Zhen-Fan Yang

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.

Linking Inflammation to Acute Rejection in Small‐For‐Size Liver Allografts: The Potential Role of Early Macrophage Activation

This study aims to investigate the immunological status of small‐for‐size liver allografts and possible mechanism that contributes to the accelerated immune response in these allografts. Eight experimental groups were: whole isografts; 40% isografts; whole allografts, no treatment; 40% allografts, no treatment; whole allografts with sodium salicylate intraperitoneal injection, D0‐3; 40% allografts with sodium salicylate, D0‐3; whole allografts with FK506 intramuscular injection D0‐3, and 40% allografts with FK506, D0‐3. The 40% allografts survived significantly shorter than whole allografts (p = 0.02). At 72 h after reperfusion, a higher number of macrophages infiltrated into the periportal area of small‐for‐size allografts than whole allografts. Remarkable up‐regulation of interleukin‐1β (IL‐1β), interleukin‐2 (IL‐2), interleukin‐10 (IL‐10) and interferon‐γ (IFN‐γ) messenger RNA (mRNA) levels were detected in small‐for‐size allografts within 24 h after reperfusion. Sodium salicylate administration reduced IL‐1β and IFN‐γ mRNA in both small‐for‐size and whole allografts, but it could decrease IL‐2 and IL‐10 mRNA levels only in small‐for‐size allografts. In vitro study revealed that CD80, CD86 and CD11b expression on macrophages was augmented after IL‐1β stimulation, whereas the up‐regulation could be blocked by sodium salicylate. In conclusion, early activation of macrophages as a result of graft injury might play an important role in the accelerated acute rejection process in small‐for‐size allografts.

Therapeutic efficacy of Traditional Chinese Medicine 319 recipe on hepatic fibrosis induced by carbon tetrachloride in rats

BACKGROUND/AIMS Hepatic fibrosis is a consequence of severe liver damage that occurs in many patients with chronic liver diseases. TCM 319 recipe is a Chinese Medicine formula which consists of six Chinese herbs. In this study, we investigated the anti-fibrotic efficacy and mechanisms of TCM 319 recipe. METHODS Hepatic fibrosis in rats was induced by carbon tetrachloride (CCl4). 34 male adult SD rats were allocated into five groups (group 1-concomitant CCl4 and TCM 319 recipe for 8 weeks; group 2-CCl4 for 4 weeks and then CCl4 and TCM 319 recipe for 4 weeks; group 3-CCl4 alone for 8 weeks; group 4-TCM 319 recipe only for 8 weeks; group 5-untreated controls). After 8 weeks of treatment, serum ALT assay, liver tissue histological examination and immunostaining were carried out to examine the liver function and fibrosis degree. The expression levels of platelet derived growth factor (PDGF-B), PDGF-Rbeta, and transforming growth factor-beta 1 (TGF-beta1) were measured by quantitative RT-PCR and western blot. RESULTS TCM 319 recipe reduced liver injury and attenuated hepatic fibrosis in group 1 compared with that in group 3. TCM 319 recipe suppressed the mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1). In addition, treatment with TCM 319 recipe significantly down-regulated mRNA expression of PDGF-B and PDGF-Rbeta, and it also suppressed protein expression of PDGF-Rbeta and TGF-beta1. CONCLUSIONS TCM 319 recipe extracts could attenuate hepatic fibrosis induced by CCl4 in rats. The anti-fibrotic effect of TCM 319 recipe is associated with the down-regulation of mRNA expression of TIMP-1, PDGF-B and PDGF-Rbeta, and with the suppression of protein expression of PDGF-Rbeta and TGF-beta1.

Long-term liver allograft survival induced by combined treatment with rAAV-hCTLA4Ig gene transfer and low-dose FK506.

Background. Recombinant adeno-associated virus vector (rAAV) is a promising vehicle for gene delivery, but few reports have documented its application in solid organ transplantation. In a rat orthotopic liver transplantation model, we investigated the efficacy of rAAV-mediated human cytotoxic T-lymphocyte-associated antigen 4 and immunoglobulin G (hCTLA4Ig) gene transfer to induce long-term allograft survival. Methods. Dark Agouti and Lewis rats were used as donors and recipients, respectively, in six experimental groups: (a) syngeneic control, (b) no treatment, (c) rAAV-green fluorescent protein, (d) rAAV-hCTLA4Ig, (e) low-dose FK506 for 7 days, and (f) rAAV-hCTLA4Ig and low-dose FK506 for 7 days. Results. The liver allografts were rejected within 10 days when no treatment was given or rAAV-green fluorescent protein was delivered. rAAV-hCTLA4Ig transduction slightly prolonged the survival time to 11 days. Long-term survival was achieved using the combined treatment of rAAV-hCTLA4Ig and low-dose FK506, whereas grafts were rejected on day 33 in the low-dose FK506 group. A sustained hCTLA4 level in plasma was detected in the combined treatment group from day 5 to day 180. On postoperative day 5, combined treatment significantly decreased the interleukin-2 and interferon-&ggr; protein levels in the grafts and the number of infiltrating B, T, CD25+, CD4+, CD8+, and NK cells. Conclusion. This study shows that rAAV-hCTLA4Ig gene transfer combined with low-dose FK506 can achieve long-term liver allograft survival.

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

Abstract 3862: The potential role of CD44 in liver regeneration

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background and Aim: Liver regeneration occurs after liver damage by ischaemia, hepatitis, or in hepatocellular carcinoma (HCC) patients receiving hepatic resection or liver transplantation. Understanding the molecular bases of this process will provide novel therapeutic approaches for patients with various liver diseases. Previous studies suggested the linkage between CD44, a multifunctional cell surface receptor, and liver regeneration. The detailed role of this receptor in liver regeneration, however, remained unclear. The present study aimed at elucidating the role of CD44 in liver regeneration. Materials and Methods: A mouse model of liver regeneration induced by partial hepatectomy (PH) was employed. Serum and liver tissues were collected at different time points after PH. ELISA was performed to measure hyaluronic acid (ligand for CD44) level in serum. Flow cytometry was done to characterize liver cell populations after PH. Liver regeneration was quantified by measurement of liver mass and by immunohistochemical staining for PCNA (a proliferation marker). Quantitative PCR was performed to compare gene expression pattern of 46 genes between CD45− CD44+ cells and their CD44− counterparts. Results: Firstly, serum hyaluronic acid level was elevated at the early stage of liver regeneration. Secondly, by flow cytometry, an increase in CD45− CD44+ cell population was found. Thirdly, immunohistochemical analysis revealed active hepatocyte proliferation during this period. Interestingly, the hepatectomy-induced cell proliferation was suppressed after treating mice with anti-CD44 antibody, suggesting a functional role of CD44 in liver regeneration. Lastly, dysregulation of genes involving in cell proliferation, angiogenesis, and metabolism of hyaluronic acid was disclosed in CD45− CD44+ cells. Conclusion: The present study indicated that CD44-mediated pathways are required for liver regeneration, which may have therapeutic implications in treating liver diseases, including HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3862. doi:1538-7445.AM2012-3862