Stratifying ICIs-responsive tumor microenvironment in HCC: from parsing out immune-hypoxic crosstalk to clinically applicable MRI-radiomics models.

BACKGROUND: We aimed to redefine Immune checkpoint inhibitors (ICIs)-responsive "hot" TME and develop a corresponding stratification model to maximize ICIs-efficacy in Hepatocellular Carcinoma (HCC). METHODS: Hypoxic scores were designed, and the relevance to immunotherapy responses were validated in pan-cancers through single cell analysis. Multi-omics analysis using the hypoxic scores and immune infiltrate abundance was performed to redefine the ICIs-responsive TME subtype in HCC patients from TCGA (n = 363) and HCCDB database (n = 228). The immune hypoxic stress index (IHSI) was constructed to stratify the ICIs-responsive TME subtype, with exploring biological mechanism in vitro and in vivo. MRI-radiomics models were built for clinical applicability. RESULTS: The hypoxic scores were lower in the dominant cell-subclusters of responders in pan-cancers. The higher immune infiltrate-normoxic (HIN) subtype was redefined as the ICIs-responsive TME. Stratification of the HIN subtype using IHSI effectively identified ICIs-responders in Melanoma (n = 122) and urological cancer (n = 22). TRAF3IP3, the constituent gene of IHSI, was implicated in ICIs-relevant "immune-hypoxic" crosstalk by stimulating MAVS/IFN-I pathway under normoxic condition. MRI-radiomics models assessing TRAF3IP3 with HIF1A expression (AUC > 0.80) screened ICIs-Responders in HCC cohort (n = 75). CONCLUSION: The hypoxic-immune stratification redefined ICIs-responsive TME and provided MRI-Radiomics models for initial ICIs-responders screening, with IHSI facilitating further identification.

Genomic evolution and the impact of SLIT2 mutation in relapsed intrahepatic cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is aggressive and has high rates of relapse, conferring poor long-term survival after curative resection. Little is known about the genomic evolution that occurs during ICC relapse. APPROACH AND RESULTS: We conducted whole-exome sequencing of 30 paired primary and relapsed tumors from 10 patients with ICC who received curative resection. We sought to identify frequently altered genes, infer tumor subclonal architectures, and track genomic evolution from primary to relapsed tumors. We examined functional effects and the mechanism of action of SLIT2, a gene specifically mutated in relapsed tumors, on tumor growth and metastasis and the tumor microenvironment. Our results indicated that relapsed ICCs were genetically derived from intrahepatic dissemination of primary tumors. However, they acquired additional mutations while maintaining most drivers, such as TP53 and IDH1. Multiregion sequencing suggested polyclonal seeding of ICC dissemination. Four of 10 relapsed ICCs acquired SLIT2 mutations that were not present in the corresponding primary tumors. Validation in an expanded sample revealed SLIT2 mutations in 2.3% (1/44) of primary ICCs and 29.5% (13/44) of relapsed ICCs. Biofunctional investigations revealed that inactivating mutation of SLIT2 resulted in activation of PI3K-Akt signaling in ICC cells, directly enhanced neutrophil chemotaxis, mediated tumor-associated neutrophil infiltration, and contributed to ICC growth and metastasis. CONCLUSIONS: We characterized genomic evolution during ICC relapse and identified SLIT2 as a driver of tumor dissemination and tumor-associated neutrophil infiltration.

Associations among the mutational landscape, immune microenvironment, and prognosis in Chinese patients with hepatocellular carcinoma.

Recent studies suggested that the immune microenvironment and mutational landscape are associated with the response to immune-based therapy in several types of cancer. The roles of those factors in Chinese HCC remain largely unknown. In this study, we obtained 182 FFPE samples of HCC cohort that were previously subjected to NGS (49 WGS, 18 WES, and 115 targeted sequencing). We performed immunohistochemistry to detect CD3, CD4, CD8, CD57, Foxp3, CD68, CD66b, and PD-L1 expression in the samples. We identified diverse associations between the mutational landscape and the immune microenvironment in the HCC samples. High mutational burden and an aristolochic acid-dominated mutational signature were both correlated with elevated tumoral PD-L1 expression and CD3+ T-cell infiltration and high numbers of CD68+ TAMs and CD66b+ TANs. CD4+ and CD8+ T cells exhibited lower infiltration levels in tumors with mutations in AXIN1/CTNNB1 and in tumors with aflatoxin-dominant mutational signatures. Moreover, tumors with TP53 mutations had less CD8+ T-cell infiltration and more Foxp3+ Treg-cell infiltration than those without TP53 mutations. Kaplan-Meier survival analysis revealed that the presence of CD8+, Foxp3+, CD66b+, or CD68+ immune cells; tumoral PD-L1 expression alone; or the presence of CD8+ or Foxp3+ cells combined with TP53 mutation were predictive of recurrence and poor overall survival after curative resection. In conclusion, the association between the mutational landscape and the immune microenvironment warrants further analysis to determine its impact on patient outcomes to guide personalized immune-based therapy for Chinese patients with HCC.

Circular RNA Sequencing Identifies CircASAP1 as a Key Regulator in Hepatocellular Carcinoma Metastasis.

BACKGROUND AND AIMS: There is growing evidence that single-stranded, circular RNA (circRNA) plays a key role in the development of certain cancers, including hepatocellular carcinoma (HCC). It is less clear, however, what role circRNA plays in HCC metastasis. APPROACH AND RESULTS: In this study, through circRNA sequencing, we identified a circRNA: circASAP1 (a circRNA derived from exons 2 and 3 of the ASAP1 gene, hsa_circ_0085616), which is associated with pulmonary metastasis after curative resection in patients with HCC. CircASAP1 was overexpressed in HCC cell lines with high metastatic potential and in metastatic HCCs. In vitro, circASAP1 promoted cell proliferation, colony formation, migration, and invasion, and in vivo, it enhanced tumor growth and pulmonary metastasis. Mechanism studies showed that circASAP1 acts as a competing endogenous RNA for microRNA 326 (miR-326) and microRNA 532-5p (miR-532-5p), both of which are tumor suppressors in HCC. We found that mitogen-activated protein kinase (MAPK) 1 and colony stimulating factor (CSF)-1 were direct common targets for microRNA 326 (miR-326) and microRNA 532-5p (miR-532-5p), which were regulated by circASAP1. CircASAP1 promotes HCC cell proliferation and invasion by regulating miR-326/miR-532-5p-MAPK1 signaling and, furthermore, mediates tumor-associated macrophage infiltration by regulating the miR-326/miR-532-5p-CSF-1 pathway. Clinical HCC samples exhibited a positive correlation between circASAP1 expression and levels of CSF-1, MAPK1, and CD68+ tumor-associated macrophages, all of which were predictive of patient outcomes. CONCLUSION: We identified circASAP1 as a key regulator of HCC metastasis that acts on miR-326/miR-532-5p-MAPK1/CSF-1 signaling and serves as a prognostic predictor in patients with HCC.

HNRNPAB-regulated lncRNA-ELF209 inhibits the malignancy of hepatocellular carcinoma.

Our previous study demonstrated that heterogeneous nuclear ribonucleoprotein AB (HNRNPAB) is a key gene that facilitates metastasis of hepatocellular carcinoma (HCC). However, the molecular mechanisms behind this relationship are not fully understood. In our study, we utilized long-noncoding RNA (lncRNA) microarrays to identify a HNRNPAB-regulated lncRNA named lnc-ELF209. Our findings from chromatin immunoprecipitation assays indicate that HNRNPAB represses lnc-ELF209 transcription by directly binding to its promoter region. We also analyzed clinical samples from HCC patients and cell lines with quantitative real-time polymerase chain reactions, RNA in situ hybridization and immunohistochemistry, and found that there is a negative relationship between HNRNPAB and lnc-ELF209 expression. Up/downregulation assays and rescue assays indicate that lnc-ELF209 inhibits cell migration, invasion and epithelial-mesenchymal transition regulated by HNRNPAB. This suggests a new regulatory mechanism for HNRNPAB-promoted HCC progression. RNA pull-down and LC-MS/MS were used to determine triosephosphate isomerase, heat shock protein 90-beta and vimentin may be involved in the tumor-suppressed function of lnc-ELF209. Furthermore, we found lnc-ELF209 could stabilize TPI protein expression. We also found that lnc-ELF209 overexpression in HCCLM3 cell resulted in a lower rate of lung metastatic, which suggested a less aggressive HCC phenotype. Collectively, these findings offer new insights into the regulatory mechanisms that underlie HNRNPAB cancer-promoting activities and demonstrate that lnc-ELF209 is a HNRNPAB-regulated lncRNA that may play an important role in the inhibition of HCC progression.

A Positive Feedback Loop Between Cancer Stem-Like Cells and Tumor-Associated Neutrophils Controls Hepatocellular Carcinoma Progression.

Tumor-associated neutrophils (TANs) play a crucial role in tumor development and progression in the cancer microenvironment. Despite increased understanding of TAN contributions to hepatocellular carcinoma (HCC) progression and prognosis, the direct interaction between TANs and HCC cells is not fully understood. In this study, we tested the effect of TANs on HCC cells in vitro and in vivo and investigated the mechanism of interaction between them. Our results showed that TANs secreted bone morphogenetic protein 2 and transforming growth factor beta 2 and triggered microRNA 301b-3p (miR-301-3p) expression in HCC cells, subsequently suppressed gene expression of limbic system-associated membrane protein (LSAMP) and CYLD lysine 63 deubiquitinase (CYLD), and increased stem cell characteristics in HCC cells. These TAN-induced HCC stem-like cells were hyperactive in nuclear factor kappa B signaling, secreted higher levels of chemokine (C-X-C motif) ligand 5 (CXCL5), and recruited more TAN infiltration, suggesting a positive feedback loop. In clinical HCC samples, increased TANs correlated with elevated miR-301b-3p, decreased LSAMP and CYLD expression, and increased nuclear p65 accumulation and CXCL5 expression, all of which predicted patient outcome. Conclusion: Our work identified a positive feedback loop governing cancer stem-like cells and TANs in HCC that controls tumor progression and patient outcome.

Peritumoral plasmacytoid dendritic cells predict a poor prognosis for intrahepatic cholangiocarcinoma after curative resection.

BACKGROUND: Plasmacytoid dendritic cells (pDCs) are present in various primary and metastatic human neoplasms; however, their clinical significance in intrahepatic cholangiocarcinoma is not clear. METHODS: To evaluate pDCs' distributions in and around tumors as well as their potential function and predictive value for prognosis in patients undergoing curative resection, we performed immunohistochemistry to examine the expression of pDC marker BDCA2, and CD3, CD4, CD8 and Foxp3 in intratumoral and peritumoral tissues from 359 patients with intrahepatic cholangiocarcinoma and compared with prognostic and clinicopathologic factors. RESULTS: Results showed that patients with high numbers of BDCA2+ pDCs in peritumoral tissues were more likely to have elevated levels of carbohydrate antigen 19-9 and gamma-glutamyl transferase, larger and more tumors, advanced tumor-node-metastasis staging, more vascular/bile duct invasion, and lymphatic metastasis in association with greater chance of recurrence and shorter overall survival. Peritumoral tissues with larger numbers of pDCs also showed increased Foxp3+ regulatory T cell infiltration, both of which were found to be independent factors for predicting time to recurrence and overall survival. By contrast, patient outcomes were not associated with the presence of intratumoral pDCs. CONCLUSIONS: Peritumoral pDC infiltration may indicate an immune tolerogenic peritumor microenvironment and can be used to predict a poor prognosis for patients undergoing curative resection for intrahepatic cholangiocarcinoma.

Prognostic factors influencing outcome in adult liver transplantation using hypernatremic organ donation after brain death.

BACKGROUND: Hypernatremic donors was regarded as the expanded criteria donors in liver transplantation. The study was to investigate the effects of donor hypernatremia on the outcomes of liver transplantation and identify the prognostic factors possibly contributing to the poor outcomes. METHODS: Donor serum sodium levels before procurement were categorized as normal sodium (< 155 mmol/L), moderate high sodium (155-170 mmol/L), and severe high sodium (≥ 170 mmol/L). Furthermore, we subdivided the 142 hypernatremic donors (≥ 155 mmol/L) into two subgroups: subgroup A, the exposure time of liver grafts from hypernatremia to reperfusion was < 36 h; and subgroup B, the exposure time was ≥ 36 h. The outcomes included initial graft function, survival rates of grafts and recipients, graft loss and early events within the first year following liver transplantation. RESULTS: There were no significant differences in the 1-year survival rates of grafts and recipients, 1-year graft loss rates and early events among the normal, moderate high and severe high sodium groups. However, the overall survival rates of grafts and recipients in subgroup A were significantly higher than those in subgroup B. Cox model showed that the exposure time (HR = 1.117; 95% CI: 1.053-1.186; P < 0.001), cold ischemia time (HR = 1.015; 95% CI: 1.006-1.024; P = 0.001) and MELD (HR = 1.061; 95% CI: 1.003-1.121; P = 0.037) were the important prognostic factors contributing to the poor outcomes of recipients with hypernatremic donors. CONCLUSIONS: The level of donor sodium immediately before organ procurement does not have negative effects on the early outcomes following adult liver transplantation. For hypernatremia liver donors, minimization of the exposure time from hypernatremia to reperfusion is critical to prevent graft loss.

Genomic sequencing identifies WNK2 as a driver in hepatocellular carcinoma and a risk factor for early recurrence.

BACKGROUND & AIMS: Early recurrence of hepatocellular carcinoma (HCC) after curative resection is common. However, the association between genetic mechanisms and early HCC recurrence, especially in Chinese patients, remains largely unknown. METHODS: We performed whole-genome sequencing (49 cases), whole-exome sequencing (18 cases), and deep targeted sequencing (115 cases) on 182 primary HCC samples. Focusing on WNK2, we used Sanger sequencing and qPCR to evaluate all the coding exons and copy numbers of that gene in an additional 554 HCC samples. We also explored the functional effect and mechanism of WNK2 on tumor growth and metastasis. RESULTS: We identified 5 genes (WNK2, RUNX1T1, CTNNB1, TSC1, and TP53) harboring somatic mutations that correlated with early tumor recurrence after curative resection in 182 primary HCC samples. Focusing on WNK2, the overall somatic mutation and copy number loss occurred in 5.3% (39/736) and 27.2% (200/736), respectively, of the total 736 HCC samples. Both types of variation were associated with lower WNK2 protein levels, higher rates of early tumor recurrence, and shorter overall survival. Biofunctional investigations revealed a tumor-suppressor role of WNK2: its inactivation led to ERK1/2 signaling activation in HCC cells, tumor-associated macrophage infiltration, and tumor growth and metastasis. CONCLUSIONS: Our results delineate genomic events that characterize Chinese HCCs and identify WNK2 as a driver of early HCC recurrence after curative resection. LAY SUMMARY: We applied next-generation sequencing and conducted an in-depth genomic analysis of hepatocellular carcinomas from a Chinese patient cohort. The results delineate the genomic events that characterize hepatocellular carcinomas in Chinese patients and identify WNK2 as a driver associated with early tumor recurrence after curative resection.

Intratumoral plasmacytoid dendritic cells as a poor prognostic factor for hepatocellular carcinoma following curative resection.

Plasmacytoid dendritic cells (pDCs) are present in various primary and metastatic human neoplasms; however, their clinical significance in hepatocellular carcinoma (HCC) is unclear. In this study, we investigated the distribution, prognostic value, and potential function of pDCs in HCC patients undergoing curative resection. We performed immunohistochemical analyses of whole tumor sections from 224 patients to assess the expression of BDCA2, CD3, CD4, CD8, Foxp3, granzyme B, IL-17, and CD34. The findings were validated using tissue microarrays from another two independent cohorts totaling 841 HCC patients undergoing curative resection. Our results demonstrated that high numbers of BDCA2+ pDCs within tumors correlated with high alpha-fetoprotein levels, greater vascular invasion, advanced tumor-node-metastasis stage, shorter overall survival, and a higher recurrence rate. However, patient outcomes were not associated with pDCs in peritumoral stromal or nontumor tissues. Furthermore, an increase in intratumoral pDCs was associated with increased intratumoral infiltration of Foxp3+ regulatory T cells and IL-17-producing cells and correlated with tumor vascular density. Univariate and multivariate analyses revealed that the presence of intratumoral pDCs alone or in combination with regulatory T and/or IL-17-producing cells was an independent predictor of time to recurrence and overall survival. In conclusion, our study demonstrated that intratumoral infiltration by pDCs is a novel indicator for poor prognosis in patients with HCC, possibly through the induction of an immune tolerogenic and inflammatory tumor microenvironment comprising regulatory T and IL-17-producing cells. An assessment of the combination of these cells represents a superior predictor of patient outcome.

Development and validation of a new tumor-based gene signature predicting prognosis of HBV/HCV-included resected hepatocellular carcinoma patients.

BACKGROUND: Due to the phenotypic and molecular diversity of hepatocellular carcinomas (HCC), it is still a challenge to determine patients' prognosis. We aim to identify new prognostic markers for resected HCC patients. METHODS: 274 patients were retrospectively identified and samples collected from Zhongshan hospital, Fudan University. We analyzed the gene expression patterns of tumors and compared expression patterns with patient survival times. We identified a "9-gene signature" associated with survival by using the coefficient and regression formula of multivariate Cox model. This molecular signature was then validated in three patients cohorts from internal cohort (n = 69), TCGA (n = 369) and GEO dataset (n = 80). RESULTS: We identified 9-gene signature consisting of ZC2HC1A, MARCKSL1, PTGS1, CDKN2B, CLEC10A, PRDX3, PRKCH, MPEG1 and LMO2. The 9-gene signature was used, combined with clinical parameters, to fit a multivariable Cox model to the training cohort (concordance index, ci = 0.85), which was successfully validated (ci = 0.86 for internal cohort; ci = 0.78 for in silico cohort). The signature showed improved performance compared with clinical parameters alone (ci = 0.70). Furthermore, the signature predicted patient prognosis than previous gene signatures more accurately. It was also used to stratify early-stage, HBV or HCV-infected patients into low and high-risk groups, leading to significant differences in survival in training and validation (P < 0.001). CONCLUSIONS: The 9-gene signature, in which four were upregulated (ZC2HC1A, MARCKSL1, PTGS1, CDKN2B) and five (CLEC10A, PRDX3, PRKCH, MPEG1, LMO2) were downregulated in HCC with poor prognosis, stratified HCC patients into low and high risk group significantly in different clinical settings, including receiving adjuvant transarterial chemoembolization and especially in early stage disease. This new signature should be validated in prospective studies to stratify patients in clinical decisions.

Downregulation of MAGE family member H1 enhances hepatocellular carcinoma progression and serves as a biomarker for patient prognosis.

AIM: The MAGE family member H1 (MAGEH1) belongs to melanoma-associated antigen (MAGE) superfamily. The role of MAGEH1 in hepatocellular carcinoma (HCC) is largely undefined. MATERIALS & METHODS: We used quantitative reverse transcription PCR and immunohistochemistry to detect MAGEH1 expression in HCC tissues. CCK-8 assay, wound healing migration assay and Transwell Matrigel invasion assay were used to measure HCC cell proliferation, migration and invasion ability. RESULTS:  MAGEH1 expression was downregulated in HCC tumor tissues compared with adjacent normal liver tissues and in samples from patients with tumor recurrence. MAGEH1 reduced HCC cell proliferation, migration and invasion ability. Low MAGEH1 expression was significantly correlated with poor prognosis in HCC patients. CONCLUSION:  MAGEH1 may serve as a potential biomarker and a new prognostic factor for HCC.

Tumor-Associated Neutrophils Recruit Macrophages and T-Regulatory Cells to Promote Progression of Hepatocellular Carcinoma and Resistance to Sorafenib.

BACKGROUND & AIMS: Neutrophils can either promote or inhibit tumor progression, depending on the tumor microenvironment, via release of cytokines. Neither the factors produced by tumor-associated neutrophils (TANs) nor their effects on tumor progression have been characterized. We investigated the roles of TANs in progression of hepatocellular carcinoma (HCC) using cell lines and immune cells isolated from patients. METHODS: We performed studies with HepG2, PLC/PRF/5, MHCC97H, and HCCLM3 human and Hepa1-6 and H22 mouse HCC cell lines; expression of chemokines and cytokines were knocked down with small hairpin RNAs. Cells were analyzed in chemotaxis assays and as growth as tumors in mice. HCC tissues and peripheral blood were collected from 20 patients undergoing curative resection or 20 healthy individuals (controls) in 2012 at Zhongshan Hospital in China. TANs and peripheral blood neutrophils (PBNs) were isolated and exposed to conditioned media from HCC cell lines; reverse-transcription polymerase chain reaction was used to quantify the expression of cytokines and chemokines. We collected neutrophils from another 60 patients undergoing curative resection for HCC in 2012 to measure the production of C-C motif chemokine ligand 2(CCL2) and CCL17. Patients were followed up until March 15, 2014. For immunohistochemical analyses, we collected HCC tissues and paired, adjacent, nontumor cirrhotic liver tissues from 832 HCC patients undergoing curative resection from 2006 through 2008. All patients were followed up until March 15, 2013. To study the effects of sorafenib, we collected clinical and pathology data from 46 patients who underwent curative resection in 2010. RESULTS: CCL2 and CCL17 were the cytokines most highly expressed by TANs and HCC cell-activated PBNs. Levels of CCL2 and CCL17 messenger RNAs and proteins were significantly higher in TANs than in PBNs, and increased in patients with HCC recurrence. CCL2 and CCL17 messenger RNA and proteins also increased when PBNs were exposed to conditioned media from HCC cell lines. Immunohistochemical analysis of a tissue microarray showed that CCL2+ and CCL17+ cells, which also expressed the neutrophil marker CD66b, were distributed throughout the HCC stroma, but not in tumor cells or the adjacent nontumor liver cells. The number of CCL2+ or CCL17+ TANs correlated with tumor size, microvascular invasion, tumor encapsulation, tumor differentiation, and stage. Patients whose tumors had lower levels of CCL2+ or CCL17+ cells had longer survival times than those with higher numbers of these cells. TAN-conditioned media, as well as recombinant CCL2 and CCL17, increased the migratory activity of the macrophages and T-regulatory (Treg) cells from patients or mice with HCC to a greater extent that PBN-conditioned media. Neutralizing antibodies against CCL2 and CCL17, or their receptors C-C chemokine receptor 2 and C-C chemokine receptor 4, reduced the migratory activities of macrophage and Treg cells. HCC cell lines injected into mice formed larger tumors when they were co-injected with TANs and formed more pulmonary metastases; these tumors were infiltrated by Ly6G+ cells, F4/80+ macrophages, and Foxp3+ Treg cells. In a phosphokinase array of human PBNs, levels of phosphorylated AKT and P38 increased after exposure to conditioned media from all 4 HCC cell types. Pharmacologic inhibitors of AKT and P38 inhibited secretion of CCL2 and CCL17 by these PBNs. In tumor-bearing mice, sorafenib increased the numbers of TANs and levels of CCL2 and CCL17 in tumors. HCC tissues from patients who received sorafenib before surgery contained more TANs than tissues from patients who did not receive sorafenib. In knockdown cells, HCC cell-derived CXCL5 was the strongest effector of neutrophil migration under hypoxic conditions. In mice, the combination of sorafenib and TAN depletion inhibited tumor growth and neovascularization to a greater extent than sorafenib alone. CONCLUSIONS: TANs recruit macrophages and Treg cells to HCCs to promote their growth, progression, and resistance to sorafenib.

miR-28-5p-IL-34-macrophage feedback loop modulates hepatocellular carcinoma metastasis.

UNLABELLED: MicroRNAs (miRNAs) play a critical role in regulation of tumor metastasis. However, the role of these molecules in hepatocellular carcinoma (HCC) has not been fully elucidated. In this study, we employed miRNA-sequencing and identified 22 miRNAs involved in HCC metastasis. One of these, miR-28-5p, was down-regulated in HCCs. This down-regulation correlated with tumor metastasis, recurrence, and poor survival. Biofunctional investigations revealed that miR-28-5p deficiency promoted tumor growth and metastasis in nude mice without altering the in vitro biological characteristics of HCC cells. Through gene expression profiles and bioinformatics analysis, we identified interleukin-34 (IL-34) as a direct target of miR-28-5p, and the effects of miR-28-5p deficiency on HCC growth and metastasis was dependent on IL-34-mediated tumor-associated macrophage (TAM) infiltration. Moreover, we found that TAMs induced by miR-28-5p-IL-34 signaling inhibit miR-28-5p expression on HCC cells by transforming growth factor beta 1, resulting in an miR-28-5p-IL-34-macrophage-positive feedback loop. In clinical HCC samples, miR-28-5p levels were inversely correlated with IL-34 expression and the number of TAMs. Patients with low miR-28-5p expression, high IL-34 levels, and high numbers of TAMs had a poor prognosis with shorter overall survival and time to recurrence. CONCLUSION: A miR-28-5p-IL-34-macrophage feedback loop modulates HCC metastasis and serves as a novel prognostic factor as well as a therapeutic target for HCC.

The elevated preoperative neutrophil-to-lymphocyte ratio predicts poor prognosis in intrahepatic cholangiocarcinoma patients undergoing hepatectomy.

A high preoperative peripheral blood neutrophil-to-lymphocyte ratio (NLR) has been reported to be a predictor of poor survival in patients with various cancers. The aim of this study was to evaluate the predictive significance of the NLR in patients undergoing hepatectomy for intrahepatic cholangiocarcinoma (ICC). From 2005 to 2011, 322 patients who underwent hepatectomy for ICC were enrolled in this retrospective study. Clinicopathological parameters, including NLR, were evaluated to identify predictors of overall and recurrence-free survival after hepatectomy. The best cutoff for NLR was 2.49, and 177 of 322 patients (54.9 %) had an NLR ≥ 2.49. The 5-year survival rate after hepatectomy was 51.1 % in patients with NLR < 2.49 and 24.8 % in those with NLR ≥ 2.49 (P = 0.0001). Univariate analyses revealed that NLR was significantly associated with recurrence-free survival (RFS) and overall survival (OS; both P < 0.05). Multivariable analyses revealed that elevated NLR independently predicted poorer OS (P = 0.003, hazard ratio [HR] = 1.600). In summary, our results indicate that elevated NLR is a promising independent predictor of poor survival after hepatectomy in patients with ICC.

Capn4 contributes to tumour growth and metastasis of hepatocellular carcinoma by activation of the FAK-Src signalling pathways.

Calpain small subunit 1 (Capn4) has been identified as a major gene that promotes metastasis of hepatocellular carcinoma (HCC). However, the mechanism by which Capn4 promotes progression of HCC is not understood. In this study, we found that Capn4 expression was increased in highly metastatic HCC cell lines and in tumour tissue from HCC patients compared to healthy patient tissue. Over-expression of Capn4 in HCC cells enhanced tumour cell growth in vitro and increased invasiveness, tumourigenicity and lung metastasis in vivo. Protein microarray analyses showed that expression of multiple proteins was regulated by Capn4. Interestingly, Capn4 was found to physically associate with FAK and promoted hyperactivity of the FAK-Src signalling pathway via increased phosphorylation of specific tyrosine residues of FAK, Src and p130Cas. Knock-down of Capn4 expression suppressed the malignant behaviour of HCC cells and inhibited the FAK-Src signalling pathway. Furthermore, Capn4-mediated invasion and metastasis of HCC cells required up-regulation of matrix metalloproteinase-2 (MMP2) through activation of this signalling pathway. Our clinical data revealed that Capn4 expression correlated well with the levels of phospho-FAK, and over-expression of both Capn4 and phospho-FAK correlates with the poorest survival outcomes in HCC. In conclusion, our data showed that Capn4 can contribute to HCC growth and metastasis via activation of the FAK-Src signalling pathway and MMP2.

CXCL5 contributes to tumor metastasis and recurrence of intrahepatic cholangiocarcinoma by recruiting infiltrative intratumoral neutrophils.

CXCL5 is a member of the CXC-type chemokine family that may play a role in carcinogenesis and cancer progression. This study investigates the biological function and clinical significance of CXCL5 in intrahepatic cholangiocarcinoma (ICC). We demonstrated that CXCL5 was overexpressed in ICC cell lines and tumor samples compared with paired normal tissues. CXCL5 had a direct chemoattractant effect on neutrophils in vitro through PI3K-Akt and extracellular signal-regulated kinase 1/2 signaling pathways. In animal studies, CXCL5 promoted tumor growth and metastasis without altering in vitro proliferative and invasive ability of ICC cells, and this effect was mediated by the recruitment of intratumoral infiltrative neutrophils by tumor-derived CXCL5. Immunohistochemical analysis of ICC samples showed that overexpression of CXCL5 correlated strongly with intratumoral neutrophil infiltration, shorter overall survival and high tumor recurrence. Multivariate analysis revealed that CXCL5 overexpression alone, or combined with the presence of intratumoral neutrophils, was an independent prognostic indicator for ICC. In conclusion, our data showed that CXCL5 promotes ICC growth and metastasis by recruiting intratumoral neutrophils. CXCL5 alone or combined with intratumoral neutrophils is a novel prognostic predictor for ICC patients and a potential therapeutic target.

[Corrigendum] Macrophage­secreted IL­8 induces epithelial­mesen-chymal transition in hepatocellular carcinoma cells by activating the JAK2/STAT3/Snail pathway.

Subsequently to the publication of the above article, an interested reader drew to the authors' attention that a possible error had been identified in the selection of images in Figs. 1 and/or 7. After having consulted their original data, the authors realized that an erroneous image appeared on p. 593, in Fig. 7F [the 'Hep­G2 / IL­8 (5 ng/ml)' data panel], where part of this figure panel was overlapping with an image on p. 589 in Fig. 1C (the 'Hep­G2 Co­cultured' data panel). After a thorough review and verification of the data by all the authors, they have confirmed that the original data presented in the paper were accurate, and the error was solely due to the selection of an incorrect image during figure arrangement. The authors confirm that this mistake in image selection did not affect the overall conclusions reported in the article. A corrected version of Fig. 7, including the correct data for the 'Hep­G2 / IL­8 (5 ng/ml)' panel in Fig. 7F, is shown on the next page. The authors are grateful to the Editor of International Journal of Oncology for granting them the opportunity to publish this Corrigendum. All the authors agree to the publication of this Corrigendum, and apologize to the readership for any inconvenience caused. [International Journal of Oncology 46: 587­596, 2015; DOI: 10.3892/ijo.2014.2761].

Characterization of tumor microbiome and associations with prognosis in intrahepatic cholangiocarcinoma.

BACKGROUND: The tumor microbiome has been characterized in several malignancies; however, no previous studies have investigated its role in intrahepatic cholangiocarcinoma (ICC). Hence, we explored the tumor microbiome and its association with prognosis in ICC. METHODS: One hundred and twenty-one ICC tumor samples and 89 adjacent normal tissues were profiled by 16S rRNA sequencing. Microbial differences between tumor and adjacent nontumoral liver tissues were assessed. Tumor microbial composition was then evaluated to detect its association with prognosis. Finally, a risk score calculated by the tumor microbiota was accessed by the least absolute shrinkage and selector operator method (Lasso) to predict prognosis of ICC. RESULTS: The tumor microbiome displayed a greater diversity than that in adjacent nontumoral liver tissues. Tumor samples were characterized by a higher abundance of Firmicutes, Actinobacteria, Bacteroidetes, and Acidobacteriota. Higher tumor microbial α diversity was associated with lymph node metastasis and predicted shortened overall survival (OS) and recurrence-free survival (RFS). A total of 11 bacteria were selected to generate the risk score by Lasso. This score showed potential in predicting OS, and was an independent risk factor for OS. CONCLUSION: In conclusion, our study characterized the tumor microbiome and revealed its role in predicting prognosis in ICC.