Comprehensive Profiling of EBV Gene Expression and Promoter Methylation Reveals Latency II Viral Infection and Sporadic Abortive Lytic Activation in Peripheral T-Cell Lymphomas.

Epstein-Barr virus (EBV) latency patterns are well defined in EBV-associated epithelial, NK/T-cell, and B-cell malignancies, with links between latency stage and tumorigenesis deciphered in various studies. In vitro studies suggest that the oncogenic activity of EBV in T-cells might be somewhat different from that in EBV-tropic B lymphoid cells, prompting us to study this much less investigated viral gene expression pattern and its regulation in nine EBV+ peripheral T-cell lymphoma (PTCL) biopsies. Using frozen specimens, RT-PCR showed 6/7 cases with a latency II pattern of EBV gene expression. Analyses of EBNA1 promoter usage and CpG methylation status in these six cases showed that only Qp was used, while Cp, Wp, and Fp were all silent. However, the remaining case showed an exceptionally unique latency III type with lytic activation, as evidenced by EBV lytic clonality and confirmed by the full usage of Cp and Qp as well as weakly lytic Fp and Wp, fully unmethylated Cp and marginally unmethylated Wp. Further immunostaining of the eight cases revealed a few focally clustered LMP1+ cells in 7/8 cases, with rare isolated LMP1+ cells detected in another case. Double immunostaining confirmed that the LMP1+ cells were of the T-cell phenotype (CD3+). In 6/8 cases, sporadically scattered Zta+ cells were detected. Double staining of EBER-ISH with T-cell (CD45RO/UCHL1) or B-cell (CD20) markers confirmed that the vast majority of EBER+ cells were of the T-cell phenotype. Predominant type-A EBV variant and LMP1 30-bp deletion variant were present, with both F and f variants detected. In summary, the EBV gene expression pattern in PTCL was found to be mainly of latency II (BART+EBNA1(Qp)+LMP1+LMP2A+BZLF1+), similar to that previously reported in EBV-infected nasopharyngeal epithelial, NK/T-cell, and Hodgkin malignancies; however, fully lytic infection could also be detected in occasional cases. Rare cells with sporadic immediate-early gene expression were commonly detected in PTCL. These findings have implications for the future development of EBV-targeting therapeutics for this cancer.

Effects of a novel immunomodulating agent, FTY720, on tumor growth and angiogenesis in hepatocellular carcinoma.

In this study, we aimed to evaluate the potential anticancer and antiangiogenic effects of FTY720 on hepatocellular carcinoma. In vitro, chemosensitivity was tested on hepatoma cells, nontumorigenic, immortalized hepatocyte cells, as well as human umbilical vein endothelial cells (HUVEC). Moreover, effect of FTY720 on cell cycle and apoptosis was analyzed. In addition, a number of angiogenesis-associated assays were carried out. The in vivo effect of the drug on hepatocellular carcinoma tumor growth on nude mice was studied. Tissues obtained were analyzed in terms of proliferation, apoptosis, tumor microvessel density, and tumor vascular permeability. Compared with the MIHA cells, the hepatoma cell lines as well as HUVECs were found to be highly sensitive to the drugs in the aspect that FTY720 could induce G(1) arrest and apoptosis in the hepatoma cells. Furthermore, FTY720 significantly decreased invasion, migration, and capillary tube formation of HUVECs at very low doses. In vivo study showed that tumor growth was significantly suppressed in the FTY720-treated animals, and staining of the tissue sections showed decreased tumor cell proliferation and increased tumor cell apoptosis in the treatment groups. Interestingly, significant reductions in tumor microvessel density and tumor vascular permeability were also found in the FTY720-treated groups. In conclusion, FTY720 not only shows potent antiangiogenic effects but is also cytotoxic toward hepatoma cells. Results from our preclinical study suggest that FTY720 can be selected as a good candidate for the treatment of hepatocellular carcinoma.

Significance of circulating endothelial progenitor cells in hepatocellular carcinoma.

This study evaluated the significance of circulating bone marrow-derived endothelial progenitor cells (EPCs) in patients with hepatocellular carcinoma (HCC), a solid tumor with rich neovasculature. Eighty patients with HCC were recruited for the study, and 16 patients with liver cirrhosis and 14 healthy subjects were also included for comparison. Blood samples were taken before treatment. Total mononuclear cells were isolated from peripheral blood, preplated to eliminate mature circulating endothelial cells, and colony-forming units (CFUs) formed by circulating EPCs were counted. To validate the CFU scores, FACS quantification of EPCs using CD133, VEGFR2, and CD34 as markers was performed in 30 cases. Our study showed significantly higher mean CFU scores in patients with HCC compared to patients with cirrhosis and healthy controls (P = .001 and .009, respectively). Furthermore, the CFU scores of patients with HCC positively correlated with levels of serum alpha-fetoprotein (r = .303, P = .017), plasma VEGF (r = .242, P = .035), and plasma interleukin-8 (IL-8) (r = .258, P = .025). Patients with unresectable HCC had higher CFU scores than patients with resectable tumors (P = .027). Furthermore, for those who underwent curative surgery, higher preoperative CFU scores were observed in patients with recurrence within 1 year compared with those who were disease-free after 1 year (P = .013). In conclusion, higher circulating levels of EPCs are seen in patients with advanced unresectable HCC as compared to patients with resectable HCC or those with liver cirrhosis. Our evidence supports the potential use of circulating level of EPCs as a prognostic marker in patients with HCC.