Hui-Chuan Sun

Plasma MicroRNA Panel to Diagnose Hepatitis B Virus–Related Hepatocellular Carcinoma

PURPOSE More than 60% of patients with hepatocellular carcinoma (HCC) do not receive curative therapy as a result of late clinical presentation and diagnosis. We aimed to identify plasma microRNAs for diagnosing hepatitis B virus (HBV) -related HCC. PATIENTS AND METHODS Plasma microRNA expression was investigated with three independent cohorts including 934 participants (healthy, chronic hepatitis B, cirrhosis, and HBV-related HCC), recruited between August 2008 and June 2010. First, we used microarray to screen 723 microRNAs in 137 plasma samples for diagnosing HCC. Quantitative reverse-transcriptase polymerase chain reaction assay was then applied to evaluate the expression of selected microRNAs. A logistic regression model was constructed using a training cohort (n = 407) and then validated using an independent cohort (n = 390). Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy. RESULTS We identified a microRNA panel (miR-122, miR-192, miR-21, miR-223, miR-26a, miR-27a and miR-801) that provided a high diagnostic accuracy of HCC (AUC = 0.864 and 0.888 for training and validation data set, respectively). The satisfactory diagnostic performance of the microRNA panel persisted regardless of disease status (AUCs for Barcelona Clinic Liver Cancer stages 0, A, B, and C were 0.888, 0.888, 0.901, and 0.881, respectively). The microRNA panel can also differentiate HCC from healthy (AUC = 0.941), chronic hepatitis B (AUC = 0.842), and cirrhosis (AUC = 0.884), respectively. CONCLUSION We found a plasma microRNA panel that has considerable clinical value in diagnosing early-stage HCC. Thus, patients who would have otherwise missed the curative treatment window can benefit from optimal therapy.

A decade’s studies on metastasis of hepatocellular carcinoma

Metastasis remains one of the major challenges before hepatocellular carcinoma (HCC) is finally conquered. This paper summarized a decade’s studies on HCC metastasis at the Liver Cancer Institute of Fudan University. We have established a stepwise metastatic human HCC model system, which included a metastatic HCC model in nude mice (LCI-D20), a HCC cell line with high metastatic potential (MHCC97), a relatively low metastatic potential cell clone (MHCC97L) and several stepwise high metastatic potential cell clones (MHCC97H, HCCLM3, and HCCLM6) from their parent MHCC97 cell. Endeavors have been made for searching human HCC metastasis-related chromosomes/proteins/genes. Monogene-based studies revealed that HCC invasion/metastasis was similar to that of other solid tumors, and the biological characteristics of small HCC were only slightly better than that of large HCC. Using comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), genotyping, cDNA microarray, and 2-dimensional gel electrophoresis, we obtained some interesting results. In particular, in collaboration with the National Institute of Health (NIH) in the United States, we generated a molecular signature that can classify metastatic HCC patients, identified osteopontin as a lead gene in the signature, and found that genes favoring metastasis progression were initiated in the primary tumors. We also found that chromosome 8p deletion, particularly in the region of 8p23, was associated with HCC metastasis. Cytokeratin 19 was identified as one of the proteins, which was found in MHCC97H, but not in MHCC97L cells. Experimental interventions using the high metastatic nude mice model have provided clues for the prevention of HCC metastasis. Translation from workbench to bedside demonstrated that serum VEGF, microvessel density, and p53 scoring may be of value for the prediction of postoperative metastatic recurrence. Interferon alpha proved effective for the prevention of recurrence both experimentally and clinically. In conclusion, HCC metastasis that probably initiated in the primary tumor is a multigene-involved, multistep, and changing process. The further elucidation of the mechanism underlying HCC metastasis will provide a more solid basis for the prediction and prevention of the metastatic recurrence of HCC.

Depletion of Tumor-Associated Macrophages Enhances the Effect of Sorafenib in Metastatic Liver Cancer Models by Antimetastatic and Antiangiogenic Effects

Purpose: To investigate the role of macrophages in tumor progression under sorafenib treatment and to explore whether combination of drugs that deplete macrophages improved the antitumor effect of sorafenib. Experimental Design: Tumor growth, lung metastasis, and tumor angiogenesis were observed in HCCLM3-R and SMMC7721, two human hepatocellular carcinoma xenograft nude mouse models, when treated with sorafenib (30 mg/kg daily, n = 6 per group) or a vehicle as control. Macrophage infiltration was measured in the peripheral blood and in sorafenib-treated tumor by immunohistochemistry and flow cytometry with F4/80 antibody and CD11b antibody. The effect of macrophage depletion on tumor angiogenesis and metastasis after sorafenib treatment, using two drug target macrophages, zoledronic acid (ZA) and clodrolip, was measured in the two models of hepatocellular carcinoma. Results: Although sorafenib significantly inhibited tumor growth and lung metastasis, it induced a significant increase in peripheral recruitment and intratumoral infiltration of F4/80- and CD11b-positive cells, which was accompanied with elevation of colony-stimulating factor-1, stromal-derived factor 1α, and vascular endothelial growth factor in the tumor and elevation of plasma colony-stimulating factor-1 and mouse vascular endothelial growth factor in peripheral blood, suggesting the role of macrophages in tumor progression under sorafenib treatment. Depletion of macrophages by clodrolip or ZA in combination with sorafenib significantly inhibited tumor progression, tumor angiogenesis, and lung metastasis compared with mice treated with sorafenib alone. ZA was more effective than clodrolip. Conclusions: Macrophages may have an important role in tumor progression under sorafenib treatment. ZA is promising when combined with sorafenib to enhance its antitumor effect. Clin Cancer Res; 16(13); 3420–30. ©2010 AACR.

Gene Expression Profiling Reveals Potential Biomarkers of Human Hepatocellular Carcinoma

Purpose: Hepatocellular carcinoma (HCC), a common cancer worldwide, has a dismal outcome partly due to the poor identification of early-stage HCC. Currently, one third of HCC patients present with low serum α-fetoprotein (AFP) levels, the only clinically available diagnostic marker for HCC. The aim of this study was to identify new diagnostic molecular markers for HCC, especially for individuals with low serum AFP. Experimental Design: We used the microarray technique to determine the expression profiles of 218 HCC specimens from patients with either high or low serum AFP. From the microarray study, we selected five candidate genes (i.e., GPC3, PEG10, MDK, SERPINI1, and QP-C), which were overexpressed in HCCs. Using quantitative real-time PCR analyses, we validated the expression of these five genes in 50 AFP-normal and 8 AFP-positive HCC specimens and 36 cirrhotic noncancerous hepatic specimens, which include 52 independent specimens not used in microarray analysis. Results: A significant increase in the expression of the five candidate genes could be detected in most of the HCC samples, including those with normal serum AFP and small tumors. GPC3, MDK, and SERPINI1 encode known serum proteins. Consistently, a significant increase in serum midkine, encoded by MDK, was associated with HCC patients, including those with normal serum AFP. Using prediction analysis of microarray, we showed that a combined score of these five genes can accurately classify noncancerous hepatic tissues (100%) and HCC (71%). Conclusions: We suggest that a diagnostic signature approach using a combined score of these five biomarkers rather than a single marker may improve the prediction accuracy of HCC patients, including those with normal serum AFP and smaller-sized tumors.

Human Hepatocellular Carcinoma Tumor–derived Endothelial Cells Manifest Increased Angiogenesis Capability and Drug Resistance Compared with Normal Endothelial Cells

Purpose: Increasing evidence indicates that tumor-derived endothelial cells (TEC) possess a distinct and unique phenotype compared with endothelial cells (NEC) from adjacent normal tissue and may be able to acquire resistance to drugs. The aim of this study was to investigate the angiogenesis activity and response to drug treatment of TECs and NECs derived from human hepatocellular carcinoma (HCC). Experimental Design: TECs or NECs were isolated from HCC or adjacent normal liver tissue using anti-CD105 antibody coupled to magnetic beads. The phenotypic and functional properties of endothelial cells were characterized by testing the expression of CD105, CD31, CD144, vascular endothelial growth factor receptor-1, vascular endothelial growth factor receptor-2, and von Willebrand factor, and the ability of DiI-Ac-LDL-uptake and tube formations. CD105+ TECs were compared with CD105+ NECs and human umbilical vein endothelial cells (HUVEC) by examining their ability to proliferate, motility, ability to adhere to tumor cells, response to tumor conditioned medium, and reactions to the chemotherapy drugs Adriamycin and 5-fluorouracil and the antiangiogenic drug sorafenib. Results: Compared with CD105+ NECs and HUVECs, CD105+ TECs showed increased apoptosis resistance and motility and proangiogenic properties. Meanwhile, CD105+ TECs had a greater ability to adhere to tumor cells and survive in the tumor environment. Moreover, CD105+ TECs acquired more resistance to Adriamycin, 5-fluorouracil, and sorafenib than CD105+ NECs and HUVECs. Conclusions: TECs possessed enhanced angiogenic activity and resistance to chemotherapeutic drugs and an angiogenesis inhibitor, and may provide a better tool for studying tumor angiogenesis and antiangiogenesis drugs in HCC.

Cytokeratin 10 and Cytokeratin 19: Predictive Markers for Poor Prognosis in Hepatocellular Carcinoma Patients after Curative Resection

Purpose: Cytokeratin 10 (CK10) was found to be expressed differently in human hepatocellular carcinoma (HCC) cell lines with different metastatic potentials in our previous research. The aim of this study was to assess the value of CK10 alone or in combination with cytokeratin 19 (CK19) in predicting tumor recurrence after curative resection in HCC patients. Experimental Design: CK10 expression in stepwise metastatic HCC cell lines and tumor tissues from 50 HCC patients was investigated using immunofluorescence assay, quantitative real-time reverse transcription-PCR, and Western blot analyses. Tumor tissue microarrays of 300 HCC patients who underwent curative resection between 1997 and 2000 were used to detect the expressions of CK10 and CK19. Clinicopathologic data for these patients were evaluated. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. Results: CK10 was overexpressed in the high metastatic HCC cell line and in tumor tissues of recurrent patients. Both univariate and multivariate analyses revealed that CK10 was a significant predictor for overall survival (OS) and disease-free survival, and that CK19 was a significant predictor for OS. CK10 expression was correlated with poor prognosis regardless of α-fetoprotein, tumor-node-metastasis stage, and vascular invasion. The 7-year OS and disease-free survival rates in CK10+ and/or CK19+ patients were 30.0% and 37.6%, respectively, which were significantly lower than that of CK10−/CK19− patients (56.1% and 60.0%, respectively; P < 0.001). Conclusion: CK10 is associated with HCC invasiveness. CK10 alone, or in combination with CK19, can be a novel predictor for poor prognosis of HCC patients after curative resection.

Activation of beta-Catenin by Hypoxia in Hepatocellular Carcinoma Contributes to Enhanced Metastatic Potential and Poor Prognosis

Purpose: Aberrant activation of β-catenin contributes to the malignant phenotype in hepatocellular carcinoma (HCC). Hypoxia is also known to promote HCC invasion and metastasis. However, the association between β-catenin and the proinvasive role of hypoxia remains unclear. We investigated the role of β-catenin in the proinvasive consequences of hypoxia in HCC. Experimental Design: We established in vitro and in vivo hypoxic models to investigate the expression of β-catenin in hypoxic HCC cells and its role in hypoxia-induced aggressiveness. The clinical significance of β-catenin and/or hypoxia-induced factor-1α (HIF-1α) was evaluated using HCC tissue microarrays. Results: Hypoxia induced β-catenin overexpression and/or intracellular accumulation in four HCC cell lines through downregulating the endogenous degradation machinery, and promoted in vitro invasion and in vivo metastasis of MHCC97 and Hep3B cells. Besides morphologic changes, hypoxic MHCC97 and Hep3B cells exhibited molecular alterations consistent with epithelial-mesenchymal transition, characterized by the loss of epithelial cell markers (E-cadherin and plakoglobin) and upregulation of mesenchymal markers (vimentin and N-cadherin), as well as the increase of matrix metalloproteinase 2. However, silencing β-catenin in these hypoxic cells reversed epithelial-mesenchymal transition and repressed metastatic potential. Positive expression of β-catenin in HCC tissue microarray was associated with the expression of HIF-1α (P = 0.034), and coexpression of β-catenin and HIF-1α in HCC was correlated with shorter overall survival and time to recurrence. Conclusion: β-Catenin in HCC is activated by hypoxia and contributes to hypoxia-induced metastatic potential. Clin Cancer Res; 16(10); 2740–50. ©2010 AACR.

Microvessel density of hepatocellular carcinoma: its relationship with prognosis.

Purpose: To elucidate the relationship between angiogenesis and prognosis after curative resection of hepatocellular carcinoma (HCC). Methods: An immunohistochemical study using anti-CD34 monoclonal antibody was carried out on surgical specimens from 78 HCC patients who had undergone curative resection; microvessel density (MVD) was counted and the overall survival and disease-free survival were analyzed retrospectively. Results: Blood vessels in the tumor were strongly stained by anti-CD34 antibody, but not those in the surrounding liver parenchyma. There were three types of tumor vessels: capillary-like (n = 59), sinusoid-like (n = 16) and mixed-type (n = 3). The median MVD count was 100 per field. The HCC were designated as hypovascular (n = 36) with an MVD count below 100, and hypervascular (n = 42) with an MVD count of 100 or more per field. The 5-year survival and disease-free survival rates were 49.7% and 42.8% respectively, and statistical analysis showed that the MVD level was not correlated with tumor size, capsule status, Edmondsons grade, α-fetoprotein level, associated cirrhosis, γ-glutamyltransferase, and serum HBsAg status. The sinusoid-like tumor vessels appeared more frequently in the more differentiated tumors (P < 0.05). No statistical difference in overall and disease-free survival between different MVD levels and microvessel types was found. Tumor size was the only predicting factor in the entire series. In patients with small HCC (≤ 5 cm, n = 40), 5-year survival and disease-free survival rates were 58.9% and 52.7% respectively, higher than the values in large HCC (39.8% and 32.0% respectively, P < 0.05). The MVD level was an independent predicting factor of disease-free survival, 5-year disease-free survival in the hypovascular group (74.6%) being better than that in the hypervascular group (34.7%, P < 0.05). Conclusions: The MVD level was not related to tumor size, capsule statuo, Edmondsons grade, α-fetoprotein level, associated cirrhosis, γ-glutamyltransferase and serum HBsAg status. In the entire series, tumor size was the only factor influencing survival after curative resection. However, in patients with small HCC, the MVD level was an independent factor of disease-free survival. The pathological and clinical implications of different types of tumor vessels in HCC remain to be studied.

Overexpression of Platelet-Derived Growth Factor Receptor α in Endothelial Cells of Hepatocellular Carcinoma Associated with High Metastatic Potential

Purpose: Little information is available on the heterogeneity of the vascular endothelium in hepatocellular carcinoma. The aim of this study was to identify the differentially expressed genes in tumor endothelial cells from highly metastatic hepatocellular carcinoma. Experimental Design: Magnetic beads conjugated with anti-CD31 antibody were used to isolate vascular endothelial cells from hepatocellular carcinoma xenografts with different metastatic potentials in nude mice. Gene expression profiles for different endothelial cells were compared by use of cDNA microarray. The up-regulated gene was confirmed by reverse transcription-PCR, real-time PCR, and immunohistochemistry. Results: cDNA microarray analysis revealed differential expression patterns in seven genes consistently presented in endothelial cells isolated from hepatocellular carcinoma with different metastatic potentials. Overexpression of platelet-derived growth factor receptor α was found only in the endothelium of highly metastatic hepatocellular carcinoma, which was confirmed by reverse transcription-PCR, real-time PCR, and immunohistochemistry. Oral administration of STI571 (imatinib mesylate or Glivec), a protein tyrosine kinase inhibitor of platelet-derived growth factor receptor, combined with s.c. injection of IFN-α not only effectively reduced tumor weight (by 81.8%) and microvessel density (by 70.2%) but also inhibited lung metastasis (by 100%). Furthermore, immunohistochemical analysis of platelet-derived growth factor receptor α in human hepatocellular carcinoma tissues revealed its correlation with postoperative recurrence, especially in patients without microvessel invasion. Conclusions: The gene expression of hepatocellular carcinoma vascular endothelium is different between tumors with different metastatic potential. Platelet-derived growth factor receptor α, which is overexpressed in endothelium of highly metastatic hepatocellular carcinoma, may serve as a biomarker for predicting metastasis and a therapeutic target for highly metastatic hepatocellular carcinoma.

Hepatic RIG-I Predicts Survival and Interferon-α Therapeutic Response in Hepatocellular Carcinoma

In hepatocellular carcinoma (HCC), biomarkers for prediction of prognosis and response to immunotherapy such as interferon-α (IFN-α) would be very useful in the clinic. We found that expression of retinoic acid-inducible gene-I (RIG-I), an IFN-stimulated gene, was significantly downregulated in human HCC tissues. Patients with low RIG-I expression had shorter survival and poorer response to IFN-α therapy, suggesting that RIG-I is a useful prognosis and IFN-α response predictor for HCC patients. Mechanistically, RIG-I enhances IFN-α response by amplifying IFN-α effector signaling via strengthening STAT1 activation. Furthermore, we found that RIG-I deficiency promotes HCC carcinogenesis and that hepatic RIG-I expression is lower in men than in women. RIG-I may therefore be a tumor suppressor in HCC and contribute to HCC gender disparity.