Background: Studies evaluating sex differences in colorectal cancer (CRC) tumor microenvironment are limited, and no previous study has focused on rectal cancer patients’ constitutive immune surveillance mechanisms. The authors aimed to assess gender-related differences in the immune microenvironment of rectal cancer patients. Methods: A systematic review and meta-analysis were conducted up to 31 May 2021, including studies focusing on gender-related differences in the CRC tumor microenvironment. Data on the mutational profile of rectal cancer were extracted from the Cancer Genome Atlas (TCGA). A subanalysis of the two IMMUNOREACT trials (NCT04915326 and NCT04917263) was performed, aiming to detect gender-related differences in the immune microenvironment of the healthy mucosa in patients with early (IMMUNOREACT 1 cohort) and locally advanced rectal cancer following neoadjuvant therapy (IMMUNOREACT 2 cohort). In the retrospective IMMUNOREACT 1 cohort (therapy naive), the authors enrolled 442 patients (177 female and 265 male), while in the retrospective IMMUNOREACT 2 cohort (patients who had neoadjuvant therapy), we enrolled 264 patients (80 female and 184 male). In the prospective IMMUNOREACT 1 cohort (therapy naive), the authors enrolled 72 patients (26 female and 46 male), while in the prospective IMMUNOREACT 2 cohort (patients who had neoadjuvant therapy), the authors enrolled 105 patients (42 female and 63 male). Results: Seven studies reported PD-L1 expression in the CRC microenvironment, but no significant difference could be identified between the sexes. In the TGCA series, mutations of SYNE1 and RYR2 were significantly more frequent in male patients with rectal cancer. In the IMMUNOREACT 1 cohort, male patients had a higher expression of epithelial cells expressing HLA class I, while female patients had a higher number of activated CD4+Th1 cells. Female patients in the IMMUNOREACT 2 cohort showed a higher infiltration of epithelial cells expressing CD86 and activated cytotoxic T cells (P=0.01). Conclusions: Male patients have more frequent oncogene mutations associated with a lower expression of T-cell activation genes. In the healthy mucosa of female patients, more Th1 cells and cytotoxic T cells suggest a potentially better immune response to the tumor. Sex should be considered when defining the treatment strategy for rectal cancer patients or designing prognostic scores.

No data is available on the molecular background of the extra-nodal extension (ENE) of lymph node metastasis (LN) in colorectal cancer (CRC). A series of 22 ENE-positive CRCs was considered and three samples per case were selected (the primary CRC, an ENE-negative and an ENE-positive metastatic LN). Samples (n = 66) were analysed by immunohistochemistry for PD-L1, CD4, CD8, CD68 and CD80. Fifteen out of twenty-two cases were further profiled through a hotspot multigene mutational custom panel, including 164 hotspot regions of AKT1, APC, BRAF, CTNNB1, KIT, KRAS, NRAS, PDGFRA, PIK3CA, PTEN and TP53 genes. A significantly higher percentage of CD4-, CD8- and CD68-positive cells was observed at the invasive front of both CRCs and in ENE in contrast with what observed at the core of both CRCs and their matched nodal metastases. ENE was also characterized by a significantly higher number of CD80-positive cells. No significant difference was observed in PD-L1 distribution among the different specimens. Fourteen out of 15 CRCs (93%) showed at least a driver mutation. The most frequently mutated gene was TP53 (n = 8 tumors), followed by APC (n = 6), BRAF (n = 4), KRAS, NRAS and PIK3CA (n = 2). In 11 out of 15 CRCs (73%) the mutational profiling of the primary tumor was consistent with what obtained from the two matched LNs. A heterogeneous intratumor immune-microenvironment has been observed in ENE-positive CRCs, which are characterized by an increased leukocytic infiltration at the ENE invasive front.

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/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.