Effects of interferon-α-transduced tumor cell vaccines and blockade of programmed cell death-1 on the growth of established tumors.

Interferon-alpha (IFN-α) has strong antitumor effects, and IFN-α gene therapy has been used clinically against some cancers. In this study, we evaluated the efficacy of the combination of IFN-α-transduced tumor cell vaccines and programmed cell death 1 (PD-1) blockade, and investigated the mechanisms of the antitumor effects of the combined therapy. A poorly immunogenic murine colorectal cancer cell line, MC38, was transduced to overexpress IFN-α. In a therapeutic model, parental tumor-bearing mice were inoculated with MC38-IFNα cells and an anti-PD-1 antagonistic antibody. Analyses of immunohistochemistry and tumor-specific lysis were performed. The outgrowth of the established tumors was significantly reduced in mice treated with the combination of IFN-α and anti-PD-1. Immunohistochemical analyses of the therapeutic model showed marked infiltration of CD4(+) cells and CD8(+) cells in the established MC38 tumors of mice treated with both IFN-α and anti-PD-1. Significant tumor-specific cytolysis was detected when splenocytes of mice that were treated with both IFN-α and anti-PD-1 were used as effector cells. These results suggest that blockade of the PD-1 PD-ligand enhanced the Th1-type antitumor immune responses induced by IFN-α. The combination of IFN-α gene-transduced tumor cell vaccines and PD-1 blockade may be a possible candidate for a cancer vaccine for clinical trials.

Lymphotropic hepatitis C virus has an interferon-resistant phenotype.

Hepatitis C virus (HCV) infects and associates with B cells, leading to abnormal B-cell activation and development of lymphoproliferative and autoimmune disorders. This immune perturbation may in turn be associated with the resistance of HCV against the host immune system. The objective of this study was to analyse the effects of HCV infection of B cells on the efficacy of interferon (IFN)-based therapy. The study enrolled 102 patients with chronic hepatitis C who were treated with pegylated IFN plus ribavirin. HCV RNA titres in B cells were compared in patients with rapid viral responder (RVR) vs non-RVR, sustained viral responder (SVR) vs non-SVR and null viral responder (NVR) vs VR. The levels of HCV RNA in B cells were significantly higher in non-RVR, non-SVR and NVR groups. Association between the therapy outcome and the positive B-cell HCV RNA was also investigated in relation to other known viral and host factors. Multivariable analyses showed that the positive B-cell HCV RNA and the minor single-nucleotide polymorphism near the IL28B gene (rs8099917) were independent factors associated with NVR in patients infected with HCV genotype 1. When these two factors were combined, the sensitivity, specificity, positive and negative predictive values for NVR were 92.3%, 98.2%, 92.3% and 98.2%, respectively. Genotype 1 and the presence of one or no mutations in the IFN-sensitivity determining region were associated with higher levels of B-cell HCV RNA. B-cell-tropic HCV appears to have an IFN-resistant phenotype. B-cell HCV RNA positivity is a predictive factor for resistance to IFN-based therapy.

Interleukin-1beta gene in esophageal, gastric and colorectal carcinomas.

Interleukin (IL)-1 gene polymorphisms are associated with development of gastric atrophy and with increased risk of gastric carcinoma. A -31C to T base transition in the promoter region of this gene is involved in carcinogenic changes within the stomach, especially in Helicobacter pylori infected individuals. We examined association between IL-1 locus polymorphisms and risk of esophageal, gastric and colorectal carcinomas in Japanese patients with H. pylori infection. IL-1B and IL-1RN polymorphisms were analyzed in 136 controls, 75 patients with esophageal carcinoma, 186 patients with gastric carcinoma, 69 patients with colorectal carcinoma, and 18 patients with ulcerative colitis (UC). For IL-1B-511 and -31 polymorphisms were determined by fluorescence-based polymerase chain reaction single-strand conformation polymorphism analysis. For IL-1 receptor antagonist gene (IL-1RN), penta-allelic variable number of tandem repeats (VNTR) was determined by PCR-standard agarose gel electrophoresis. For gastric carcinoma, IL-1B-511 heterozygotes (OR, 0.48; 95% CI, 0.3-0.9; p=0.0115) and T carriers (OR, 0.52; 95% CI, 0.3-1.0; p=0.0185) had a significantly reduced risk of carcinoma. For colorectal carcinoma, IL-1B-511 heterozygotes (OR, 0.34; 95% CI, 0.2-0.7; p=0.0028) and T carriers (OR, 0.43; 95% CI, 0.2-0.9; p=0.0015) had a significantly low risk of carcinoma. No significant difference was observed in the frequencies of IL-1B-31C/T and IL-1RN genotypes between controls and the esophageal carcinoma patients. Our results shows that IL-1B-511C/T and T carrier state may indicate less risk for gastric and colorectal carcinoma in the Japanese population.

Study of p53 gene alteration as a biomarker to evaluate the malignant risk of Lugol-unstained lesion with non-dysplasia in the oesophagus.

Mutations of the p53 gene are detected frequently in oesophageal dysplasia and cancer. It is unclear whether Lugol-unstained lesions (LULs) with non-dysplastic epithelium (NDE) are precursors of oesophageal squamous cell carcinoma (ESCC). To study the genetic alterations of NDE in the multistep process of oesophageal carcinogenesis, we determined the relationship between p53 mutations and LULs-NDE. Videoendoscopy with Lugol staining was performed prospectively in 542 oesophageal cancer-free subjects. Lugol-unstained lesions were detected in 103 subjects (19%). A total of 255 samples, including 152 LULs (NDE, 137; dysplasia, 15) and 103 paired samples of normal staining epithelium, were obtained from 103 subjects. After extraction of DNA and polymerase chain reaction analysis, direct sequencing method was applied to detect mutations of the p53 gene. The p53 mutation was detected in five of 137 samples with LULs-NDE (4%) and in five of 15 samples with dysplasia (33%). A hotspot mutation was found in 20% of LULs-NDE with p53 mutation and in 40% of dysplasia with p53 mutation. In contrast, no p53 mutations were found in 103 paired NDE samples with normal Lugol staining. In biopsy samples from oesophageal cancer-free individuals, the p53 missense mutations containing a hotspot mutation were found in NDE, which was identified as an LUL. These findings suggest that some LULs-NDE may represent the earliest state of oesophageal squamous cell carcinoma in Japanese individuals.

Evaluation of paraumbilical vein as a prognostic index of severe liver cirrhotic patients with portal-systemic shunts.

AIM: The aim of this study was to predict the outcome in severe liver cirrhotic patients with portal-systemic shunts. METHODS: One-hundred and sixteen patients with liver cirrhosis diagnosed as Child-Pugh class B and C with portal-systemic shunts confirmed by abdominal ultrasonography, computed tomography and magnetic resonance imaging were enrolled in this study. Twenty-three factors were evaluated concerning clinical laboratory parameters and extracted prognostic factors using the Cox proportional hazards model, and the prognostic index (PI) was prepared by combining these factors. RESULTS: The cumulative survival rates after admission were 64.6%, 35.6% and 25% after 1, 3 and 5 years, respectively. Using multivariate analysis, age, the presence of hepatocellular carcinoma (HCC), portal vein tumor thrombosis (PVTT) and paraumbilical vein (PUV) shunt were selected as significant prognostic factors that contributed independently to the prognosis of severe liver cirrhotic patients with portal-systemic shunts. The PI was calculated with the following formula using these 4 factors. PI = 0.042 x Age + 0.913 x HCC + 0.989 x PVTT + 1.079 x PUV shunt. The group with a high score for PI was found to die with significantly higher frequency than the group with a low score. CONCLUSIONS: It was found that tumor related factors and PUV shunt were the most important factors for severe liver cirrhotic patients with portal-systemic shunts. The PI is suggested to be an appropriate index to predict the prognosis for these patients.

Prognostic factors of primary biliary cirrhosis detected by health screening.

AIM: The liver cirrhosis is likely to differ in the Japanese and Western populations. Thus, we performed a retrospective cohort analysis by a review of clinical records to clarify prognostic factors after the onset of primary biliary cirrhosis (PBC) detected by health screening. METHODS: The subjects were 52 patients with PBC. Thirty-nine factors were evaluated concerning clinical data and extracted prognostic factors using the Cox proportional hazard model. RESULTS: The mean duration of the follow-up period was 5.1 years, during which 6 (11.5%) of the patients died. The cumulative survival rate after the onset of PBC was 93.4% after 5 year, and 67.8% after 10 years. Multivariate analysis indicated 2 factors, i.e. the body mass index (BMI), and IgG, as independent prognostic factors. Their hazard ratios were 0.399 (per 1 kg/m2 of BMI) and 1.282 (per 100 mg/dL of IgG). The prognostic index (PI) was calculated by the following formula using these 2 factors. PI = 0.919 x BMI+0.249 x IgG. CONCLUSIONS: The prediction of the outcome using PI based on the 2 factors provides additional information for the determination of the therapeutic approach in PBC after health screening.

BRAF mutations and phosphorylation status of mitogen-activated protein kinases in the development of flat and depressed-type colorectal neoplasias.

Although some molecular differences between flat-depressed neoplasias (FDNs) and protruding neoplasias (PNs) have been reported, it is uncertain if the BRAF mutations or the status of phosphorylated mitogen-activated protein kinase (p-MAPK) are different between theses two groups. We evaluated the incidence of BRAF and KRAS mutations, high-frequency microsatellite instability (MSI-H), and the immunohistochemical status of p-MAPK in the nonserrated neoplasias (46 FDNs and 57 PNs). BRAF mutations were detected in four FDNs (9%) and none of PNs (P=0.0369 by Fisher's exact test). KRAS mutations were observed in none of FDNs and in 14 PNs (25%; P=0.0002 by Fisher's exact test). MSI-H was detected in seven out of 44 FDNs (16%) and in one out of 52 of PNs (2%) (P=0.022 by Fisher's exact test). Type B and C immunostaining for p-MAPK was observed in 34 out of 46 FDNs (72%), compared with 24 out of 55 PNs (44%; P=0.0022 by chi(2) test). There was no significant difference in the type B and C immunostaining of p-MAPK between FDNs with and without BRAF mutations. BRAF and KRAS mutations are mutually exclusive in the morphological characteristics of colorectal nonserrated neoplasia. Abnormal accumulation of p-MAPK protein is more likely to be implicated in the tumorigenesis of FDNs than of PNs. However, this abnormality in FDNs might occur via the genetic alteration other than BRAF or KRAS mutation.

Dendritic cell therapy with interferon-alpha synergistically suppresses outgrowth of established tumors in a murine colorectal cancer model.

Both dendritic cell (DC)-based immunotherapy and interferon (IFN)-alpha therapy have been proved to have potent long-lasting antitumor effects. In anticipation of synergistic antitumor effects, we performed combination therapy with DCs and IFN-alpha gene-transduced murine colorectal cancer MC38 cells (MC38-IFN-alpha). DCs incubated with MC38-IFN-alpha, but not neomycin-resistance gene-transduced MC38 cells (MC38-Neo), effectively enhanced proliferation of allogeneic splenocytes in vitro. In 12 of 17 mice, DCs in combination with MC38-IFN-alpha prevented the development of a parental tumor, while DCs and MC38-Neo did in only three of 17 mice (P=0.008). In a therapeutic model of an established parental tumor, inoculation of DCs and MC38-IFN-alpha suppressed the growth of the established parental tumors significantly compared with the administration of DCs with MC38-Neo or naive splenocytes with MC38-IFN-alpha (P=0.016 and 0.024, respectively). Analyses of immunohistochemistry and tumor-infiltrating mononuclear cells showed that CD8(+), CD11c(+), and NK1.1(+) cells markedly infiltrated the established tumors of mice treated with DCs and MC38-IFN-alpha. From the results of observation of parental tumor outgrowth in immune cell-depleted mice, CD8(+) cells, and asialo-GM-1(+) cells were thought to contribute to the antitumor effects induced by the combination therapy. Furthermore, MC38-specific cytolysis was detected when splenocytes of mice inoculated with DCs and MC38-IFN-alpha cells were stimulated with MC38-IFN-alpha cells in vitro. Since DC-based immunotherapy in combination with IFN-alpha-expressing tumor cells induces potent antitumor cellular immune responses, it should be considered for clinical application.

Interleukin-4 gene transduced tumor cells promote a potent tumor-specific Th1-type response in cooperation with interferon-alpha transduction.

To investigate antitumor mechanisms in interleukin (IL)-4 therapy, we established an IL-4-overexpressing MC38 murine colorectal cancer cell line (MC38-IL4). As a therapy against established tumors, MC38-IL4 cells were inoculated contralaterally 7 days after wild-type (MC38-WT) cells had been injected, significantly reducing growth of wild-type tumors (P=0.030). Immunohistochemical analysis showed numerous granulocytes infiltrating wild-type tumors of MC38-IL4-inoculated mice. Injection of MC38-IL4 cells in leukocyte-depleted mice confirmed that granulocytes were involved in IL-4-related primary antitumor effects. Inoculation of MC38-WT in leukocyte-depleted mice initially injected with MC38-IL4 suggested that T cells contributed to the antitumor effects. To investigate tumor-specific responses, we stimulated splenocytes of MC38-immune mice with MC38-IL4 cells in vitro, resulting in MC38-specific lysis (57.5+/-7.2%, effector to target ratio=20). Treatment of established wild-type tumors with MC38-IL4 in combination with interferon (IFN)-alpha-overexpressing MC38 cells (MC38-IFNalpha) significantly reduced the growth of wild-type tumors (P=0.009). In vitro IFN-gamma production by splenocytes from mice injected with both MC38-IL4 and -IFNalpha was greatly enhanced in comparison with MC38-IL4 alone, while IL-10 production was not increased. Thus, granulocytes concern early antitumor effects of IL-4 therapy. Subsequently, IL-4 induces long-lasting, tumor-specific immune responses. IL-4 appears to promote a T-helper 1-type antitumor immune response, which is enhanced in cooperation with IFN-alpha.

Pathological features and genetic alterations in colorectal carcinomas with characteristics of nonpolypoid growth.

We sought to clarify pathological features and genetic alterations in colorectal carcinomas with characteristics of nonpolypoid growth. Colorectal carcinomas resected at Showa University Hospital in Tokyo included 86 with characteristics of polypoid growth (PG) and 21 with those of nonpolypoid growth (NPG). Mutations of APC, Ki-ras, and p53 genes, as well as microsatellite instability (MSI), were analysed using fluorescence-based polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Carcinomas with an NPG pattern were smaller than PG tumours (P<0.0001). Carcinomas with a PG pattern were more likely to harbour Ki-ras mutations (36%) than NPG tumours (0%; P<0.0001). Mutation types in the APC gene differed significantly between PG and NPG carcinomas (P=0.0189), including frameshift mutations in 66% of PG carcinomas but no NPG carcinomas. Presence of a p53 mutation at a 'hot spot' also was more likely in PG carcinomas (37%) than in NPG carcinomas (0%; P=0.0124). No significant difference in presence of MSI was evident between carcinomas with PG and NPG patterns. In conclusion, significant genetic differences were evident between carcinomas with PG and NPG patterns. Genetic changes in NPG carcinomas differed from those of the conventional adenoma-carcinoma sequence. Assuming that some nonpolypoid growth lesions transform rapidly into advanced carcinomas, 20% of all colorectal carcinomas may progress in this manner.

Hepatitis B core antigen stimulates interleukin-10 secretion by both T cells and monocytes from peripheral blood of patients with chronic hepatitis B virus infection.

In chronic hepatitis B virus (HBV) infection, immune responses to hepatitis B core antigen (HBcAg) are weak. Interleukin (IL)-10 is a potent immunosuppressive cytokine which we reported recently to be secreted in response to HBcAg by peripheral blood mononuclear cells (PBMCs) from patients with chronic HBV infection or healthy controls. Using an enzyme-linked immunospot assay, we compared the ability of HBcAg to stimulate IL-10 production by PBMC with that of lipopolysaccharide (LPS), phytohaemagglutinin-P and hepatitis C virus-derived antigens in 16 patients with chronic HBV infection and six healthy controls. Frequencies of IL-10 spot-forming cells (SFC) in response to HBcAg were comparable to those obtained with LPS in patients with chronic HBV infection. Frequencies of IL-10 SFC in response to HBcAg or to LPS were significantly higher in patients with chronic HBV infection than in healthy controls. IL-10 SFC in response to HBcAg consisted of 26-35% T cells, 62-70% monocytes and less than 1% B cells in patients with chronic HBV infection. Only monocytes contributed to IL-10 production in controls. Frequencies of HBcAg stimulated IL-10 SFC representing T cells and monocytes were significantly higher in patients with elevated serum alanine aminotransferase (ALT) and detectable HBV DNA than in patients with normal ALT and undetectable HBV DNA. The potent ability of HBcAg to stimulate IL-10 production by PBMC may contribute importantly to immune tolerance toward HBV.

[Interferon-treated hepatitis C virus(HCV) patients with sustained biochemical response without eradication of HCV(asymptomatic HCV carrier)].

Patients with hepatitis C virus(HCV) responding differently to interferon(IFN) therapy were speculated to have different incidence of disease progression to cirrhosis and of the development of hepatocellular carcinoma(HCC). However, the background and prognosis of the patients with sustained biochemical response without eradication of HCV (BR) (asymptomatic HCV carrier) has not been revealed so far. Review of recent studies suggest that the characteristics of the patients with BR are lower HCV RNA load, higher rate of HCV subtype-2 and lower score of liver fibrosis when compared with those with NR. The IFN therapy in patients who have not cleared HCV and showed normal ALT retards progression of fibrosis and reduces the incidence of cirrhosis and HCC.

Contrast-enhanced intraductal ultrasonography for thickened bile duct wall.

PURPOSE: We carried out this study to evaluate the usefulness of contrast-enhanced intraductal ultrasonography (ceIDUS) in the differentiation of thickened bile duct wall at the hepatic bifurcation caused by malignant tumor from that caused by cholangitis. METHODS: Seven patients (two with primary sclerosing cholangitis [PSC], one with secondary sclerosing cholangitis [SSC], and four with bile duct carcinomas [BDC] at the hepatic bifurcation underwent endoscopic ceIDUS, in which we used Levovist. The recorded images of echo-brightness were analyzed histographically. RESULTS: The bile duct wall, in PSC and SSC, but not in BDC, was enhanced by Levovist. CONCLUSION: ceIDUS with histographic analysis may be useful for distinguishing thickened bile duct wall caused by malignant tumor from that caused by cholangitis.

Rapid decrease of plasma HCV RNA in early phase of twice daily administration of 3 MU doses interferon-beta in patients with genotype 1b hepatitis C infection: a multicenter randomized study.

Virological response to interferon (IFN) is poor in patients with plasma levels of HCV RNA higher than 1 Meq/ml and genotype 1b hepatitis C viral infection. In 60 patients, a randomized control study was conducted to compare 3 MU of IFN-beta twice daily for four weeks (group A) and 6 MU once a day for four weeks (group B) followed by a four-week administration of 6 MU once a day. The plasma levels of HCV RNA, determined by an amplicore-monitor method, for patients in group A were significantly lower than those for group B at the fourth and eighth day of IFN administration, and complete virological responses were noted in two patients from group A but none in group B. It is concluded that twice daily administration of 3 MU IFN-beta is more effective than once a day 6 MU in the early phase of IFN therapy.

HCV-core protein accelerates recovery from the insensitivity of liver cells to Fas-mediated apoptosis induced by an injection of anti-Fas antibody in mice.

BACKGROUND/AIMS: Hepatitis C virus (HCV) is a major etiologic agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The aim of this study was to elucidate pathological effects of HCV-core protein on liver cells. METHODS: We have generated transgenic mice carrying HCV-core cDNA (Px-core) and pathologically examined livers of Px-core mice. RESULTS: HCV-core protein was detectable in livers from lines 5 (C5) and 8 (C8) of Px-core transgenic mice. Since chronic hepatitis and cirrhosis precede hepatocellular carcinoma in patients with HCV infection, we tried to examine the effect of repetitive injection of a small dose of anti-Fas antibody in the transgenic mice. Surprisingly, an initial injection of anti-Fas antibody induced resistance of liver cells to the second injection of anti-Fas antibody in both Px-core and littermate control mice. The insensitivity of liver cells induced in the control mice continued for more than 24 weeks after the first injection but was broken within 1 week after partial hepatectomy. However, the sensitivity was restored in the Px-core mice within 12 weeks after the injection. CONCLUSION: HCV-core protein in liver cells may affect persistence of Fas-mediated liver cell injury.

Duodenal stenosis after endoscopic biliary metallic stent placement for malignant biliary stenosis.

BACKGROUND: Some patients who undergo endoscopic insertion of biliary metallic stents for malignant biliary stenosis later develop symptomatic duodenal stenosis due to tumor invasion. METHODS: We compared the development of symptomatic duodenal stenosis in patients who had undergone endoscopic biliary metallic stent insertion (metallic stent group) with that in patients who had undergone either endoscopic biliary drainage or percutaneous transhepatic biliary drainage with a plastic stent (nonmetallic stent group). Fourteen patients in the metallic stent group were matched with 14 patients in a nonmetallic stent group. All patients had a Karnofsky performance status score of greater than 90% and were clinical stage IV when they underwent biliary decompression. RESULTS: Although there was no difference in survival time between the 2 groups, 5 of 14 patients in the metallic stent group developed symptomatic duodenal stenosis due to tumor invasion during the observation period whereas this occurred in only 1 of 14 patients in the nonmetallic stent group. Multiple logistic regression analysis indicates that the type of stent (p = 0.022) and survival time (p = 0.002) are 2 independent prognostic factors for the development of symptomatic duodenal stenosis. CONCLUSIONS: Patients treated with endoscopic biliary metallic stent insertion are prone to develop symptomatic duodenal stenosis due to tumor invasion compared with those treated with either endoscopic retrograde biliary drainage or percutaneous transhepatic biliary drainage with a plastic stent.

Fatal hepatic failure caused by chemotherapy-induced reactivation of hepatitis B virus in a patient with hematologic malignancy.

A patient with hematologic malignancy and hepatitis B virus (HBV) infection received chemotherapy containing a glucocorticoid. The patient developed severe hepatitis after chemotherapy and, despite achieving complete remission of the malignancy, died of hepatic failure. We carried out a retrospective study of changes in the serological markers of HBV in this patient. Both serum hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (HBsAb) were negative on admission. During the course of chemotherapy, HBsAg gradually became positive, but no liver dysfunction was apparent until after completion of the chemotherapy. The patient showed no initial evidence of being a latent HBV carrier. Therefore, we believe that screening for HBsAg is insufficient for detecting latent HBV carriers, and that investigation for hepatitis B core antibody (HBcAb) is essential.