Landscape and prognostic values of lymphocytes in patients with hepatocellular carcinoma undergoing transarterial embolization.

Transarterial embolization (TACE), the first-line treatment for hepatocellular carcinoma (HCC), does not always lead to promising outcomes in all patients. A better understanding of how the immune lymphocytes changes after TACE might be the key to improve the efficacy of TACE. However, there are few studies evaluating immune lymphocytes in TACE patients. Therefore, we aimed to evaluate the short- and long-term effects of TACE on lymphocyte subsets in patients with HCC to identify those that predict TACE prognosis. Peripheral blood samples were collected from 44 HCC patients at the following time points: one day before the initial TACE, three days after the initial TACE, and one month after the initial TACE and subjected to peripheral blood mononuclear cell isolation and flow cytometry. Dynamic changes in 75 lymphocyte subsets were recorded and their absolute counts were calculated. Tumor assessments were made every 4-6 weeks via computed tomography or magnetic resonance imaging. Our results revealed that almost all lymphocyte subsets fluctuated three days after TACE, but only Tfh and B cells decreased one month after TACE. Univariate and multivariate Cox regression showed that high levels of Th2 and conventional killer Vδ2 cells were associated with longer progressive-free survival (PFS) after TACE. Longer overall survival (OS) after TACE was associated with high levels of Th17 and viral infection-specific Vδ1 cells, and low levels of immature NK cells. In conclusion, TACE has a dynamic influence on the status of lymphocytes. Accordingly, several lymphocyte subsets can be used as prognostic markers for TACE.

PD-L1 blockade liberates intrinsic antitumourigenic properties of glycolytic macrophages in hepatocellular carcinoma.

OBJECTIVE: Patients with increased PD-L1+ host cells in tumours are more potent to benefit from antiprogrammed death-1/programmed death ligand-1 (PD-L1) treatment, but the underlying mechanism is still unclear. We aim to elucidate the nature, regulation and functional relevance of PD-L1+ host cells in hepatocellular carcinoma (HCC). DESIGN: A total of untreated 184 HCC patients was enrolled randomly. C57BL/6 mice are given injection of Hepa1-6 cells to form autologous hepatoma. ELISpot, flow cytometry and real-time PCR are applied to analyse the phenotypic characteristics of PD-L1+ cells isolated directly from HCC specimens paired with blood samples or generated from ex vivo and in vitro culture systems. Immunofluorescence and immunohistochemistry are performed to detect the presence of immune cells on paraffin-embedded and formalin-fixed samples. The underlying regulatory mechanisms of metabolic switching are assessed by both in vitro and in vivo studies. RESULTS: We demonstrate that PD-L1+ host macrophages, which constructively represent the major cellular source of PD-L1 in HCC tumours, display an HLA-DRhighCD86high glycolytic phenotype, significantly produce antitumourigenic IL-12p70 and are polarised by intrinsic glycolytic metabolism. Mechanistically, a key glycolytic enzyme PKM2 triggered by hepatoma cell derived fibronectin 1, via a HIF-1α-dependent manner, concurrently controls the antitumourigenic properties and inflammation-mediated PD-L1 expression in glycolytic macrophages. Importantly, although increased PKM2+ glycolytic macrophages predict poor prognosis of patients, blocking PD-L1 on these cells eliminates PD-L1-dominant immunosuppression and liberates intrinsic antitumourigenic properties. CONCLUSIONS: Selectively modulating the 'context' of glycolytic macrophages in HCC tumours might restore their antitumourigenic properties and provide a precise strategy for anticancer therapy.

Nano-enabled coordination platform of bismuth nitrate and cisplatin prodrug potentiates cancer chemoradiotherapy via DNA damage enhancement.

The combination of chemotherapy and radiotherapy (chemoradiotherapy) is a promising strategy, extensively studied and applied clinically. Meanwhile, radiosensitizers play an important role in improving clinical radiotherapy therapeutic efficacy. There are still some disadvantages in practical applications, because radiosensitizers and drugs are difficult to deliver spatio-temporally to tumor sites and work simultaneously with low efficiency for DNA damage and repair inhibition, leading to an inferior synergistic effect. Herein, a suitable radiosensitizer of nano-enabled coordination platform (NP@PVP) with bismuth nitrate and cisplatin prodrug is developed by a simple synthetic route to improve the effectiveness of chemo-radiation synergistic therapy. When NP@PVP is internalized by a tumor cell, the bismuth in NP@PVP can sensitize radiation therapy (RT) by increasing the amount of reactive oxygen species generation to enhance DNA damage after X-ray radiation; meanwhile, the cisplatin in NP@PVP can inhibit DNA damage repair with spatio-temporal synchronization. NP@PVP is demonstrated to exhibit higher sensitization enhancement ratio (SER) of 2.29 and excellent tumor ablation capability upon irradiation in vivo in comparison with cisplatin (SER of 1.78). Our strategy demonstrates that the RT sensitization effect of bismuth and cisplatin based NP@PVP has great anticancer potential in chemo-radiation synergistic therapy, which is promising for clinical application.

Activation of MET promotes resistance to sorafenib in hepatocellular carcinoma cells via the AKT/ERK1/2-EGR1 pathway.

Sorafenib is an oral multikinase inhibitor that has become an established therapeutic approach in advanced hepatocellular carcinoma (HCC). However, the benefit of sorafenib in clinical therapy is often affected by drug resistance. Therefore, it is important to explore the mechanisms underlying sorafenib resistance and to develop individualized therapeutic strategies for coping with this problem. In this study, we found that addition of HGF to sorafenib-treated HCC cells activated MET and re-stimulated the downstream AKT and ERK1/2 pathways. Thereby, restored sorafenib-treated HCC cells proliferation, migration and invasion ability, and rescued cells from apoptosis. In addition, we found that HGF treatment of HCC cells induced early growth response protein (EGR1) expression, which is involved in sorafenib resistance. Importantly, the HGF rescued effect in sorafenib-treated HCC cells could be abrogated by inhibiting MET activation with PHA-665752 or by downregulating EGR1 expression with small interfering RNA (siRNA). Therefore, inhibition of the HGF/MET pathway may improve response to sorafenib in HCC, and combination therapy should be further investigated.

Knockdown of metadherin inhibits cell proliferation and migration in colorectal cancer.

Metadherin (MTDH) is a multifunctional oncogene involved in tumor cell migration and metastasis through regulating a number of oncogenic signaling pathways in various human malignancies. Previous studies have demonstrated that MTDH is overexpressed in human colorectal cancer (CRC) and associated with cancer progression and a poor prognosis. However, the underlying mechanisms remain largely unknown. The present study investigated the expression and role of MTDH in CRC cells as well as the underlying mechanism of this. Western blot analysis and quantitative polymerase chain reaction were conducted to determine protein and mRNA expression of MTDH in three human CRC cell lines. A short hairpin RNA (shRNA) targeting MTDH was introduced into CRC HCT116 cells to stably inhibit MTDH expression. A Cell Counting Kit­8 assay, colony formation assay, Transwell assay and flow cytometry were used to investigate the effect of MTDH­knockdown on cell proliferation, migration, apoptosis and cell cycle arrest. Western blotting was performed to examine the protein expression levels of cell growth­ and apoptosis­associated genes. The results demonstrated that MTDH was commonly expressed in CRC cell lines. MTDH silencing significantly suppressed cell growth, colony forming ability and migration while inducing the apoptosis of HCT116 cells. In addition, MTDH depletion induced S phase cell cycle arrest in HCT116 cells. Mechanistically, knockdown of MTDH markedly downregulated the expression of phosphorylated protein kinase B, c­Myc, proliferating cell nuclear antigen and B­cell lymphoma 2 (Bcl­2) protein in HCT116 cells, and the expression of p53 and Bcl­2­associated X protein was significantly increased compared with the negative control shRNA group (P<0.05), suggesting that MTDH may function through the expression of numerous types of apoptosis­associated and signaling channel proteins in CRC cells. Taken together, these data indicated that MTDH may serve as a biomarker and candidate therapeutic target for CRC.

Six2 is negatively correlated with good prognosis and decreases 5-FU sensitivity via suppressing E-cadherin expression in hepatocellular carcinoma cells.

This work aims to study the roles and related mechanisms of six2 in 5-FU sensitivity of hepatocellular carcinoma (HCC) cells. KM-Plotter analysis showed that HCC patients with higher six2 expression levels had shorter overall survival. Six2 expression was higher in clinical HCC tissues than in normal tissues, and was negatively correlated with E-cadherin expression. Additionally, six2 overexpression decreased the sensitivity of HCC cells to 5-Fu, characterized as attenuating 5-FU-induced cell apoptosis and downregulation of cell viability, and promoted HCC cells stemness. Mechanistically, six2 overexpression repressed E-cadherin expression via stimulating promoter methylation of the E-cadherin. And E-cadherin overexpression rescued six2-induced decrease of 5-FU sensitivity and promotion on HCC cells stemness. Therefore, our results suggest that Six2 is negatively correlated with good prognosis and decreases 5-FU sensitivity via suppressing E-cadherin expression in HCC cells.

Combination of the neutrophil to lymphocyte ratio and the platelet to lymphocyte ratio as a useful predictor for recurrence following radiofrequency ablation of hepatocellular carcinoma.

The aim of the present study was to investigate the prognostic potential of a novel inflammation-based system, the combination of the neutrophil to lymphocyte ratio (NLR) and the platelet to lymphocyte ratio (PLR) (CNP), for predicting the survival time of patients with hepatocellular carcinoma (HCC) who had received radiofrequency ablation (RFA). A total of 287 HCC patients treated with RFA were enrolled in the study. Patients with an elevated NLR (>2.58) and an elevated PLR (>131.78) were allocated a score of 2, and patients exhibiting one or neither of these characteristics were allocated a score of 1 or 0, respectively. The association between the CNP and various HCC clinicopathological factors, patterns of recurrence and prognoses were analyzed. The CNP was associated with liver cirrhosis (P=0.015), Child-Pugh class (P=0.024), total bilirubin level (P=0.028), neutrophil count (P<0.001), lymphocyte count (P<0.001) and platelet count (P<0.001). Compared with their low-CNP counterparts, patients with an elevated CNP were more likely to develop distant intrahepatic recurrence [52.3% (CNP 2) vs. 33.9% (CNP 0) and 34.6% (CNP 1), P=0.015; CNP 0 vs. CNP 1, P=0.922; CNP 1 vs. CNP 2, P=0.020] and extrahepatic metastasis [25.0% (CNP 2) vs. 7.6% (CNP 0) and 18.5% (CNP 1), P=0.003; CNP 0 vs. CNP 1, P=0.020; CNP 1 vs. CNP 2, P=0.309], and had shorter overall survival (OS) time (CNP 0 vs. CNP 1, P<0.001; CNP 1 vs. CNP 2, P<0.001) and recurrence-free survival (RFS; CNP 0 vs. CNP 1, P=0.012; CNP 1 vs. CNP 2, P=0.004). Moreover, multivariate analysis revealed that the CNP was superior to the NLR and the PLR as an independent prognostic marker of OS and RFS. Therefore, it was concluded that the CNP may represent a useful predictor for recurrence and prognosis in patients with HCC treated with RFA.

The relationship of kinase insert domain receptor gene polymorphisms and clinical outcome in advanced hepatocellular carcinoma patients treated with sorafenib.

Kinase insert domain receptor (KDR) is the principal receptor that promotes the pro-angiogenic action of vascular endothelial growth factor and has been the principal target of anti-angiogenic therapies. Our aim was to determine whether single-nucleotide polymorphisms (SNPs) in KDR gene are associated with clinical outcomes after first-line sorafenib therapy in advanced hepatocellular carcinoma (HCC). The SNPs in KDR were tested in 78 advanced HCC patients receiving first-line sorafenib. Correlations with clinicopathological features and survival were analyzed. Patients with AA genotype of rs1870377 and AA genotype of rs2305948 were significantly associated with a better response and longer time to progression (TTP) (5.8 vs 4.0 months, P=0.001; 5.8 vs 4.5 months, P=0.016, respectively). Patients harboring AA genotype in rs1870377 and TT/TC genotype in rs2071559 had a longer overall survival (OS) (15.0 vs 9.6 months, P=0.001; 13.0 vs 9.0 months, P=0.007, respectively). At multivariate analysis, major vascular invasion and rs1870377 were independent factors in TTP and performance status, rs1870377, and rs2071559 were independent factors in OS. Our results suggest that SNPs in KDR gene can predict clinical outcome in advanced HCC patients receiving first-line sorafenib.

Genetic variants in the KDR gene is associated with the prognosis of transarterial chemoembolization treated hepatocellular carcinoma.

Kinase insert domain receptor (KDR) is the principal receptor that promotes the proangiogenic action of vascular endothelial growth factor and is involved in the tumorigenesis and progression of many malignancies, including hepatocellular carcinoma (HCC). Single-nucleotide polymorphisms (SNPs) of KDR have been reported to be with the risk and prognosis of several malignancies. Our aim was to determine whether SNPs in KDR gene are associated with clinical outcomes in HCC patients treated with transcatheter arterial chemoembolization. A total of 192 HCC patients were tested for KDR SNPs, and the SNP results were correlated with progression-free survival (PFS) and overall survival (OS). The association of the SNPs with the overall survival (OS) of patients was assessed by Kaplan-Meier method, and then Cox proportional hazards model was used to assess the variables resulted significant at univariate analysis. No significant differences were found in correlation between KDR SNPs and patients' PFS. Our data showed that genotype AA+TA of rs1870377 and genotype CC+TC of rs2071559 were significantly associated with overall survival of HCC patients (P<0.001 and P<0.001, respectively) and remained as significant predictors for OS adjusting for high level of serum AFP (>400 µg/L), existence of portal vein tumor thrombus, and high BCLC stage (HR=0.61; 95% CI, 0.36-0.88; P=0.003 and HR=0.54; 95% CI, 0.40-0.94; P=0.002, respectively). Our results suggest that SNPs rs1870377 and rs2071559 in the KDR gene may serve as independent prognosis biomarkers for unresectable HCC patient, which warranted further validating investigation.

Sorafenib continuation after first disease progression could reduce disease flares and provide survival benefits in patients with hepatocellular carcinoma: a pilot retrospective study.

BACKGROUND: Sorafenib is a promising drug for advanced hepatocellular carcinoma (HCC); however, treatment may be discontinued for multiple reasons, such as progressive disease, adverse events, or the cost of treatment. The consequences of sorafenib discontinuation and continuation are uncertain. MATERIALS AND METHODS: We retrospectively analyzed 88 HCC patients treated with sorafenib from July 2007 to January 2013. Overall survival (OS), post-disease progression overall survival (pOS), and time to disease progression (TTP) were compared for survival analysis. Cox proportional hazard regression was performed to assess the effect of important factors on OS in the overall patient population and on pOS in patients who continued sorafenib treatment. RESULTS: Sorafenib was discontinued and continued in 24 and 64 patients, respectively. The median OS (355 vs 517 days respectively; p=0.015) and median post-PD OS (260 vs 317 days, respectively; p=0.020) were statistically different between the discontinuation and continuation groups. Neither the median time to first PD nor the time to second PD were significantly different between the 2 groups. In the discontinuation group, 3 of the 24 patients (12.5%) suffered disease outbreaks. In Cox proportional hazard regression analysis after correction for confounding factors, BCLC stage (p=0.002) and PD site (p=0.024) were significantly correlated with pOS in patients who continued sorafenib treatment. CONCLUSIONS: Sorafenib discontinuation may cause HCC flares or outbreaks. It is advisable to continue sorafenib treatment after first PD, particularly in patients with Barcelona Clinic Liver Cancer stage B disease or only intrahepatic PD.

[Expression of serum microRNAs (miR-222, miR-181, miR-216) in human hepatocellular carcinoma and its clinical significance].

OBJECTIVE: To investigate the abnormal expression of microRNAs (miR-216, miR-222, miR-181) in the serum of patients with hepatocellular carcinoma (HCC) and its clinical significance. METHODS: Serum miRNAs expression was investigated in 49 patients with HCC and 25 healthy normal controls by using real-time PCR technique, and then correlations between miRNAs expression and the clinicopathological features and prognosis of HCC patients were evaluated. RESULTS: No differences were observed between the HCC patients and healthy controls with respect to the expressions of serum miR-181 and miR-216 (P > 0.05), while miR-222 was found to be significantly overexpressed in HCC serum samples (6.1 ± 6.6 vs 1.2 ± 0.6, P < 0.01). According to the median fold change of miR-222 (3-fold) in all HCC serum samples, we divided the 49 patients into two groups:a low expression group (25 patients) and a high expression group (24 patients).High level of miR-222 expression was correlated with cirrhosis (P < 0.01), tumor number (P = 0.013), portal vein tumor thrombosis (P = 0.002) and TNM stage (P = 0.020), while not correlated with serum alpha-fetoprotein level, tumor size and HbsAg. Kaplan-Meier survival analysis showed that the median overall survival in the high miR-222 expression group was significantly shorter than that in the low expression group (8.7 months vs 16.5 months, P = 0.036). In multivariate Cox analysis, TNM stage and serum miR-222 expression were two independent prognostic factors. CONCLUSION: Serum miR-222, upregulated in HCC, maybe helpful in prognosis of HCC patients.

[Prognostic significance of serum level of vascular endothelial growth factor receptor-2 in hepatocellular carcinoma patients after transcatheter arterial chemoembolization].

OBJECTIVE: To explore the prognostic significance of serum vascular endothelial growth factor receptor-2 (VEGFR-2) in patients with hepatocellular carcinoma (HCC) after transcatheter arterial chemoembolization (TACE). METHODS: A total of 69 HCC patients undergoing TACE from October 2008 to April 2012 were recruited and examined. Their serum level of VEGFR-2 level was measured by enzyme-linked immunosorbent assay (ELISA). The relationship between VEGFR-2 and their clinicopathologic features were observed. Prognostic significance of VEGFR-2 was assessed for their survival. RESULTS: Significant differences existed when the serum level of VEGFR-2 was categorized by tumor number and liver cirrhosis (P = 0.021, P = 0.049). The post-treatment serum level of VEGFR-2 was significant higher than that at pre-treatment (P = 0.045). When the mean pre-treatment serum level of VEGFR-2 (8709 ng/L) was used as a cut-off point, the patients with a low serum level of VEGFR-2 had better overall and progression-free survival than those with a high serum level of VEGF (17 vs. 28 months, P = 0.001 and 10 vs. 15 months P = 0.031 respectively). As revealed by multivariate Cox analysis, the pre-treatment serum level of VEGFR-2 was an independent and significant prognostic factor of survival for HCC patients at post-TACE. CONCLUSION: The pre-treatment serum level of VEGFR-2 may predict the post-TACE prognosis in HCC patients.