Long non-coding RNA HOTAIR up-regulates chemokine (C-C motif) ligand 2 and promotes proliferation of macrophages and myeloid-derived suppressor cells in hepatocellular carcinoma cell lines.

Accumulating evidence demonstrated that Hox antisense intergenic RNA (HOTAIR) serves essential roles in the development and metastasis of several types of cancer. In hepatocellular carcinoma (HCC), high expression of HOTAIR is associated with poor prognosis, and HOTAIR regulates cell migration and proliferation. However, the downstream molecular targets of HOTAIR depend on the cancer cell types, and little is known about the precise molecular mechanisms of HOTAIR involved in cancer development. The present study investigated the role of HOTAIR in HCC cell lines. Notably, the overexpression of HOTAIR in HCC cell lines did not affect cell migration and proliferation capability. In the microarray analysis, C-C motif chemokine ligand (CCL)2 was identified to be differentially expressed in HOTAIR-overexpressing cells, and it was confirmed that HOTAIR promotes the secretion of CCL2. Furthermore, it was revealed that the proportion of macrophages and myeloid-derived suppressor cells (MDSCs) were increased when peripheral blood mononuclear cells were co-cultured with HOTAIR-overexpressing cells. Collectively, these data suggest that HOTAIR regulates CCL2 expression, which may be involved in the recruitment of macrophages and MDSCs to the tumor microenvironment.

Identification of Genotype 2 HCV in Serotype-1 Hepatitis C Patients Unresponsive to Daclatasvir plus Asunaprevir Treatment.

It is important to determine the genotypes or serotypes of hepatitis C virus (HCV) in patients before treatment with direct-acting antiviral agents (DAAs), because the effects of DAAs differ among genotypes. In Japan, two tests for HCV typing are available clinically, but only serotyping, not genotyping, is approved by the public health insurance. Although most serotype-1 Japanese patients are infected with genotype 1b HCV, it is known that a small proportion of patients show different results from two typing methods. This study focused on such patients and the effectiveness of treatment with daclatasvir plus asunaprevir (DCV/ASV) was evaluated. We analyzed 644 DCV/ASV-treated patients with serotype 1 or genotype 1b, and among them, 166 serotype-1 patients received a commercial-based direct sequencing (DS) test for resistant-associated variants of genotype 1b HCV. We found four patients (2.4%) with DS test failure, suggesting that the PCR primers targeting genotype 1b may not match. Importantly, none of the four patients achieved a sustained virological response. Our in-house DS test analyzing the 5'-untranslated region and coding regions for NS4 and NS5B of HCV showed that three of the four patients were infected with genotype 2 HCV, and one patient was infected with genotype 1a HCV. No recombinant virus of different genotypes was found. This study indicates that a subset of serotype-1 hepatitis C patients is infected with HCV of genotype 2 or 1a in Japan and that DCV/ASV is not effective for such patients. Thus, attention should be paid to DAA treatment without HCV genotyping.

PD-L1+MDSCs are increased in HCC patients and induced by soluble factor in the tumor microenvironment.

Myeloid-derived suppressor cells (MDSCs) could have important roles in immune regulation, and MDSCs can be induced in patients with various malignant tumors. The immune-suppressive functions of MDSCs in hepatocellular carcinoma (HCC) patients have not been clarified. Therefore, we tried to analyze the biological significance of MDSCs in HCC patients. We quantified PD-L1+MDSCs of HCC patients in various conditions by using multi-color flow cytometry analysis. PBMCs from HCC patients contained significantly higher percentages of PD-L1+MDSCs in comparison to those from healthy subjects (p < 0.001). The percentages of PD-L1+MDSCs were reduced by curative treatment for HCC (p < 0.05), and the percentages of PD-L1+MDSCs before treatment were inversely correlated with disease-free survival time. After we cocultivated PBMCs and several liver cancer cell lines in a transwell coculture system, the percentages of PD-L1+MDSCs were significantly increased compared with control (p < 0.05). The expression of M-CSF and VEGFA was higher in the cell lines that strongly induced PD-L1+MDSCs. Peripheral blood from HCC patients had significantly higher percentages of PD-L1+MDSCs in comparison to those of healthy subjects, and the percentages of PD-L1+MDSCs were reduced by HCC treatment, suggesting that we might use PD-L1+MDSCs as a new biomarker of HCC.

Reactivation of resolved hepatitis B virus infection with immune escape mutations after long-term corticosteroid therapy.

Hepatitis B virus (HBV) reactivation from resolved infection is a serious problem which can frequently lead to severe hepatitis. Generally, it occurs several months after the start of immunosuppressive therapy; however, it sometimes occurs a few years later, even after cessation of therapy. Here we report a patient with de novo HBV infection who had received corticosteroid therapy for pemphigus vulgaris for 6 years. Full-genome HBV sequence analysis using serial serum samples revealed that the patient was infected with HBV subgenotype C2, which had the G1896R mixed mutation in the precore region. Interestingly, it had the immune escape mutations P120A and G145R in the S gene. Because both hepatitis B surface antigen and antibodies to hepatitis B surface antigen (HBsAb) were positive at the onset of the de novo infection, it was considered that HBV with these mutations escaped from neutralization by the pre-existing HBsAbs. This case indicates that HBV reactivation with an immune escape mutant can occur long after immunosuppressive therapy.

Shifting hepatitis B virus genotypes of acute hepatitis B patients in northeast Japan.

It has been reported that acute hepatitis B (AHB) patients with genotype A HBV (HBV/A) have been increasing since the 1990s in metropolitan areas in Japan. However, little is known about the trends of HBV genotypes in AHB patients in northeast Japan where genotype B HBV (HBV/B) prevails more than in other areas. In this study, we aimed to clarify the changes in the HBV genotypes and clinical characteristics of AHB patients in this area. HBV genotypes were determined by direct sequencing (n = 125) or enzyme immunoassay (n = 9) using serum samples from AHB patients including fulminant hepatitis in 1987-2014. Among 134 patients, 26 (19%), 33 (25%), and 75 (56%) patients were infected with HBV of genotypes A, B, and C, respectively. HBV/A emerged from 2001 and the percentage was increased gradually up to 48% in 2010-2014, whereas HBV/B was reduced from 40% in 1987-1994 to 10% in 2010-2014. Phylogenetic analysis showed that three major subgenotype A2 strains had come into this area serially. The levels of HBV DNA and prothrombin time were higher in HBV/A patients than other genotypes. This study could not show significant difference in the HBsAg-positive period among genotypes nor the effect of nucleoside analogues to shorten the HBsAg-positive period. A higher level of initial HBV DNA was associated with late disappearance of HBsAg. In conclusion, the percentage of HBV/A in AHB patients has been increasing in northeast Japan since 2001, which is later than metropolitan areas, whereas that of HBV/B is decreasing.

Radiation Therapy Is a Reasonable Option for Improving the Prognosis in Hepatocellular Carcinoma.

Radiation therapy (RT) may be suitable for treating patients with hepatocellular carcinoma (HCC) who are difficult to treat with any other option. However, it remains unclear whether RT extends survival in these patients. Among the 957 HCC patients treated at Tohoku University Hospital from January 2007 to December 2013, only 49 patients received RT. We therefore retrospectively analyzed the outcomes of these patients; they were divided into three groups based on the reasons for choosing RT: 27 patients at Stage IV A (67.1 ± 1.6 years, 50.5 ± 2.1 Gy), 9 patients with alternative therapy (72.2 ± 2.4 years, 58.9 ± 1.1 Gy), and 13 patients who received RT after transarterial chemoembolization (TACE) (75.6 ± 2.1 years, 56.5 ± 1.5 Gy). RT was employed to ensure the local control of the lesion. The patients at Stage IV A were treated with radical RT (n = 16) or with palliative RT (n = 11). In radical RT group, the response rate was 37.5% and the complete response rate was 25%. The survival rate was 12.5 ± 2.6 months after radical RT. This is considered relatively good for Stage IV A. The disease-free survival rate was 13.0 ± 2.8 months after RT. This excellent disease-free survival indicates that RT is an alternative to other treatments. In the TACE group, patients who received the RT had the significantly long disease-free survival rate than only-TACE (18.0 ± 3.8 months vs. 11.2 ± 0.58 months). We propose that RT is effective and safe for HCC.

Differential Expression of CX3CL1 in Hepatitis B Virus-Replicating Hepatoma Cells Can Affect the Migration Activity of CX3CR1+ Immune Cells.

UNLABELLED: In addition to stellate cells and immune cells, inflamed hepatocytes and hepatoma cells express various kinds of chemokines that attract various kinds of immune cells. Previously, we reported that hepatitis B virus (HBV) replication can induce physiological stress. The aim of this study was to analyze the effect of chemokines produced by HBV-infected hepatocytes and hepatoma cells. A real-time PCR array targeting genes related to chemokines and enzyme-linked immunosorbent assay (ELISA) were carried out to detect the specific chemokines produced by Huh7 cells and HepG2 cells infected with various HBV genotypes. A migration assay, flow cytometry analysis, and immunohistochemistry were carried out to analyze the candidate immune cells that can affect the immunopathogenesis of HBV infection. The expressions of CX3CL1 mRNA and protein were significantly different among HBV genotypes A, B, and C and control cells (mock) (P < 0.05). CD56(+) NK cells and CD8(+) T cells migrated to the hepatoma cells with HBV replication. Moreover, the migration activity of both immune cells was partially cancelled after the treatment of CX3CL1 neutralizing antibody. The expression level of NKG2D on CX3CR1(+) NK cells in HCC with HBV infection was significantly lower than that in hepatocellular carcinoma (HCC) with HCV infection and chronic hepatitis B and C patients (P < 0.05). On the other hand, the frequency of PD-1(high) CX3CR1(+) CD8(+) T cells in HCC with HBV infection was significantly higher than that in HCC with HCV infection and chronic hepatitis B and C (P < 0.05). The expression of CX3CL1 in HBV-replicating hepatocytes and hepatoma cells could contribute to the immunopathogenesis of HBV infection. IMPORTANCE: The progressions of the disease are significantly different among HBV genotypes. However, it has not been clear that how different HBV genotypes could induce different inflammatory responses. Here, we first report that the levels of expression of CX3CL1 mRNA and protein were significantly different among HBV genotypes A, B, and C and mock. Not only the differential expression of CX3CL1 among the genotypes but also the phenotype of CX3CR1(+) NK cells and T cells were gradually changed during the progression of the disease status. In addition to in vitro study, the analysis of immunohistochemistry with human samples and NOG mice with human lymphocytes and hepatoma cells supports this phenomenon. The quantification of CX3CL1 could contribute to better understanding of the disease status of HBV infection. Moreover, modifying CX3CL1 might induce an immune response appropriate to the disease status of HBV infection.

[A case of liver cirrhosis due to non-alcoholic steatohepatitis complicated by myotonic dystrophy].

A female in her 50s with a four-year history of myotonic dystrophy was admitted to our hospital with hematochezia. She was diagnosed with synchronous colonic cancer of the transverse and sigmoid colon, for which she underwent partial transverse and sigmoid colectomy, respectively. Postoperative respiratory failure resulted in prolonged stay in the intensive care unit. Her liver and renal function gradually deteriorated, and she eventually died from these sequelae on postoperative day 26. Intraoperative liver biopsy revealed cirrhosis arising from non-alcoholic steatohepatitis (NASH). Although myotonic dystrophy is believed to be a multisystem disease, its association with cirrhosis has not been reported in Japan. We therefore report this rare case of liver cirrhosis arising from NASH in a patient with myotonic dystrophy.

[A case of huge papillary adenocarcinoma of the gallbladder with marked necrosis].

A 71-year-old woman was admitted to our hospital for evaluation of a right upper abdominal tumor. A contrast-CT scan demonstrated a huge tumor extending from the hepatic hilum to the pelvic space. The rim of tumor was enhanced. The center of the tumor was not enhanced and thus considered to consist of mucus or necrotic tissues. Preoperative diagnosis as gallbladder carcinoma without infiltrating to peripheral organ was made and subsequent cholecystectomy with full-thickness dissection has been performed. The tumor itself was in a swollen gallbladder, 18 cm in diameter, consisting of necrotic tissues in the lumen. Pathologic diagnosis was papillary adenocarcinoma, classified as pHinf1a, revealing fStage II. In many cases with undifferentiated carcinoma of the gallbladder, the neoplasms grow expansively to become large tumors with marked necrosis. We report a rare case of papillary adenocarcinoma of the gallbladder presenting both a clinical course and radiologic findings indistinguishable from undifferentiated carcinoma of the gallbladder.