Localization and density of tertiary lymphoid structures associate with molecular subtype and clinical outcome in colorectal cancer liver metastases.

BACKGROUND: Tertiary lymphoid structures (TLSs) have been proposed to assess the prognosis of patients with cancer. Here, we investigated the prognostic value and relevant mechanisms of TLSs in colorectal cancer liver metastases (CRCLM). METHODS: 603 patients with CRCLM treated by surgical resection from three cancer centers were included. The TLSs were categorized according to their anatomic subregions and quantified, and a TLS scoring system was established for intratumor region (T score) and peritumor region (P score). Differences in relapse-free survival (RFS) and overall survival (OS) between groups were determined. Multiplex immunohistochemical staining (mIHC) was used to determine the cellular composition of TLSs in 40 CRCLM patients. RESULTS: T score positively correlated with superior prognosis, while P score negatively associated with poor survival (all p<0.05). Meanwhile, T score was positively associated with specific mutation subtype of KRAS. Furthermore, TLSs enrichment gene expression was significantly associated with survival and transcriptomic subtypes of CRCLM. Subsequently, mIHC showed that the densities of Treg cells, M2 macrophages and Tfh cells were significantly higher in intratumor TLSs than in peritumor TLSs (p=0.029, p=0.047 and p=0.041, respectively), and the frequencies of Treg cells and M2 macrophages were positively correlated with P score, while the frequencies of Tfh cells were positively associated with T scores in intratumor TLSs (all p<0.05). Next, based on the distribution and abundance of TLSs, an Immune Score combining T score and P score was established which categorized CRCLM patients into four immune classes with different prognosis (all p<0.05). Among them, patients with higher immune class have more favorable prognoses. The C-index of Immune Class for RFS and OS was higher than Clinical Risk Score statistically. These results were also confirmed by the other two validation cohorts. CONCLUSIONS: The distribution and abundance of TLSs is significantly associated with RFS and OS of CRCLM patients, and a novel immune class was proposed for predicting the prognosis of CRCLM patients.

Programmed death ligand 1 expression in human intrahepatic cholangiocarcinoma and its association with prognosis and CD8+ T-cell immune responses.

BACKGROUND: Agents targeting the programmed death ligand 1 (PD-L1)/programmed death receptor 1 immune checkpoint exhibited promising clinical outcomes in a variety of malignant tumors, including intrahepatic cholangiocarcinoma (ICC). However, the relationship between PD-L1 expression and CD8+ T-cell immune responses is not well defined in ICC. PATIENTS AND METHODS: We investigated PD-L1 expression immunohistochemistry in formalin-fixed, paraffin-embedded tissues from 192 ICC patients undergoing curative resection and correlated our results with the clinicopathologic features and prognosis. We also quantified CD8+ T-cell infiltration in ICC specimens and evaluated the relationship between PD-L1 expression and CD8+ T-cell infiltration. After incubating human ICC cell lines (HCCC9810 and RBE) with interferon (IFN)-γ, we measured the PD-L1 expression of these ICC cells by Western blot and flow cytometry. RESULTS: Only 34 patients (17.7%) showed ≥5% membranous PD-L1 expression on tumor cells, and tumoral PD-L1 overexpression (≥5%) was significantly associated with superior overall survival (P=0.012) and disease-free survival (P=0.018). A significant positive association was found between PD-L1 expression and the presence of CD8+ T-cells. In fresh frozen ICC specimens, IFN-γ was found to be significantly correlated with PD-L1 and CD8A gene expression, as evaluated by reverse transcription-polymerase chain reaction. Moreover, stimulation of the HCCC9810 and RBE cells with recombinant IFN-γ, secreted by CD8+ T-cells rapidly induced PD-L1 upregulation in these cell lines in vitro. CONCLUSION: Tumor PD-L1 overexpression is mainly stimulated by activated CD8+ T-cells pre-existing in the ICC microenvironment, and PD-L1 is a favorable prognostic factor for the patients. These observations suggest that anti-PD-L1/programmed death receptor 1 therapy may benefit ICC patients with tumor cell PD-L1 expression and the presence of CD8+ T-cells.

microRNA-26a induces a mitochondrial apoptosis mediated by p53 through targeting to inhibit Mcl1 in human hepatocellular carcinoma.

AIM: We have previously found that microRNA-26a (miR-26a) is a potential tumor suppressor in hepatocellular carcinoma (HCC). In this study, we further explored the roles of miR-26a in HCC apoptosis. METHODS: miR-26a expression levels were detected in HCC tissues by real-time PCR. Statistical analysis was performed to explore the correlation between miR-26a expression and apoptotic cells and the antiapoptotic protein levels. In vitro assays were performed to investigate the roles of miR-26a in HCC apoptosis. The immunohistochemical staining analysis, Western blot, and luciferase reporter assay were performed to evaluate the relationship between miR-26a and its potential upstream regulating and downstream target genes. The potential mechanism of the combination treatment of interferon-α1b (IFN-α1b) and 5-fluorouracil (5-FU) was explored by in vitro and in vivo assays. RESULTS: miR-26a levels were significantly associated with the number of apoptotic cells and inversely correlated with the protein levels of Bcl-2, Bcl-xL, and Mcl1 in HCC tissues. Furthermore, miR-26a was proved to induce the mitochondrial apoptosis in vitro by directly targeting to inhibit Mcl1 in HCC cells. Moreover, p53 was demonstrated to mediate miR-26a-induced apoptosis, by activating its promoter in HCC. Meanwhile, the combination treatment of IFN-α1b and 5-FU could induce the expression of p53, which then upregulated miR-26a and downregulated Mcl1 levels, and finally promoted the apoptosis of HCC cells through a mitochondrial pathway. CONCLUSION: These findings highlight the important and related molecular mechanism of miR-26a in the regulation of apoptosis and implicate the potential application of combination of IFN-α1b and 5-FU in HCC treatment.