Purpose: We developed a novel systemic immune-inflammation index (SII) based on lymphocyte, neutrophil, and platelet counts and explored its prognostic value in hepatocellular carcinoma (HCC). Experimental Design: The SII was developed based on a retrospective study of 133 patients with HCC undergoing resection between 2005 and 2006, and validated in a prospective study of 123 patients enrolled from 2010 to 2011. The circulating tumor cell (CTC) level in the validation cohort was measured using the CellSearch system. Prediction accuracy was evaluated with area under the receiver operating characteristic curve (AUC). Results: An optimal cutoff point for the SII of 330 × 109 stratified the patients with HCC into high (≥330) and low SII (<330) groups in the training cohort. Univariate and multivariate analyses revealed the SII was an independent predictor for overall survival and relapse-free survival, and prognostic for patients with negative α-fetoprotein and Barcelona Clinic Liver Cancer stage 0+A. The AUCs of the SII for survival and recurrence were higher than other conventional clinical indices. An SII ≥ 330 was significantly associated with vascular invasion, large tumors, and early recurrence. CTC levels were significantly higher in the SII ≥ 330 group (1.71 ± 0.34 vs. 4.37 ± 1.04, P = 0.029). In patients with detectable CTCs, those with SII ≥ 330 had higher recurrence rates and shorter survival time than patients with SII < 330. Conclusion: The SII was a powerful prognostic indicator of poor outcome in patients with HCC and is a promising tool for HCC treatment strategy decisions. The dismal outcome in patients with high SII scores might be related to higher CTC levels. Clin Cancer Res; 20(23); 6212–22. ©2014 AACR.

Epithelial cell adhesion molecule–positive (EpCAM+) hepatocellular carcinoma (HCC) cells may constitute a tumor-initiating subpopulation in tumorigenic cell lines and HCC specimens. In the present study, EpCAM+ circulating tumor cells (CTCs) were identified prospectively in HCC patients undergoing curative resection, and the prognostic significance and their stem cell–like characteristics were investigated further. Blood samples from 123 HCC patients were tested prior to resection and 1 month thereafter. CTCs were present in 66.67% of patients, and the cell count measured in 7.5 mL of blood (CTC7.5) ranged between 1 and 34. Fifty-one patients had CTC7.5 of ≥2 preoperatively, and these patients developed tumor recurrence earlier than those with CTC7.5 of <2 CTCs ( P < 0.001). A preoperative CTC7.5 of ≥2 was an independent prognostic factor for tumor recurrence ( P < 0.001). Its prognostic significance also applied to patients with alpha-fetoprotein (AFP) levels of ≤400 ng/mL or subgroups with low recurrence risk (all P < 0.05). A significant decrease of CTC-positive rates (66.67% to 28.15%, P < 0.05) and CTC7.5 values (2.60 ± 0.43 to 1.00 ± 0.36, P < 0.05) was observed 1 month after resection. Patients with consistent CTC7.5 <2 had lower recurrence rates than those with values consistently ≥2 (15.5% versus 87.50%, P < 0.001). EpCAM+ CTCs displayed cancer stem cell biomarkers (CD133 and ABCG2), epithelial-mesenchymal transition, Wnt pathway activation, high tumorigenic potential, and low apoptotic propensity. Conclusion : Stem cell–like phenotypes are observed in EpCAM+ CTCs, and a preoperative CTC7.5 of ≥2 is a novel predictor for tumor recurrence in HCC patients after surgery, especially in patient subgroups with AFP levels of ≤400 ng/mL or low tumor recurrence risk. EpCAM+ CTCs may serve as a real-time parameter for monitoring treatment response and a therapeutic target in HCC recurrence.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide because of rapid progression and high incidence of metastasis or recurrence. Accumulating evidence shows that CD73-expressing tumor cell is implicated in development of several types of cancer. However, the role of CD73 in HCC cell has not been systematically investigated and its underlying mechanism remains elusive. CD73 expression in HCC cell was determined by RT-PCR, Western blot, and immunohistochemistry staining. Clinical significance of CD73 was evaluated by Cox regression analysis. Cell counting kit-8 and colony formation assays were used for proliferation evaluation. Transwell assays were used for motility evaluations. Co-immunoprecipitation, cytosolic and plasma membrane fractionation separation, and ELISA were applied for evaluating membrane localization of P110β and its catalytic activity. NOD/SCID/γc(null) (NOG) mice model was used to investigate the in vivo functions of CD73. In the present study, we demonstrate that CD73 was crucial for epithelial-mesenchymal transition (EMT), progression and metastasis in HCC. CD73 expression is increased in HCC cells and correlated with aggressive clinicopathological characteristics. Clinically, CD73 is identified as an independent poor prognostic indicator for both time to recurrence and overall survival. CD73 knockdown dramatically inhibits HCC cells proliferation, migration, invasion, and EMT in vitro and hinders tumor growth and metastasis in vivo. Opposite results could be observed when CD73 is overexpressed. Mechanistically, adenosine produced by CD73 binds to adenosine A2A receptor (A2AR) and activates Rap1, which recruits P110β to the plasma membrane and triggers PIP3 production, thereby promoting AKT phosphorylation in HCC cells. Notably, a combination of anti-CD73 and anti-A2AR achieves synergistic depression effects on HCC growth and metastasis than single agent alone. CD73 promotes progression and metastasis through activating PI3K/AKT signaling, indicating a novel prognostic biomarker for HCC. Our data demonstrate the importance of CD73 in HCC in addition to its immunosuppressive functions and revealed that co-targeting CD73 and A2AR strategy may be a promising novel therapeutic strategy for future HCC management.

Purpose: The spatial heterogeneity of phenotypic and molecular characteristics of CTCs within the circulatory system remains unclear. Herein, we mapped the distribution and characterized biological features of CTCs along the transportation route in hepatocellular carcinoma (HCC). Experimental Design: In 73 localized HCC patients, blood was drawn from peripheral vein (PV), peripheral artery (PA), hepatic veins (HV), infrahepatic inferior vena cava (IHIVC), and portal vein (PoV) before tumor resection. Epithelial and mesenchymal transition (EMT) phenotype in CTCs were analyzed by a 4-channel immunofluorescence CellSearch assay and microfluidic quantitative RT-PCR. The clinical significance of CTCs from different vascular sites was evaluated. Results: The CTC number and size gradient between tumor efferent vessels and postpulmonary peripheral vessels was marked. Tracking the fate of CTC clusters revealed that CTCs displayed an aggregated–singular-aggregated manner of spreading. Single-cell characterization demonstrated that EMT status of CTCs was heterogeneous across different vascular compartments. CTCs were predominantly epithelial at release, but switched to EMT-activated phenotype during hematogeneous transit via Smad2 and β-catenin related signaling pathways. EMT activation in primary tumor correlated with total CTC number at HV, rather than epithelial or EMT-activated subsets of CTCs. Follow-up analysis suggested that CTC and circulating tumor microemboli burden in hepatic veins and peripheral circulation prognosticated postoperative lung metastasis and intrahepatic recurrence, respectively. Conclusions: The current data suggested that a profound spatial heterogeneity in cellular distribution and biological features existed among CTCs during circulation. Multivascular measurement of CTCs could help to reveal novel mechanisms of metastasis and facilitate prediction of postoperative relapse or metastasis pattern in HCC. Clin Cancer Res; 24(3); 547–59. ©2017 AACR.

Hydrogen sulfide (H₂S) is now recognized as a third gaseous mediator along with nitric oxide (NO) and carbon monoxide (CO), though it was originally considered as a malodorous and toxic gas. H₂S is produced endogenously from cysteine by three enzymes in mammalian tissues. An increasing body of evidence suggests the involvement of H₂S in different physiological and pathological processes. Recent studies have shown that H₂S has the potential to protect the heart against myocardial infarction, arrhythmia, hypertrophy, fibrosis, ischemia-reperfusion injury, and heart failure. Some mechanisms, such as antioxidative action, preservation of mitochondrial function, reduction of apoptosis, anti-inflammatory responses, angiogenic actions, regulation of ion channel, and interaction with NO, could be responsible for the cardioprotective effect of H₂S. Although several mechanisms have been identified, there is a need for further research to identify the specific molecular mechanism of cardioprotection in different cardiac diseases. Therefore, insight into the molecular mechanisms underlying H₂S action in the heart may promote the understanding of pathophysiology of cardiac diseases and lead to new therapeutic targets based on modulation of H₂S production.

Background: In the present study, we assessed the clinical value of circulating tumor cells (CTC) with stem-like phenotypes for diagnosis, prognosis, and surveillance in hepatitis B virus (HBV)–related hepatocellular carcinoma (HCC) by an optimized qPCR-based detection platform. Methods: Differing subsets of CTCs were investigated, and a multimarker diagnostic CTC panel was constructed in a multicenter patient study with independent validation (total n = 1,006), including healthy individuals and patients with chronic hepatitis B infection (CHB), liver cirrhosis (LC), benign hepatic lesion (BHL), and HBV-related HCC, with area under the receiver operating characteristic curve (AUC-ROC) reflecting diagnostic accuracy. The role of the CTC panel in treatment response surveillance and its prognostic significance were further investigated. Results: The AUC of the CTC panel was 0.88 in the training set [sensitivity = 72.5%, specificity = 95.0%, positive predictive value (PPV) = 92.4, negative predictive value (NPV) = 77.8] and 0.93 in the validation set (sensitivity = 82.1%, specificity = 94.2%, PPV = 89.9, NPV = 89.3). This panel performed equally well in detecting early-stage and α-fetoprotein–negative HCC, as well as differentiating HCC from CHB, LC, and BHL. The CTC load was decreased significantly after tumor resection, and patients with persistently high CTC load showed a propensity of tumor recurrence after surgery. The prognostic significance of the CTC panel in predicting tumor recurrence was further confirmed [training: HR = 2.692; 95% confidence interval (CI), 1.617–4.483; P < 0.001; and validation: HR = 3.127; 95% CI, 1.360–7.190; P = 0.007]. Conclusions: Our CTC panel showed high sensitivity and specificity in HCC diagnosis and could be a real-time parameter for risk prediction and treatment monitoring, enabling early decision-making to tailor effective antitumor strategies. Clin Cancer Res; 24(9); 2203–13. ©2018 AACR.