Serial circulating tumor DNA profiling predicts tumor recurrence after liver transplantation for liver cancer.

BACKGROUND: Minimal residual disease (MRD) is proposed to be responsible for tumor recurrence. The role of circulating tumor DNA (ctDNA) to detect MRD, monitor recurrence, and predict prognosis in liver cancer patients undergoing liver transplantation (LT) remains unrevealed. METHODS: Serial blood samples were collected to profile ctDNA mutational changes. Baseline ctDNA mutational profiles were compared with those of matched tumor tissues. Correlations between ctDNA status and recurrence rate (RR) and recurrence-free survival (RFS) were analyzed, respectively. Dynamic change of ctDNA was monitored to predict tumor recurrence. RESULTS: Baseline mutational profiles of ctDNA were highly concordant with those of tumor tissues (median, 89.85%; range 46.2-100%) in the 74 patients. Before LT, positive ctDNA status was associated with higher RR (31.7% vs 11.5%; p = 0.001) and shorter RFS than negative ctDNA status (17.8 vs 19.4 months; p = 0.019). After LT, the percentage of ctDNA positivity decreased (17.6% vs 47.0%; p < 0.001) and patients with positive ctDNA status had higher RR (46.2% vs 21.3%; p < 0.001) and shorter RFS (17.2 vs 19.2 months; p = 0.010). Serial ctDNA profiling demonstrated patients with decreased or constant negative ctDNA status had lower RR (33.3% vs 50.0%; p = 0.015) and favorable RFS (18.2 vs 15.0 months, p = 0.003) than those with increased or constant positive ctDNA status. Serial ctDNA profiling predicted recurrence months ahead of imaging evidence and serum tumor biomarkers. CONCLUSIONS: ctDNA could effectively detect MRD and predict tumor recurrence in liver cancer patients undergone LT.

Single-cell and spatial architecture of primary liver cancer.

Primary liver cancer (PLC) poses a leading threat to human health, and its treatment options are limited. Meanwhile, the investigation of homogeneity and heterogeneity among PLCs remains challenging. Here, using single-cell RNA sequencing, spatial transcriptomic and bulk multi-omics, we elaborated a molecular architecture of 3 PLC types, namely hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular-cholangiocarcinoma (CHC). Taking a high-resolution perspective, our observations revealed that CHC cells exhibit internally discordant phenotypes, whereas ICC and HCC exhibit distinct tumor-specific features. Specifically, ICC was found to be the primary source of cancer-associated fibroblasts, while HCC exhibited disrupted metabolism and greater individual heterogeneity of T cells. We further revealed a diversity of intermediate-state cells residing in the tumor-peritumor junctional zone, including a congregation of CPE+ intermediate-state endothelial cells (ECs), which harbored the molecular characteristics of tumor-associated ECs and normal ECs. This architecture offers insights into molecular characteristics of PLC microenvironment, and hints that the tumor-peritumor junctional zone could serve as a targeted region for precise therapeutical strategies.

Autophagy suppression facilitates macrophage M2 polarization via increased instability of NF-κB pathway in hepatocellular carcinoma.

The tumor microenvironment is a highly heterogeneous circumstance composed of multiple components, while tumor-associated macrophages (TAMs) are major innate immune cells with highly plastic and are always educated by tumor cells to structure an advantageous pro-tumor immune microenvironment. Despite emerging evidence focalizing the role of autophagy in other immune cells, the regulatory mechanism of autophagy in macrophage polarization remains poorly understood. Herein, we demonstrated that hepatocellular carcinoma (HCC) cells educated macrophages toward M2-like phenotype polarization under the condition of coculture. Moreover, we observed that inhibition of macrophage autophagy promoted M2-like macrophage polarization, while the tendency was impeded when autophagy was motivated. Mechanistically, macrophage autophagy inhibition inactivates the NF-κB pathway by increasing the instability of TAB3 via ubiquitination degradation, which leads to the M2-like phenotype polarization of macrophages. Both immunohistochemistry staining using human HCC tissues and experiment in vivo verified autophagy inhibition is correlated with M2 macrophage polarization. Altogether, we illustrated that macrophage autophagy was involved in the process of HCC cells domesticating M2 macrophage polarization via the NF-κB pathway. These results provide a new target to interfere with the polarization of macrophages to M2-like phenotype during HCC progression.

Inhibition of autophagy in macrophage promotes IL-1ß-mediated hepatocellular carcinoma progression via inflammasome accumulation and self-recruitment.

Hepatocellular carcinoma (HCC) has a complex and changeable tumor microenvironment. Despite emerging evidence focusing on autophagy process within immune cells, the function and regulatory mechanism of macrophage autophagy in tumor progression remains unclear. Our results of multiplex-immunohistochemistry and RNA-sequencing identified the reduced levels of autophagy in tumor macrophages in the HCC microenvironment, associated with a poor prognosis and increased microvascular metastasis in HCC patients. Specifically, HCC suppressed the macrophage autophagy initiation through the up-regulation of mTOR and ULK1 phosphorylation at Ser757. Knockdown of autophagy-related proteins to further inhibit autophagy significantly boosted the metastatic potential of HCC. Mechanistically, the accumulation of NLRP3 inflammasome mediated by autophagy inhibition promoted the cleavage, maturation, and release of IL-1ß, which facilitated the HCC progression, eventually accelerating HCC metastasis via the epithelial-mesenchymal transition. Autophagy inhibition provoked macrophage self-recruitment through the CCL20-CCR6 signaling was also a crucial account of HCC progression. Recruited macrophages mediated the cascade amplification of IL-1ß and CCL20 to form a novel pro-metastatic positive feedback loop through promoting HCC metastasis and increased macrophage recruitment, respectively. Notably, targeting IL-1ß/IL-1 receptor signaling impaired lung metastasis induced by macrophage autophagy inhibition in a mice HCC lung metastasis model. In summary, this study highlighted that inhibition of tumor macrophage autophagy facilitated HCC progression by increasing IL-1ß secretion via NLRP3 inflammasome accumulation and by macrophage self-recruitment through the CCL20 signaling pathway. Interruption of this metastasis-promoting loop by IL-1ß blockade may provide a promising therapeutic strategy for HCC patients.

Effectiveness and Safety of Avatrombopag in Liver Cancer Patients with Severe Thrombocytopenia: Real-World Data and Challenges.

Background: Avatrombopag has been approved in patients who have severe thrombocytopenia (<50 × 109/L) and chronic liver disease (CLD) while receiving invasive procedures. The real-world application and effectiveness of avatrombopag in the subgroup patients with liver cancer remain unknown. Methods: Liver cancer patients (including primary liver cancer and colorectal cancer liver metastasis) who had severe thrombocytopenia and received avatrombopag were retrospectively enrolled. Avatrombopag dose, peak and absolute platelet count increase, combination treatment with other thrombopoietic agents, responder (peak count ≥50 × 109/L with absolute increase ≥20 × 109/L) rate, and anticancer treatment effect were analyzed. Thrombosis and bleeding events were assessed. Results: In total, 93 patients were enrolled, with 72 and 21 in the CLD and non-CLD groups, respectively. Patients with CLD had hepatitis B or C, larger spleen volume, and a higher cirrhosis degree. Baseline platelet counts were similar between two groups (median, 37.0 × 109/L vs. 39.0 × 109/L; P=0.594), while patients without CLD had higher peak platelet (median, 134.0 × 109/L vs. 74.0 × 109/L; P=0.015) and absolute increase (median, 101.0 × 109/L vs. 41.0 × 109/L; P=0.020) after avatrombopag treatment. The responder rate was higher in patients without CLD (100% vs. 76.4%; P=0.010). Combined avatrombopag with other thrombopoietic agents significantly increased platelet count; repeated use of avatrombopag produced similar effects with that of initial treatment. Concerning anticancer treatment effect, patients who responded to avatrombopag had a higher disease control rate. No thrombosis or hemorrhagic events were observed, even in patients with portal vein tumor thrombosis. Conclusion: Avatrombopag was safe and effective and ensured successful implementation of anticancer treatment in liver cancer patients with severe thrombocytopenia, accompanied with or without CLD.

Conversion therapy for initially unresectable hepatocellular carcinoma using a combination of toripalimab, lenvatinib plus TACE: real-world study.

BACKGROUND: Combination conversion therapies afforded curative surgery chance for initially unresectable hepatocellular carcinoma (uHCC). This study aimed to evaluate the conversion rate and clinical outcomes of a first-line conversion regimen of lenvatinib combined with transarterial chemoembolization (TACE) plus immunotherapy for initial uHCC by interpreting real-world data. METHODS: Conversion therapy data of patients with uHCC from November 2018 to January 2021 were analysed. The regimens included triple combination therapy (t-CT: lenvatinib, TACE, plus toripalimab) and dual combination therapy (d-CT: lenvatinib plus TACE). Another study population diagnosed with hepatocellular carcinoma of macrovascular invasion disease were included as the upfront surgery cohort. Treatment responses and conversion rate were primary outcomes. Survival and adverse events were analysed. RESULTS: Fifty-one patients receiving t-CT (n = 30) and d-CT (n = 21) were enrolled. Higher overall response rates (76.7 per cent versus 47.6 per cent, P = 0.042) and disease control rates (90.0 per cent versus 57.1 per cent, P = 0.042) were observed via t-CT than d-CT. Both median overall survival and event-free survival were not reached in the t-CT cohort. A higher rate of curative conversion resection was achieved through t-CT than d-CT (50.0 per cent versus 19.0 per cent, P = 0.039). The disease-free survival of patients undergoing conversion resection in the t-CT cohort (n = 15) was higher than that in the upfront surgery cohort (n = 68, P = 0.039). Both t-CT and d-CT regimens were tolerable. CONCLUSIONS: Better treatment responses and conversion rate for patients with uHCC were obtained with first-line t-CT. Neoadjuvant t-CT before surgery should be recommended for patients with macrovascular invasion.

Integration of Inflammation-Immune Factors to Build Prognostic Model Predictive of Prognosis and Minimal Residual Disease for Hepatocellular Carcinoma.

Background: Tumor recurrence after hepatectomy is high for hepatocellular carcinoma (HCC), and minimal residual disease (MRD) could be the underlying mechanism. A predictive model for recurrence and presence of MRD is needed. Methods: Common inflammation-immune factors were reviewed and selected to construct novel models. The model consisting of preoperative aspartate aminotransferase, C-reactive protein, and lymphocyte count, named ACLR, was selected and evaluated for clinical significance. Results: Among the nine novel inflammation-immune models, ACLR showed the highest accuracy for overall survival (OS) and time to recurrence (TTR). At the optimal cutoff value of 80, patients with high ACLR (> 80) had larger tumor size, higher Edmondson's grade, more vascular invasion, advanced tumor stage, and poorer survival than those with low ACLR (≤ 80) in the training cohort (5-year OS: 43.3% vs. 80.1%, P < 0.0001; 5-year TTR: 74.9% vs. 45.3%, P < 0.0001). Multivariate Cox analysis identified ACLR as an independent risk factor for OS [hazard ratio (HR) = 2.22, P < 0.001] and TTR (HR = 2.36, P < 0.001). Such clinical significance and prognostic value were verified in validation cohort. ACLR outperformed extant models, showing the highest area under receiver operating characteristics curve for 1-, 3-, and 5-year OS (0.737, 0.719, and 0.708) and 1-, 3-, and 5-year TTR (0.696, 0.650, and 0.629). High ACLR correlated with early recurrence (P < 0.001) and extremely early recurrence (P < 0.001). In patients with high ACLR, wide resection margin might confer survival benefit by decreasing recurrence (median TTR, 25.5 vs. 11.4 months; P = 0.037). Conclusions: The novel inflammation-immune model, ACLR, could effectively predict prognosis, and the presence of MRD before hepatectomy and might guide the decision on resection margin for patients with HCC.

KRAS acting through ERK signaling stabilizes PD-L1 via inhibiting autophagy pathway in intrahepatic cholangiocarcinoma.

BACKGROUND: While the correlation between PD-L1 expression and KRAS mutation has been previously reported in other solid tumors such as non-small cell lung cancer (NSCLC), whether PD-L1 can be modulated by ERK signaling downstream of KRAS in intrahepatic cholangiocarcinoma (iCCA) and the underlying molecular regulatory mechanism remain unclear. METHODS: The expression of ERK, p-ERK, PD-L1 and autophagy markers following KRAS knockdown or Ras/Raf/MEK/ERK signaling inhibitors treatment was examined in two human iCCA cell lines (HuCCT1 and RBE) using western blotting and immunofluorescence. Both pharmacological autophagy inhibitors and short-interfering RNA against ATG7 were applied to inhibit autophagy. The apoptosis rates of iCCA cell lines were detected by flow cytometry and CCK-8 after co-culturing with CD3/CD28-activated human CD8+ T lymphocytes. Immunohistochemistry was applied to detect the correlation of ERK, p-ERK and PD-L1 in 92 iCCA tissues. RESULTS: The present study demonstrated that the PD-L1 expression level was distinctly reduced in KRAS-mutated iCCA cell lines when ERK signaling was inhibited and ERK phosphorylation levels were lowered. The positive association between p-ERK and PD-L1 was also verified in 92 iCCA tissue samples. Moreover, ERK inhibition induced autophagy activation. Both inhibiting autophagy via autophagy inhibitors and genetically silencing the ATG7 expression partially reversed the reduced PD-L1 expression caused by ERK inhibition. In addition, ERK-mediated down-regulation of PD-L1 via autophagy pathways induced the apoptosis of iCCA cells when co-cultured with CD3/CD28-activated human CD8+ T lymphocytes in vitro. CONCLUSIONS: Our results suggest that ERK signaling inhibition contributes to the reduction of PD-L1 expression through the autophagy pathway in iCCA. As a supplement to anti-PD-1/PD-L1 immunotherapy, ERK-targeted therapy may serve as a potentially novel treatment strategy for human KRAS-mutated iCCA.

Laparoscopic vs. Open Repeat Hepatectomy for Recurrent Liver Tumors: A Propensity Score-Matched Study and Meta-Analysis.

Background: It remains unclear whether the short-term benefits of laparoscopic repeat hepatectomy (LRH) accrue to patients with recurrent liver tumors. The present study aimed to report our own center's experience and perform a meta-analysis to evaluate the safety and feasibility of LRH in comparison with open repeat hepatectomy (ORH) for treating recurrent liver tumors. Patients and Methods: A propensity score-matched study was performed including 426 patients receiving LRH or ORH for recurrent hepatocellular carcinoma between January 2017 and December 2018. Surgical outcomes and perioperative inflammation-based markers, including monocyte-to-lymphocyte ratio, neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, and systemic immune-inflammation index were collected from medical records and analyzed. Additionally, a systematic literature review was performed to identify relevant studies in PubMed, EMBASE, Web of Science, and Cochrane library databases up to October 1, 2020. Information including patient demographics, pathologic characteristics, and short-term outcomes was extracted and analyzed using random- or fixed-effects models. Results: Of 68 LRHs, 57 were matched with an ORH finally. Our study demonstrated that LRH was significantly associated with less intraoperative blood loss (50 vs. 100 mL; P < 0.001), lower rate of hepatic inflow occlusion (10.52 vs. 33.3%; P = 0.003), and shorter postoperative hospital stay (5 vs. 6 days; P = 0.001) after 1:1 propensity score matching. The operation time, rate of blood transfusion, and postoperative complications were similar between the two groups. Moreover, all four inflammation-based markers were significantly lower in LRH group on postoperative day 1. In the meta-analysis, a total of 12 studies comprising 1,315 patients receiving repeat hepatectomy met the selection criteria. Similar to our own study, the meta-analysis showed shorter hospital stay [standard mean difference (SMD) = -0.51, 95% confidence interval (CI) = -0.79 to -0.22, P < 0.001], less intraoperative blood loss (SMD = -0.79, 95% CI = -1.11 to -0.47, P < 0.001), and lower rate of major postoperative complications [odds ratio (OR) = 0.35, 95% CI = 0.19-0.66, P = 0.001] in the LRH group. There was no difference in the field of overall postoperative complication and operation time between LRH and ORH groups. Conclusion: Compared with ORH, LRH results in relatively better surgical outcomes and faster postoperative recovery. It could be considered a feasible and effective option for the treatment of recurrent liver tumors.

Laparoscopic hepatectomy enhances recovery for small hepatocellular carcinoma with liver cirrhosis by postoperative inflammatory response attenuation: a propensity score matching analysis with a conventional open approach.

BACKGROUND: The concurrent presence of liver cirrhosis and hepatocellular carcinoma (HCC) poses a challenge for laparoscopic surgeons to establish a routine practice. The aim of this study was to gather evidence and produce recommendations on the safe and effective practice of laparoscopic hepatectomy for patients with solitary HCC (≤ 5 cm) and liver cirrhosis. METHODS: Between October 2013 and October 2014, 356 curative hepatectomies were performed for patients pathologically diagnosed with solitary HCC (≤ 5 cm) accompanied by cirrhosis (stage 4 fibrosis). To overcome selection bias, a 1:2 match using propensity score matching analysis was conducted between laparoscopic and open hepatectomy. Perioperative outcomes were compared between the groups, including hospitalization, operation time, blood loss, and surgical complications. Perioperative inflammation-based markers, including systemic immune-inflammation index (SII), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR) were collected from medical records and analyzed. RESULTS: There were 43 and 77 patients in the laparoscopic and open groups, respectively. The laparoscopic group had less hepatic inflow occlusion (16.3% vs. 61%; P < 0.001), shorter operation time (155 vs. 170 min; P = 0.004), and shorter postoperative hospital stay (4 vs. 7 days; P < 0.001). Although the difference was not significant (P = 0.154), the rate of postoperative complications tended to be lower in the laparoscopic group (2.3%) compared with the open group (9.1%). The increase in postoperative SII, NLR, and LMR for laparoscopic hepatectomy were significantly lower than for open hepatectomy. NLR < 5.8 on postoperative day 3 was significantly correlated with shorter hospital stay (P < 0.001). CONCLUSIONS: Compared with open hepatectomy, laparoscopic hepatectomy for selected HCC patients, even in the presence of cirrhosis, might result in better perioperative outcomes and postoperative inflammatory response attenuation, and ultimately promote faster recovery. This provides evidence for considering routine laparoscopic hepatectomy through careful selection of patients with HCC.

Laparoscopic Versus Open Left Lateral Segmentectomy for Large Hepatocellular Carcinoma: A Propensity Score-Matched Analysis.

BACKGROUND: With the advancement of endoscopic technology, laparoscopic liver resection has become the standard procedure for left lateral segmentectomy. The aim of this study was to compare perioperative and oncological outcomes between laparoscopic and open left lateral segmentectomy for hepatocellular carcinoma (HCC) >5 cm. PATIENTS AND METHODS: A total of 66 patients underwent left lateral segmentectomy for HCC (>5 cm) during the period spanning between 2013 and 2015. To overcome selection bias, 1:3 match using propensity score-matched analysis was performed between laparoscopic and open liver resection. RESULTS: Relatively smaller tumor size (6.0 vs. 7.0 cm; P=0.030) and more frequent incidence of complete tumor capsule (93.3% vs. 58.8%; P=0.013) were observed in the laparoscopic group compared with the open group before matching. Although the longer operation time (195 vs. 150 min; P=0.022) was consumed in the laparoscopic procedure after matching, the laparoscopic group had shorter postoperative hospital stay (6 vs. 7 d; P=0.002) and less blood loss volume (50 vs. 100 mL; P=0.022). The Pringle maneuver for hepatic inflow occlusion was more likely to be applied in patients who underwent open surgery. The incidence of postoperative complication seemed to be lower in the laparoscopic group (6.7%) compared with that in the open group (11.8%) before matching. On the basis of propensity score-matched analysis, the complication rates were comparable between the 2 groups (7.1% vs. 6.7%, P=0.953). No difference in the 1-year and 3-year overall and recurrence-free survival rates was found between the laparoscopic and open groups. CONCLUSION: Laparoscopic left lateral segmentectomy for large HCC patients showed better perioperative outcomes and equivalent oncologic outcomes as the open procedure, providing evidence for considering as a standard laparoscopic practice through careful selection.

Tumor-associated macrophages modulate resistance to oxaliplatin via inducing autophagy in hepatocellular carcinoma.

BACKGROUND: Oxaliplatin-based chemotherapy is widely used to treat hepatocellular carcinoma (HCC). Recent studies suggested that therapeutic resistance of tumors was affected by tumor microenvironment (TME). As a major component of TME, the role of tumor-associated macrophages (TAMs) on drug resistance in HCC is largely unknown. METHODS: 26 HCC samples were obtained from patients who had underwent transarterial chemoembolization (TACE) within 3 months before receiving curative resections. Immunohistochemistry was applied to detect the density of TAMs in these tissues. SMMC-7721 and Huh-7 cell lines were used to co-culture with THP-1 derived macrophages. Under oxaliplatin treatment, cell death was measured using MTT and annexin V/propidium iodide assays. Autophagy activation was evaluated by GFP-LC3 redistribution and LC3 conversion in SMMC-7721 and Huh-7. Short-interfering RNA against ATG5 gene was applied to inhibit autophagy. In vivo validation was conducted in Huh-7 with or without macrophages using an HCC xenograft model in nude mice after oxaliplatin administration. RESULTS: We found that the density of TAMs in HCC samples was associated with the efficacy of TACE. Macrophages inhibited cell death induced by oxaliplatin in HCC cells. Autophagy was functionally activated in HCC cells after co-culturing with macrophages. Suppression of autophagy using RNA interference of ATG5 in HCC cells promoted the oxaliplatin cytotoxicity in the co-culture system. Critically, co-implantation with macrophages in HCC xenografts weakens cytotoxic effect of oxaliplatin through inducing autophagy to avoid apoptosis. CONCLUSIONS: Our results suggest that TAMs induce autophagy in HCC cells which might contribute to oxaliplatin resistance. Targeting TAMs is a promising therapeutic strategy to enhance the effects of chemotherapy oxaliplatin in HCC patients.

Autophagy activation contributes to glutathione transferase Mu 1-mediated chemoresistance in hepatocellular carcinoma.

Glutathione transferase Mu 1 (GSTM1) induces cancer drug resistance by hydrolyzing cancer chemotherapeutics or activating the anti-apoptosis pathway. However, the chemoresistance-inducing mechanism of GSTM1 in hepatocellular carcinoma (HCC) remains unknown. In the present study, the expression of GSTM1 was examined in three HCC cell lines. Oxaliplatin and sorafenib were selected as chemotherapeutic agents. Small interfering RNA was used to decrease GSTM1 expression. Cell death was measured using MTT and annexin V/propidium iodide assays. Activation of autophagy was evaluated by green fluorescent protein-light chain 3 redistribution and analysis of autophagy-related 5 expression in MHCC97-H and Huh-7 cells. A stepwise increase in GSTM1 expression with increasing metastatic potential of HCC cell lines was revealed. Cell death induced by oxaliplatin and sorafenib was significantly increased following GSTM1-knockdown in MHCC97-H and Huh-7 cells. Activation of autophagy was significantly inhibited by silencing GSTM1 expression. The results of the present study suggest that GSTM1 may protect HCC cells against the effect of oxaliplatin treatment through activating autophagy. The present study provides a novel perspective on HCC drug-resistance.

MicroRNA-30a suppresses autophagy-mediated anoikis resistance and metastasis in hepatocellular carcinoma.

MiRNA-30a (miR-30a) was previously reported as one of metastatic hepatocellular carcinoma (HCC)-related microRNAs. However, the function of miR-30a on enhancing our biological understanding of HCC metastasis is not clear. This study demonstrated that miR-30a was significantly down-regulated in HCC tissues and cell lines, and was associated with vascular invasion, metastasis potential and recurrent disease in HCC. Functional studies confirmed that miR-30a could inhibit the metastasis of HCC in a well-established nude mouse model of lung metastasis. Moreover, miR-30a was proved to prevent anoikis inhibition of HCC cells in vivo and in vitro. Mechanically, autophagy related protein Beclin 1 and Atg5 were direct downstream targets of miR-30a, and mediated autophagy activity influence of miR-30a in HCC. Taken together, downregulated miR-30a in metastatic HCC mediates Beclin 1 and Atg5-dependent autophagy, which confers anoikis resistance in HCC cells. The molecular basis of autophagy action during this process partly contributes to the HCC metastasis, suggesting that targeting autophagy via miR-30a may have therapeutic implications for the prevention of HCC recurrence/metastasis.

Association of hepatitis status with surgical outcomes in patients with dual hepatitis B and C related hepatocellular carcinoma.

BACKGROUND: The conception that serological hepatitis markers determined surgical prognosis of hepatocellular carcinoma (HCC) associated with hepatitis B (HBV) or hepatitis C (HCV) has been well defined. However, little is known about the relationship between surgical outcomes and serological hepatitis markers in patients with dual HBV and HCV related HCC. METHODS: A retrospective analysis of the clinical data of 39 HCC patients with HBV-HCV coinfection who underwent curative hepatectomy between 2001 and 2011 was performed. HBV DNA quantification, expression of HBV antigens, anti-HCV signal-to-cutoff ratio (S/CO) and some clinicopathological characteristics were investigated to show the potential relationship among them and the surgical prognosis. RESULTS: The Cox proportional hazards model identified that HBV DNA quantification of 1,000 IU/mL or higher, HBeAg seropositivity, tumor size of greater than 5 cm, multiple tumors, and vascular invasion were risk factors for HCC prognosis. Thus, HBV DNA quantification, HBsAg level, HBeAg status and HCV-Ab level which may reveal the hepatitis status were further analyzed. The overall survival time in the group with high (≥1,000 IU/mL) HBV DNA quantification was significantly lower than the group with low (<1,000 IU/mL) HBV DNA quantification. Similarly, the high HBsAg level (≥1,000 IU/mL) was associated with poor survival compared with the low HBsAg level. Moreover, HBeAg seropositivity determined a higher cumulative risk for death. However, no significant difference was observed in overall survival time between the groups with low (<10.9 S/CO) and high (≥10.9 S/CO) HCV-Ab level. Compared to HCV-Ab high-level group, the serological HBsAg level was observed significantly higher in HCV-Ab low-level group. Furthermore, the data we analyzed showed these 4 serological hepatitis markers were not correlated with cumulative recurrence rate. On multivariate analysis, none of serological hepatitis markers was an independent prognostic factor for HCC patients with dual hepatitis B and C. CONCLUSION: Among HCC patients with HBV-HCV coinfection, those who with preoperatively high HBV DNA quantification or HBeAg seropositivity had a short survival time and served as poor survival indicators. Serological expression of HBV status rather than HCV status might potentially dominate the surgical outcomes of the Chinese HCC patients with HBV-HCV coinfection.

Lamp2a is required for tumor growth and promotes tumor recurrence of hepatocellular carcinoma.

Exploring the function of chaperone-mediated autophagy (CMA) in cancer has promoted progress in cancer treatment through the regulation of CMA pathways. However, CMA status and function in hepatocellular carcinoma (HCC) by focusing on the regulatory role of lyso-some-associated membrane protein type 2a (Lamp2a) remain to be clarified. We examined Lamp2a in a normal human liver cell line, 6 HCC cell lines, 10 normal liver samples as well as 42 HCC tissue and para-tumor tissues samples, and then validated it in 228 HCC patients to assess the relationship between Lamp2a and clinical prognosis. Gain and loss of Lamp2a function were also explored in HCC cell lines and xenograft models. Significantly lower level of Lamp2a expression was found in HCC cells and tissues compared with normal hepatic cells, para-tumor tissues and normal livers. Although no differences in HCC cell morphology or function were observed in relation to Lamp2a expression under normal culture or short-term starvation conditions, Lamp2a blockage significantly inhibited HCC cell viability under prolonged starvation. Critically, Lamp2a is required for HCC xenograft growth in vivo by helping cells to avoid apoptosis and promoting cell proliferation. Furthermore, a significant correlation between Lamp2a expression and tumor size or cumulative recurrence was uncovered in HCC patients. Collectively, the present study shows that impaired Lamp2a expression in HCC contributes to tumor cell viability and promotes tumor growth and recurrence. Targeting chaperone-mediated autophagy through Lamp2a may also imply a potentially novel treatment strategy for HCC.

Macrophage-secreted IL-8 induces epithelial-mesenchymal transition in hepatocellular carcinoma cells by activating the JAK2/STAT3/Snail pathway.

Macrophages are a major component of the leukocyte infiltrate of tumors and play a pivotal role in the progression of hepatocellular carcinoma (HCC). However, the molecular mechanisms by which macrophages promote HCC invasion are poorly understood. The present study was undertaken to investigate the relationship between macrophages and epithelial-mesenchymal transition (EMT) of HCC. Double-staining immunohistochemistry was used to observe the association between macrophages and EMT markers in clinical HCC samples and it showed that EMT primarily occurred at the edge of the tumor nest, in which infiltrating macrophages were always observed. This indicated that CD68 which is a marker of macrophages, was correlated with EMT marker levels. In addition, after being cultured with macrophages for 24 h, the ability of HCC cells to migrate and invade increased, Snail and N-Cadherin expression was upregulated, and E-Cadherin was downregulated. An antibody array assay was applied to analyze the supernatant of these cultures and it demonstrated IL-8 increased significantly in the macrophage co-culture system. Finally, the role of macrophage-derived IL-8 in the invasion of HCC cells was assayed, and downstream signaling pathways were also investigated. We found that IL-8: i) may induce EMT and promote HCC cell migration and invasion and ii) is associated with the JAK2/STAT3/Snail signaling pathway. Taking together, these findings revealed that macrophages that have infiltrated tumors may induce epithelial-mesenchymal transition of HCC cells via the IL-8 activated JAK2/STAT3/Snail pathway. Thus, this may offer a potential target for developing new HCC therapies.

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.

Overexpression of HnRNP A1 promotes tumor invasion through regulating CD44v6 and indicates poor prognosis for hepatocellular carcinoma.

Heterogeneous ribonucleoprotein (hnRNP) A1 is a member of the A/B subfamily of ubiquitously expressed hnRNPs, which have a wide variety of functions in gene expression and signal transduction. To investigate the biological function and clinical significance of hnRNP A1 in hepatocellular carcinoma (HCC), we measured hnRNP A1 expression in four HCC cell lines and two independent cohorts of HCC patients. We found that hnRNP A1 was overexpressed in the highly metastatic HCC cell lines and in tumor tissues of patients with recurrent HCC. Knockdown of hnRNP A1 in highly metastatic HCC cells caused a significant decrease in cell invasion, while upregulation of hnRNP A1 in poorly metastatic HCC cells led to a significant increase in their invasive capacity. We found that this effect may occur through the regulation of CD44v6 expression by hnRNP A1 in HCC cells. Both quantitative reverse transcription-polymerase chain reaction (qRT-RCR) and immunohistochemistry revealed that hnRNP A1 was upregulated in HCC tissues and coincided with overexpression of CD44v6. HCC patients with high hnRNP A1 tended to have higher levels of CD44v6, shorter overall survival (OS) and higher rates of tumor recurrence. Multivariate analyses revealed that hnRNP A1 alone or in combination with CD44v6 were independent prognostic indicators for OS and time to recurrence and have potential as therapeutic targets. In conclusion, overexpression of hnRNP A1 promotes HCC invasion by regulating the level of CD44v6 and indicates a poor prognosis for HCC patients after curative resection.

Validity of plasma macrophage migration inhibitory factor for diagnosis and prognosis of hepatocellular carcinoma.

We performed our study to determine whether plasma macrophage migration inhibitory factor (MIF) levels have diagnostic and prognostic value in hepatocellular carcinoma (HCC) patients. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were used to measure the expression of MIF in plasma and tissues, respectively. Plasma MIF levels were compared to HCC occurrence, clinicopathological features and outcomes. Cutpoints of plasma MIF levels for diagnosis and prognosis were, respectively, determined by receiver operating characteristic analysis and X-tile in corresponding training cohort, and then were confirmed in the validation cohort. The postoperative plasma MIF levels of HCC patients were detected in an independent cohort (80 HCC patients). As a result, MIF expression in situ was mainly observed in the cytoplasm of HCC cells. Intratumoral MIF expression was positively correlated with plasma MIF levels (r = 0.759, p < 0.001). Compared to serum α-fetoprotein (AFP), plasma MIF had a higher diagnostic value for discrimination of HCC from controls at 35.3 ng/ml. With determined cutpoints, plasma MIF levels demonstrated a significant association with overall survival (OS) and disease-free survival (DFS) of HCC patients even in patients with normal serum AFP levels and Tumor Node Metastasis (TNM) stage I. In addition, the plasma MIF levels were identified as an independent factor for OS [hazard ratio (HR) = 1.754; p = 0.012] and DFS (HR = 2.121; p < 0.001). Plasma MIF levels decreased markedly within 30 days after tumor resection (p < 0.001). Therefore, plasma MIF levels have potential as a diagnostic and prognostic factor for HCC.