients, with reduced DNA repair, proliferative, metabolic, angiogenic, and immune pathway activity. Post-NAT FH⁺ and FDR⁺ patients showed higher mutational ratios. In FH⁻, but not FH⁺, higher mutational ratio was associated with improved disease-free survival and immune activation.Discussion: Familial aggregation may define a biologically distinct MSS rectal cancer subgroup, characterised by altered epithelial–immune coordination and dissociation between therapy-induced genomic stress and immune surveillance.​

Background: Anastomotic leaks (ALs) remain a critical complication after rectal cancer surgery. Emerging evidence suggests that local immune dysregulation may play a key role in anastomotic healing. We investigated the immune microenvironment of histologically normal, tumor-adjacent rectal mucosa-a tumor-conditioned field-as a potential substrate for AL predisposition. Methods: IMMUNOREACT 4 is a sub-analysis of the IMMUNOREACT project (clinicaltrials.gov NCT04915326 and NCT04915326), a multicenter translational study evaluating immune features of histologically normal, tumor-adjacent rectal mucosa of patients undergoing colorectal anastomosis. A prospective cohort (n = 121) was analyzed using flow cytometry, in addition to a retrospective cohort (n = 262) using immunohistochemistry. Immune markers of epithelial activation and lymphocyte subsets were compared between patients with and without postoperative ALs. Exploratory predictive models combining immune and clinical variables were developed and evaluated using discrimination, calibration and decision curve analyses. Results: At flow cytometry, the CK+HLAabc+ MFI (AUC 0.66, 95% CI 0.52-0.80), CD8+CD38+ cell rate (AUC 0.65, 95% CI 0.52-0.78) and CD3+CTLA4+ cell rate (AUC 0.65, 95% CI 0.51-0.80) showed moderate predictive potential for ALs. In immunohistochemistry, CD3+ (AUC 0.57, 95% CI 0.54-0.60), CD8+ (AUC 0.57, 95% CI 0.52-0.62), CD8β+ (AUC 0.59, 95% CI 0.53-0.65) and Tbet+ (AUC 0.60, 95% CI 0.56-0.64) showed some predictive ability for ALs. The model including CD8β+, the BMI, neutrophile/lymphocyte ratio and tumor location had an AUC of 0.67 (95% CI 0.62-0.72). Conclusions: Immune activation within histologically normal, tumor-adjacent rectal mucosa-characterized by epithelial HLA upregulation and cytotoxic or Th1 T cell infiltration-is associated with postoperative ALs. Although predictive accuracy is limited, these findings support the concept that a tumor-conditioned immune microenvironment may predispose patients to impaired anastomotic healing. Integration of mucosal immune profiling with clinical variables represents a promising exploratory approach that warrants further prospective validation.

Background: Transanal excision of rectal cancer can be considered the definitive surgical treatment if the depth spread is T1 or lower, and the lesion is completely included within the resection margin. This study aims to analyze the immune microenvironment in healthy rectal mucosa as a possible predictor of tumor infiltration depth, lateral tumor spread, and recurrence of rectal cancer after transanal local excision. Methods: This study is a subanalysis of data from the IMMUNOREACT 1 and 2 trials (NCT04915326 and NCT04917263, respectively) including all the patients who underwent transanal excision of rectal cancer. This multicentric study collected healthy mucosa surrounding the neoplasms of patients with rectal cancer. A panel of immune markers was investigated at immunohistochemistry: CD3, CD4, CD8, CD8(3, Tbet, FoxP3, PD-L1, MSH6, and PMS2 and CD80. Flow cytometry determined the proportion of epithelial cells expressing CD80, CD86, CD40, HLA ABC or HLA DR and the proportion of activated CD8+ T cells, CD4+ Th1 cells, and Treg. Results: Receiver operating characteristic curve analysis for predicting deep tumor spread showed an area under the curve of 0.70 (95% confidence interval: 0.60-0.80) for CD25+FoxP3+ cell rate and 0.74 (95% confidence interval: 0.53-0.92) for CK+CD86+ cell rate. Receiver operating characteristic curve analysis for predicting lateral tumor spread showed an area under the curve of 0.82 (95% confidence interval: 0.61-0.99) for CD8+CD38+ MFI, 0.96(95% confidence interval: 0.85-0.99) for CD8(3 infiltration, and 0.97 (95% confidence interval: 0.87-0.99) for CK+HLAabc+ cell rate. Receiver operating characteristic curve analysis for predicting recurrence showed an area under the curve of 0.93 (95% confidence interval: 0.76-0.99) for CD8+CD38+ MFI and 0.94 (95% confidence interval: 0.78-0.99) for CD8+CD28+ MFI. Low CD8+CD38+ MFI and low CD8+CD28+ MFI were associated with shorter disease-free survival (P = .025 and P = .021, respectively). Conclusion: Our study showed that the association between the high proportion of epithelial cells acting as presenting cells and deep or lateral tumor spread may be explained by the presence of a greater tumor load at the site. Moreover, it showed that weak activation of CD8+ T cells within the rectal mucosa is associated with lateral tumor spread and eventually a higher recurrence rate. The mucosal level of CD8(3 infiltration detected at immunohistochemistry might be tested as a marker of lateral tumor spread and potentially translated into clinical practice. (c) 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

BackgroundLong-term daily use of aspirin reduces incidence and mortality due to colorectal cancer (CRC). This study aimed to analyze the effect of aspirin on the tumor microenvironment, systemic immunity, and on the healthy mucosa surrounding cancer.MethodsPatients with a diagnosis of CRC operated on from 2015 to 2019 were retrospectively analyzed (METACCRE cohort). Expression of mRNA of immune surveillance-related genes (PD-L1, CD80, CD86, HLA I, and HLA II) in CRC primary cells treated with aspirin were extracted from Gene Expression Omnibus-deposited public database (GSE76583). The experiment was replicated in cell lines. The mucosal immune microenvironment of a subgroup of patients participating in the IMMUNOREACT1 (ClinicalTrials.gov NCT04915326) project was analyzed with immunohistochemistry and flow cytometry.ResultsIn the METACCRE Cohort, 12% of 238 patients analyzed were aspirin users. Nodal metastasis was significantly less frequent (p = .008) and tumor-infiltrating lymphocyte infiltration was higher (p = .02) among aspirin users. In the CRC primary cells and selected cell lines, CD80 mRNA expression was increased following aspirin treatment (p = .001). In the healthy mucosa surrounding rectal cancer, the ratio of CD8/CD3 and epithelial cells expressing CD80 was higher in aspirin users (p = .027 and p = .034, respectively).ConclusionsThese data suggested that regular aspirin use may have an active role in enhancing immunosurveillance against CRC. In patients with colorectal cancer, nodal metastasis was significantly less frequent, and tumor-infiltrating lymphocytes were higher among aspirin users. In the colorectal cancer primary cells and selected cell lines, CD80 mRNA expression was increased following aspirin treatment. In the healthy mucosa surrounding rectal cancer, the ratio of CD8/CD3 and epithelial cells expressing CD80 was higher in aspirin users.

BACKGROUND: The diagnosis of celiac disease (CD) is still challenging and tests that show an activation of the immune system against gluten are required. IgA antiendomysial antibodies detection in the supernatant of intestinal biopsies by immunofluorescence technique (AEA-biopsy) is a promising diagnostic tool. The aim of the present study was to evaluate the diagnostic accuracy of AEA-biopsy in a pediatric population with suspected CD.METHODS: All children who underwent upper gastrointestinal endoscopy at the Unit of Pediatrics of Treviso Hospital were enrolled and divided into 4 groups: classical CD, CD excluded, potential CD and control group. For each patient, serum autoantibodies and histological evaluation were determined. Two additional biopsy samples were taken to test for presence of AEA. RESULTS: A total of 92 patients were enrolled. All the classical CD cases (38) had a positive AEA-biopsy. In the CD excluded group (10 in total) AEA-biopsy was negative in all patients except 1. Among potential CD patients (which were 14), AEA-biopsy was negative in 4. In the control group (30 patients) AEA-biopsy was negative in all patients except 1. The sensitivity and specificity of AEA-biopsy were 100% and 96% respectively.CONCLUSIONS: AEA-biopsy has an excellent diagnostic accuracy in a routine clinical setting.

Lynch syndrome is rarely associated with rectal cancer (RC) and thus, metachronous RC has been scarcely investigated. This study aimed to analyze the mucosal immune microenvironment in sporadic and metachronous RC. We analyzed the mucosal immune microenvironment in the 25 metachronous RCs present in the IMMUNOREACT 1 and 2 multicentre observational studies (624 patients). A panel of immune markers was retrospectively investigated at immunohistochemistry: CD3, CD4, CD8, CD8b, Tbet, FoxP3, PD-L1, MSH6, and PMS2 and CD80. Single-cell suspensions were subjected to flow-cytometry to determine the proportion of epithelial cells (pan-cytokeratin) acting as antigen-presenting cells (expressing CD80, CD86, HLA-ABC) and the proportion of activated CD8 + T cells (CD8 + positive for CD28, CD38), inhibitory T cells (CD3 + CTLA-4+) of activated CD4 + T helper cells (CD4 + CD25+) and activated T regulatory cells (CD4 + CD25 + FoxP3+). No mismatch repair gene deficiencies were observed in the patients. The previous history of colorectal adenoma was significantly more frequent in metachronous RC. In healthy epithelial cells, HLA-ABC expression was significantly higher in patients with metachronous RC. In therapy-na & iuml;ve metachronous RC patients, a significantly lower level of circulating lymphocytes and CD3 + T-cell infiltration in the healthy mucosa surrounding the RC was observed compared to patients with non-metachronous cancer. Our study supports the hypothesis that metachronous RC can occur in a cancerization field in patients with weak systemic and local immune systems. The peculiar site of RC makes the mismatch-repair genes deficiency in metachronous cancer onset less relevant.

BACKGROUND:The DNA mismatch repair (MMR) system is a highly preserved protein complex recognizing short insertions, short deletions, and single base mismatches during DNA replication and recombination. MMR protein status is identified using immunohistochemistry. Deficit in one or more MMR proteins, configuring deficient MMR status (dMMR), leads to frameshift mutations particularly clustered in microsatellite repeats. Thus, microsatellite instability (MSI) is the epiphenomenon of dMMR. In colorectal cancer (CRC), MMR/MSI status is a biomarker with prognostic and predictive value of resistance to 5-fluorouracil and response to immune checkpoint inhibitor therapy.SUMMARY:In this Review, we describe the challenges the practicing pathologist may face in relation to the assessment of MMR/MSI status and any open issues which still need to be addressed, focusing on pre-analytic issues, pitfalls in the interpretation, and technical aspects of the different assays.KEY MESSAGES:The current methods of detecting dMMR/MSI status have been optimized for CRCs, and whether these techniques can be applied to all tumor and specimen types is still not fully understood. Following the Food and Drug Administration (FDA), tissue/site agnostic drug approval of pembrolizumab for advanced/metastatic MSI tumors, MMR/MSI status in gastrointestinal tract is a common request from the oncologist. In this setting, several issues still need to be addressed, including criteria for sample adequacy.