Umeshita K

Treatment of hepatocellular carcinoma with major portal vein thrombosis by combined therapy with subcutaneous interferon-alpha and intra-arterial 5-fluorouracil; role of type 1 interferon receptor expression.

We previously reported the beneficial effects of combination therapy of interferon (IFN)-α/5-fluorouracil (FU) for advanced hepatocellular carcinoma (HCC) with tumour thrombi in the major portal branches. This report describes the results of longer follow-up and includes more than double the number of patients relative to the original report, and evaluates the role of IFN-α/type 2 interferon receptor (IFNAR2) expression on the response to the combination therapy. The study subjects were 55 patients with advanced HCC and tumour thrombi in the major branches of the portal vein (Vp3 or 4). They were treated with at least two courses of IFN-α/5-FU without major complication. In the 55 patients, 24 (43.6%) showed objective response (eight (14.5%) showed complete response, 16 (29.1%) partial response), four (7.3%) showed no response, and 27 (49.1%) showed progressive disease. Immunohistochemically, IFNAR2 expression was detected in nine out of 13 (69.2%) patients. There was significant difference in the time-to-progression survival (P=0.0002) and the overall survival (P<0.0001) between IFNAR2-positive and -negative cases. There was a significant correlation between IFNAR2 expression and response to IFN-α/5-FU combination therapy in univariate analysis (P=0.0070). IFN-α/5-FU combination therapy is a promising modality for advanced HCC with tumour thrombi in the major portal branches and could significantly depend on IFNAR2 expression.

MicroRNA-21 induces resistance to the anti-tumour effect of interferon-α/5-fluorouracil in hepatocellular carcinoma cells

Background:We reported recently the clinical efficiency of interferon (IFN)-α/5-fluorouracil (5-FU) combination therapy in advanced hepatocellular carcinoma (HCC). However, prediction of the response to the combination therapy remains unsatisfactory. The aim of this study was to investigate the anti-tumour effects of microRNA (miR)-21 on the sensitivity of HCC cells to IFN-α/5-FU and whether miR-21 can be used as a predictor of the response to such therapy in HCC.Methods:Changes in the sensitivity of HCC cells (PLC/PRF/5 and HepG2) to IFN-α/5-FU were examined after transfection with pre-miR-21 or anti-miR-21. The correlation between miR-21 expression level, evaluated by qRT–PCR, and response to the therapy was also investigated in clinical HCC specimens.Results:Hepatocellular carcinoma cells transfected with pre-miR-21 were significantly resistant to IFN-α/5-FU. Annexin V assay showed that the percentage of apoptotic cells was significantly lower in cells transfected with pre-miR-21 than control cells. Transfection of anti-miR-21 rendered HCC cells sensitive to IFN-α/5-FU, and such sensitivity was weakened by transfection of siRNAs of target molecules, PETN and PDCD4. miR-21 expression in clinical HCC specimens was significantly associated with the clinical response to the IFN-α/5-FU combination therapy and survival rate.Conclusions:The miR-21 in HCC cell lines and clinical HCC samples is a significant modulator of the anti-tumour effect of IFN-α and 5-FU. This suggests that miR-21 is a potentially suitable marker for the prediction of the clinical response to the IFN-α/5-FU combination therapy.

Activation of Wnt/ β -catenin signalling pathway induces chemoresistance to interferon- α /5-fluorouracil combination therapy for hepatocellular carcinoma

Type I IFN receptor type 2 (IFNAR2) expression correlates significantly with clinical response to interferon (IFN)-α/5-fluorouracil (5-FU) combination therapy for hepatocellular carcinoma (HCC). However, some IFNAR2-positive patients show no response to the therapy. This result suggests the possibility of other factors, which would be responsible for resistance to IFN-α/5-FU therapy. The aim of this study was to examine the mechanism of anti-proliferative effects of IFN-α/5-FU therapy and search for a biological marker of chemoresistance to such therapy. Gene expression profiling and molecular network analysis were used in the analysis of non-responders and responders with IFNAR2-positive HCC. The Wnt/β-catenin signalling pathway contributed to resistance to IFN-α/5-FU therapy. Immunohistochemical analysis showed positive epithelial cell adhesion molecule (Ep-CAM) expression, the target molecule of Wnt/β-catenin signalling, only in non-responders. In vitro studies showed that activation of Wnt/β-catenin signalling by glycogen synthesis kinase-3 inhibitor (6-bromoindirubin-3′-oxime (BIO)) induced chemoresistance to IFN-α/5-FU. BrdU-based cell proliferation ELISA and cell cycle analysis showed that concurrent addition of BIO and IFN-α/5-FU significantly to hepatoma cell cultures reduced the inhibitory effects of the latter two on DNA synthesis and accumulation of cells in the S-phase. The results indicate that activation of Wnt/β-catenin signalling pathway induces chemoresistance to IFN-α/5-FU therapy and suggest that Ep-CAM is a potentially useful marker for resistance to such therapy, especially in IFNAR2-positive cases.

The status of Fas and Fas ligand expression can predict recurrence of hepatocellular carcinoma

The status of Fas and Fas ligand (Fas L) expression was investigated in this study for 103 hepatocellular carcinomas (HCC). We studied the expression of the following three factors, Fas and Fas L expression in carcinoma cells and Fas L expression in stromal mononuclear cells (defined as stromal Fas L index). Fas expression in HCC cells was significantly decreased in cases with poor differentiation (P< 0.0001) and of larger size (P = 0.0058). Fas L expression in carcinoma cells was observed exclusively in moderately or poorly differentiated cases. Furthermore, each factor had prognostic significance for disease-free survival (DFS) (P< 0.0001, P = 0.0222 and 0.0027 respectively). We then scored the results of each factor and defined the total score as ‘Fas-Fas L risk score’. The P -value of the score for DFS was even lower than that of the clinical stage by multivariate analysis. These results suggest that the evaluation of Fas and Fas ligand expression potentially has a significant prognostic value for DFS of HCC patients, in addition to the clinical stage, and can be regarded as a new prognostic marker.

Both cell proliferation and apoptosis significantly predict shortened disease-free survival in hepatocellular carcinoma

In this study, we investigated the proliferating cell index by the percentage of Ki-67 expressing cells (Ki-67 LI) and the apoptotic index (AI) by the number of morphologically apoptotic cells per 1000 carcinoma cells in haematoxylin and eosin sections of 76 hepatocellular carcinomas (HCC). Both indices showed excellent correlation with each other (P < 0.0001) and were significantly higher in cases of poor differentiation, of advanced stages, with portal invasion and with intrahepatic metastasis. Furthermore, cases with higher Ki-67 LI or higher AI displayed poor outcomes for disease-free survival (P = 0.0001 and P = 0.0005) by univariate analysis. By multivariate analysis, both indices could be regarded as independent prognostic factors. These results strongly suggest that Ki-67 LI and AI have very similar clinical significance, reflecting the existence of biologically aggressive phenotypes and poor disease-free survival rate in HCC.

Protection of islet allografts transplanted together with Fas ligand expressing testicular allografts

Summary Fas ligand (FasL) is highly expressed in testicular tissues and thought to be responsible for protection from allograft rejection by inducing apoptosis of anti-graft activated T cells. FasL-expressing islets have been shown to induce a granulocyte-mediated inflammatory reaction. We investigated whether a graft can be protected from alloimmune responses by manipulating the Fas/FasL-system. We transplanted allogeneic islets under the kidney capsule of streptozotocin-induced diabetic mice together with testicular tissue. Significant prolongation of survival of C3H islet allograft was observed in C57BL/6 (B6) recipients transplanted with C3H testicular tissue, but not in those transplanted with C3H-gld testicular tissue expressing non-functional FasL. No significant prolongation was observed in B6-lpr recipients expressing non-functional Fas. Immunohistochemical staining of C3H testicular tissue in the composite graft showed a high expression of FasL, but not that of the C3H-gld testicular tissue. In situ terminal deoxynucleotidyl transferase-mediated dUDP-biotin catalysed DNA nick-end labelling (TUNEL) staining of a composite graft of C3H islet and testicular tissue in B6 recipients demonstrated extensive apoptosis of infiltrating mononuclear cells around the graft. The protective effect of C3H testicular tissue was abrogated when anti-FasL monoclonal antibody was administered i. p. postoperatively. Our results suggest that FasL-positive testicular allografts protect composite islet allografts and indicate that manipulation of Fas/FasL mediated apoptosis is a suitable strategy for controlling rejection of islet allografts. [Diabetologia (1998) 41: 315–321]

Relationship between pathologic prognostic factors and abnormal levels of des-γ-carboxy prothrombin and α-fetoprotein in hepatocellular carcinoma

Abstract The relationship between pathologic prognostic factors and abnormal levels of des-γ-carboxy prothrombin and α-fetoprotein was investigated in 42 patients with resectable hepatocellular carcinoma. The frequencies of macroscopic massive type, intrahepatic metastasis, and portal vein tumor thrombus were significantly higher in patients with positive des-γ-carboxy prothrombin (p

Insulin-like growth factor-binding protein 7 alters the sensitivity to interferon-based anticancer therapy in hepatocellular carcinoma cells

Background:A striking efficiency of interferon (IFN)-based anticancer therapy for advanced hepatocellular carcinoma (HCC) has been reported. Because its clinical efficiency greatly depends on each patients local response, prediction of local response is crucial.Methods:Continuous exposure of IFN-α to parental PLC/PRF/5 cells (PLC-P) and a limiting dilution method resulted in the establishment of IFN-resistant cell clones (PLC-Rs). Microarray analyses of PLC-P and PLC-Rs identified insulin-like growth factor-binding protein 7 (IGFBP7) as one of the most significantly downregulated genes in PLC-Rs. Changes in anticancer effects of IFN-α were examined in HCC cells after genetic manipulation of IGFBP7 expression. The correlation between immunohistochemically determined IGFBP7 expression and the response to IFN-α/5-fluorouracil (5-FU) therapy was investigated in surgically resected HCC specimens.Results:PLC-R cells showed a remarkable downregulation of IGFBP7 and resistance to IFN-α, compared with PLC-P. Parental PLC/PRF/5 cells transfected with short hairpin RNA against IGFBP7 showed a significant resistance to IFN-α relative to control cells (IC50 fold increase=14.38 times). Insulin-like growth factor-binding protein 7 transfection into PLC-R restored sensitivity to IFN-α. In resected specimens, IGFBP7 expression significantly correlated with the response to IFN-α/5-FU therapy.Conclusion:IGFBP7 could be a useful predictor of the response to IFN-based therapy in advanced HCC.

Age as an independent risk factor for surgical site infections in a large gastrointestinal surgery cohort in Japan

A large scale survey was conducted to examine risk factors for surgical site infections (SSIs) among Japanese patients undergoing gastrointestinal surgery. The purposes of the study were: (i) to investigate age as a risk factor for SSIs in gastrointestinal surgery; and (ii) to examine the differences in risk factors for SSIs between laparoscopic cholecystectomy and open cholecystectomy. Surveillance data were prospectively collected from 20 participating hospitals in Japan between July 2003 and November 2007. SSIs were identified by use of the Centers for Disease Control and Prevention criteria. SSIs were identified in 1471 of 12 015 available cases, with an overall incidence of 12.2%. In the final logistic regression model, age was a risk factor in open cholecystectomy, gastrectomy and appendicectomy. Length of operation was a risk factor for SSIs for six surgical procedures, and wound class and drain use were also risk factors in most procedures. When comparing laparoscopic surgery against open procedure, use of silk sutures was a risk factor for SSIs in laparoscopic cholecystectomy. Drain use, wound class, operation duration, male gender and age were additional risk factors for SSIs in open cholecystectomy. In summary, patient age is a significant predictor for SSIs in some gastrointestinal procedures, although risk factors for SSIs in laparoscopic procedures appear quite different from those in open procedures.

Clinical significance of hepatic resection in hepato-cellular carcinoma (HCC), analyzed by disease-free survival (DFS) curves

Hypothesis: The clinical significance of hepatectomy for hepatocellular carcinoma (HCC) is still controversial because of frequent intrahepatic recurrence, which results from either recurrence due to residual intrahepatic metastasis (Rim) or recurrence due to metachronous, multicentric liver carcinogenesis (Rmc). Design: Retrospective review. Disease-free survival curves were obtained by the Kaplan-Meier method and the rates of Rim and Rmc were analyzed using 2 regression lines, based on the evidence that Rmc occurs at a constant rate throughout follow-up, whereas Rim occurs only in the early postoperative period. Setting: University hospital.