BackgroundCD3 +  and CD8 + T-cell infiltration were reported as positive predictive markers of survival in colorectal cancer (CRC) patients. Here, we demonstrate the prognostic significance of CD4 + and FOXP3 + T-cell densities in CRC.MethodsWe quantified the intratumoural densities of CD3 + , CD8 + , CD4 +  and FOXP3 + T cells by immunohistochemistry and digital pathology in 342 CRC patients who underwent curative resection. Microsatellite instability was also assessed in 322 specimens. Patient demographics, clinicopathological features and survival rates were analysed.ResultsHigh CD3 + , CD4 +  and FOXP3 + T-cell densities were associated with improved relapse-free survival (RFS); high CD8 + , CD4 +  and FOXP3 + T-cell densities were associated with improved disease-specific survival (DSS). Patients with low CD4 + and low FOXP3 + T-cell densities exhibited extremely poor prognoses. T stage, vascular/lymphatic invasion and CD4 + T-cell density were independent prognostic indicators for DSS. The distributions of CD4 + and FOXP3 + T-cell densities were not significantly different between the high microsatellite instability group and other groups, in contrast to those of CD3 + and CD8 + T-cell densities.ConclusionsIntratumoural CD4 + T-cell density and combined CD4 + and FOXP3 + T-cell densities were stronger prognostic indicators than other clinicopathological features. These results may facilitate the establishment of novel prognostic factors and therapeutic strategies for CRC.

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Cetuximab has activity against colorectal cancers. Recent studies demonstrated that cetuximab induces antibody‐dependent cell‐mediated cytotoxicity via immune cells, and a new immune‐related mechanism of inducing immunogenic cell death. This study aimed to evaluate the immune responses induced by cetuximab in tumor microenvironments at liver metastasis sites of metastatic colorectal cancer patients. We assessed immune cell infiltration in the liver metastatic sites of 53 colorectal cancer patients. These patients were divided into three groups according to the treatment before operation: chemotherapy with cetuximab, chemotherapy without cetuximab, and no chemotherapy. The inflammatory cells in the liver metastatic sites were assessed by hematoxylin–eosin staining, focusing on the invasive margin. The overall inflammatory reaction and number of lymphoid cells were assessed with a four‐point scoring system. We then assessed immune cell infiltration (CD3, CD8 and CD56) in 15 liver metastatic sites. Hematoxylin–eosin staining demonstrated more inflammatory cells in the chemotherapy with cetuximab group than in the other groups (P < 0.001). Of note, inflammatory cells were found in intratumoral areas, and the destruction of cancer cell foci was observed in the chemotherapy with cetuximab group. Moreover, a higher infiltration of CD3+ (P = 0.003), CD8+ (P = 0.003) and CD56+ (P = 0.001) cells was observed in the chemotherapy with cetuximab group than in the other groups. These results suggest that cetuximab might have an immune‐enhancing effect. As such, the immune‐related mechanism of action of cetuximab may enhance the efficacy of combination therapy, such as chemotherapy and immunotherapy using therapeutic peptides.

The cancer stroma is important in cancer development, however, whether the aberrant expression of microRNAs (miRNAs) in the cancer stroma is associated with cancer progression remains to be fully elucidated. The aim of the present study was to identify the miRNAs associated with liver metastasis in the cancer stroma of human colorectal cancer (CRC). Using laser capture microdissection, cancer stroma was obtained from the primary lesion of six patients with CRC with liver metastasis (CRCwLM) and six patients with CRC without liver metastasis (CRCwoLM), and miRNA microarray analysis was performed. Candidate miRNA expression status in the stroma was validated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis in 40 CRC cases (wLM, n=20; woLM, n=20), and the association between miRNA expression and clinicopathological factors was assessed in 101 advanced CRC samples. The localization of candidate miRNAs in CRCs was analyzed using in situ hybridization analysis (ISH). The microarray analysis identified six miRNAs with expression differing between the CRCwLM and CRCwoLM cancer stroma. Validation using RT‑qPCR analysis of the stroma showed that the expression levels of miR‑221 and miR‑222 in the cancer stroma were significantly higher in CRCwLM than in CRCwoLM. The RT‑qPCR analysis of 101 CRC samples showed that a high expression level of miR‑221 or miR‑222 in the cancer stroma was associated with liver metastasis, distant metastasis, and shorter overall survival rate of patients with CRC (P<0.05). Increased levels of miR‑221 and miR‑222 were observed in cancer cells and in fibroblasts in the stromal tissue in the ISH analysis. The results suggested that the overexpression of miR‑221 and miR‑222 in the cancer stroma is associated with the metastatic activity and malignant potential in patients with CRC.

Many clinical trials of peptide vaccines have been conducted. However, these vaccines have provided clinical benefits in only a small fraction of patients. The purpose of the present study was to explore microRNAs (miRNAs) as novel predictive biomarkers for the efficacy of vaccine treatment against colorectal cancer. First, we carried out microarray analysis of pretreatment cancer tissues in a phase I study, in which peptide vaccines alone were given. Candidate miRNAs were selected by comparison of the better prognosis group with the poorer prognosis group. Next, we conducted microarray analysis of cancer tissues in a phase II study, in which peptide vaccines combined with chemotherapy were given. Candidate miRNAs were further selected by a similar comparison of prognosis. Subsequently, we carried out reverse‐transcription PCR analysis of phase II cases, separating cancer tissues into cancer cells and stromal tissue using laser capture microdissection. Treatment effect in relation to overall survival (OS) and miRNA expression was analyzed. Three miRNA predictors were negatively associated with OS: miR‐125b‐1 in cancer cells (P = 0.040), and miR‐378a in both cancer cells (P = 0.009) and stromal cells (P < 0.001). Multivariate analysis showed that expression of miR‐378a in stromal cells was the best among the three predictors (HR, 2.730; 95% CI, 1.027–7.585; P = 0.044). In conclusion, miR‐125b‐1 and miR‐378a expression might be considered as novel biomarkers to predict the efficacy of vaccine treatment against colorectal cancer.