Wun-Ching Yu

Tumor Microvessel Density as a Predictor of Recurrence After Resection of Hepatocellular Carcinoma: A Prospective Study

PURPOSE This study prospectively evaluated the correlation of tumor microvessel density (MVD) with clinicopathologic features and postoperative recurrence in patients undergoing resection of hepatocellular carcinoma (HCC). PATIENTS AND METHODS Tumor MVD was assessed in 100 patients with resection of HCC using a computer image analyzer after immunostaining for CD34 (MVD-CD34) and von Willebrand factor (MVD-vWF), respectively. Patients were prospectively followed for recurrence. RESULTS Mean tumor MVD-CD34 (236/0.74 mm(2)) was higher than mean tumor MVD-vWF (87/0.74 mm(2)) (P <.001). By multiple regression analysis, tumor size was the only pathologic feature significantly related to tumor MVD-CD34. The median MVD-CD34 was 316/0.74 mm(2) in HCCs < or = 5 cm (n = 46) and 146/0.74 mm(2) in HCCs more than 5 cm (n = 54) (P <.001). Among patients with HCCs < or = 5 cm, those with higher than median MVD-CD34 had worse disease-free survival (at 3 years, 13%) than those with a lower MVD-CD34 (at 3 year, 74%) (P =.002). Multivariate analysis showed that tumor MVD-CD34 was the only significant factor predictive of disease-free survival in patients with HCC < or = 5 cm. For HCCs more than 5 cm, MVD-CD34 did not have a significant prognostic influence. MVD-vWF did not have a significant prognostic influence on disease-free survival in either HCCs < or = 5 cm or more than 5 cm. CONCLUSION This study shows that a high MVD-CD34 was predictive of early postresection recurrence in patients with HCCs < or = 5 cm and, therefore, may be a novel prognostic marker in this subset of patients.

Macrophage migration inhibitory factor: Roles in regulating tumor cell migration and expression of angiogenic factors in hepatocellular carcinoma

Macrophage migration inhibitory factor (MIF) may contribute to multiple aspects of tumor progression, including control of cell proliferation, differentiation, cell survival and angiogenesis. However, the potential roles of MIF in regulating hepatocellular carcinoma (HCC) tumor cell migration and the expression of angiogenic factors by HCC tumor cells have not been studied yet. In our study, we reported that intracellular MIF mRNA and protein were overexpressed in HCC tissues compared to nontumor tissues by using in situ hybridization and immunohistochemic staining. HCC tumor cell lines also secreted large amounts of MIF into the supernatants of tumor cell culture. To assess the role of MIF in HCC, we employed the transwell invasion chamber to study the effect of MIF on tumor cell migration. Our results showed that recombinant MIF and the supernatants of tumor cell line culture could enhance the invasion and migration of HCC cells. This effect can be inhibited by the addition of a neutralizing anti‐MIF antibody. We observed that increased MIF serum levels correlated with higher levels of interleukin‐8 (IL‐8) in the sera of patients with HCC than in normal volunteers. We therefore hypothesized that MIF may regulate the production of angiogenic factors by HCC cells. To test this hypothesis, we examined the effect of MIF treatment on vascular endothelial growth factor (VEGF) and IL‐8 expression by HCC cell lines. MIF induced a significant dose‐dependent increase in IL‐8 and VEGF production. Taken together, our results indicated that MIF may act as an autocrine‐acting factor that stimulates angiogenesis and metastasis in HCC by promoting expression of angiogenic factors and migration of tumor cells. A more detailed understanding of the MIF regulatory mechanisms involved may provide insight into new direction in the treatment of HCC.

Correlation of serum basic fibroblast growth factor levels with clinicopathologic features and postoperative recurrence in hepatocellular carcinoma

BACKGROUND Basic fibroblast growth factor (bFGF) is an important positive regulator of tumor angiogenesis. This study evaluated the role of serum bFGF as a biological marker of tumor invasiveness and postresection recurrence in hepatocellular carcinoma (HCC). METHODS Concentrations of bFGF in preoperative serum samples in 88 patients undergoing resection of HCC were measured by a quantitative enzyme-linked immunosorbent assay. A single pathologist performed histopathologic examination of all tumor specimens. All patients were prospectively monitored for tumor recurrence. RESULTS The preoperative serum bFGF levels ranged from <0.22 to 71.2 pg/mL (median 10.8 pg/mL). There was significant correlation between high serum bFGF levels and large tumor >5 cm, presence of venous invasion or advanced pTNM stage. Patients with a serum bFGF level >10.8 pg/mL had worse disease-free survival than those with a level <10.8 pg/mL (median disease-free survival 11.2 versus 20 months, P = 0.044). Serum bFGF level >10.8 pg/mL (P = 0.035) and tumor size >5 cm (P = 0.004) were independent preoperative factors that predicted early recurrence after resection of HCC. CONCLUSIONS This study supports a role of bFGF in tumor growth and invasion in HCC. A high preoperative serum bFGF level appears to be predictive of invasive tumor and early postoperative recurrence. The clinical implications of serum bFGF level in HCC warrant further investigation.

Long‐term oral branched chain amino acids in patients undergoing chemoembolization for hepatocellular carcinoma: a randomized trial

Background : Patients undergoing transarterial chemoembolization for hepatocellular carcinoma have advanced tumour or severe cirrhosis and frequently have associated protein‐calorie malnutrition. The role of nutritional supplements for such patients is unclear.

SPARC and Hevin expression correlate with tumour angiogenesis in hepatocellular carcinoma

Both Secreted Protein Acidic and Rich in Cysteine (SPARC) and Hevin are multifunctional matricellular glycoproteins. Recent experimental studies suggested that Hevin and SPARC together diminish angiogenesis, but their significance in hepatocellular carcinoma (HCC) remains unclear. This study aimed to correlate SPARC and Hevin expression with angiogenesis and clinicopathological features in HCC. SPARC and Hevin protein and mRNA expression in HCC specimens were assessed by immunostaining, immunoblotting, and quantitative reverse transcriptase‐polymerase chain reaction. Tumour microvessel density (MVD) was assessed by CD34 immunostaining. The role of SPARC and Hevin in HCC was further assessed in an in vivo nude mice xenograft model. Both SPARC and Hevin mRNA levels were significantly higher in tumours than in non‐tumourous livers. A significant correlation between tumour SPARC and Hevin mRNA levels was found. Moreover, SPARC protein localized in the tumour sinusoidal area correlated significantly with Hevin protein localized in HCC cells. Truncated forms of SPARC and Hevin proteins were detected in clinical samples. Truncated SPARC protein localized in the tumour sinusoidal area correlated significantly with tumour MVD. On the other hand, overexpression of full‐length SPARC in tumour xenografts in athymic nude mice significantly delayed tumour growth, and this delay was related to a decrease in tumour angiogenesis. Expression of Hevin protein within HCC cells was related to the presence of tumour encapsulation and the absence of hepatitis B surface antigen in clinical samples. Overexpression of Hevin in tumour xenografts also significantly delayed tumour growth. In conclusion, this study has shown that SPARC and Hevin are upregulated in HCC compared with non‐tumourous liver, and that they are inter‐related at both mRNA and protein levels. Moreover, both SPARC and Hevin were related to HCC angiogenesis and tumour progression. Copyright

T lymphocyte function in patients with malignant biliary obstruction

Abstract The T lymphocyte function in 59 patients with malignant biliary obstruction undergoing pre‐operative endoscopic drainage (group Ia, n= 24) or surgery (group Ib, n= 35) was evaluated by mitogen stimulation test with phytohaemagglutinin. The T lymphocyte function before endoscopic or surgical intervention was found to be impaired as compared with patients with gastric cancer (group II, n= 27) and with normal persons (group III, n= 19). Regression analysis showed a significant negative correlation between T lymphocyte function and the serum bilirubin level (correlation coefficient ‐ 0.3, P= 0.01) and a positive correlation with serum albumin level (correlation coefficient 0.34, P= 0.01) and serum transferrin level (correlation coefficient 0.45, P= 0.001). After 18 ± 3 days of endoscopic biliary drainage, the T lymphocyte function of group Ia patients did not change substantially. At postoperative day 14, there were more patients in both groups Ia and Ib having deterioration of T lymphocyte function than those with improvement. The incidence of postoperative sepsis was found to be significantly higher in patients with deterioration than those with improvement of T lymphocyte function (18/31 vs 7/26, P= 0.036). It is concluded that endoscopic biliary drainage and surgery could not reverse the T lymphocyte dysfunction in patients with malignant biliary obstruction.

Abstract 5346: Identification of essential genes for the development of hepatitis B virus-associated hepatocellular carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background and Aim: Hepatocellular carcinoma (HCC) is a highly lethal and prevalent cancer, posing a grave threat to human health globally. Hepatitis B virus (HBV) infection is considered as a major risk factor for this cancer, especially in the Asia-Pacific region. Unfortunately, the molecular mechanisms of hepatocarcinogenesis remain obscure, which hinders the development of effective therapies for the disease. In the present study, we attempted to elucidate the molecular details of HBV-induced hepatocarcinogenesis by investigating differentially regulated genes at multiple developmental stages of HCC in a HBV transgenic mouse model. Materials and Methods: The transgenic mice which overproduced HBV large envelope polypeptide in hepatocytes and developed liver tumors spontaneously were used in this study. To unravel transcriptomics dynamics underlying hepatocarcinogenesis, RNA prepared from livers of both transgenic and wild type mice of different ages (at months 2, 12, 18 and 19) were subjected to RNA sequencing. Selected target genes were first validated by quantitative PCR (qPCR) using a larger set of mouse liver tissues (n=96) collected from 8 time points. Clinical implications of the selected genes were then explored in a set of human liver samples comprising 18 normal, 29 cirrhosis and 96 pairs of HCC. RNA and protein expression levels were determined by qPCR, immunohistochemical staining and Western blotting, respectively. Results: Upon analysis of 20,209 gene transcripts, 2574 and 1035 transcripts were found to be up-regulated (≥2 folds) and down-regulated (≤2 folds) in tumors, respectively, when compared with the wild type controls. Among these, 133 most prominent genes that exhibited concordant differential expression throughout the stages of tumor progression were chosen for validation in mouse liver tissues. Correlation analysis showed a high correlation between RNA sequencing and qPCR data (r=0.7495; P<0.0001), indicating a high validity of the data. Forty-six biologically informative genes were further validated in human liver samples. By Gene Ontology analysis, the target genes were revealed to play roles in a variety of biological processes including stress and inflammation responses, metabolic and apoptotic processes. Immunohistochemical staining and Western blotting demonstrated significant differential expression of these genes between HCC and non-tumorous livers. Statistical analyses revealed their significant correlation with clinicopathological parameters including venous infiltration, tumor size and overall survival, implicating their roles in hepatocarcinogenesis. Conclusion: This study has demonstrated a systematic strategy for identifying crucial genes for HBV-associated HCC, which may have profound implications in combating this deadly cancer. Citation Format: CT Lam, ZF Yang, JC Lau, MN Ng, WC Yu, DW Ho, ST Fan. Identification of essential genes for the development of hepatitis B virus-associated hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5346. doi:10.1158/1538-7445.AM2014-5346