Masatoshi Imamura

Genome-wide association of IL28B with response to pegylated interferon-α and ribavirin therapy for chronic hepatitis C

The recommended treatment for patients with chronic hepatitis C, pegylated interferon-α (PEG-IFN-α) plus ribavirin (RBV), does not provide sustained virologic response (SVR) in all patients. We report a genome-wide association study (GWAS) to null virological response (NVR) in the treatment of patients with hepatitis C virus (HCV) genotype 1 within a Japanese population. We found two SNPs near the gene IL28B on chromosome 19 to be strongly associated with NVR (rs12980275, P = 1.93 × 10−13, and rs8099917, 3.11 × 10−15). We replicated these associations in an independent cohort (combined P values, 2.84 × 10−27 (OR = 17.7; 95% CI = 10.0–31.3) and 2.68 × 10−32 (OR = 27.1; 95% CI = 14.6–50.3), respectively). Compared to NVR, these SNPs were also associated with SVR (rs12980275, P = 3.99 × 10−24, and rs8099917, P = 1.11 × 10−27). In further fine mapping of the region, seven SNPs (rs8105790, rs11881222, rs8103142, rs28416813, rs4803219, rs8099917 and rs7248668) located in the IL28B region showed the most significant associations (P = 5.52 × 10−28–2.68 × 10−32; OR = 22.3–27.1). Real-time quantitative PCR assays in peripheral blood mononuclear cells showed lower IL28B expression levels in individuals carrying the minor alleles (P = 0.015).

Interferon Therapy after Tumor Ablation Improves Prognosis in Patients with Hepatocellular Carcinoma Associated with Hepatitis C Virus

Context Hepatocellular carcinoma often follows hepatitis C virus infection. Currently available treatments for hepatocellular carcinoma are unsatisfactory. Percutaneous ethanol injection therapy into tumor nodules shows some promise, but recurrence rates are high. Contribution In a carefully selected group of 74 patients with multicentric hepatocellular carcinoma, mild hepatitis C, and mild cirrhosis, patients randomly assigned to receive interferon in addition to ethanol injections showed improved survival at 5 and 7 years, particularly among patients with a sustained virologic response. Cautions Combined treatment of multicentric hepatocellular carcinoma offers the possibility of enhanced survival for carefully selected patients; this study is small, however, and enrolled only patients with low virus levels and mild cirrhosis. The Editors Chronic hepatitis C virus (HCV) infection is a common, frequently asymptomatic disease. Despite the clinically quiescent course of HCV infection, it may slowly progress to cirrhosis and, eventually, to hepatocellular carcinoma (1, 2). Cirrhosis is a major risk factor for the development of hepatocellular carcinoma (3, 4), and 70% to 80% of patients with hepatocellular carcinoma in Japan have HCV infection (5). Current strategies for treating hepatocellular carcinoma include surgical resection, transarterial embolization, percutaneous ethanol injection therapy, radiofrequency wave ablation, and chemotherapy (6-9). Recent studies have shown that percutaneous ethanol injection therapy is effective for hepatocellular carcinoma when the tumors are small (<3 to 5 cm in diameter) and limited in number; survival rates are similar to those obtained with surgery (10-12). Five-year survival rates, however, are poor (30% to 60% for both hepatectomy and percutaneous ethanol injection therapy). Poor prognosis may be the result of the high incidence of tumor recurrence; the cumulative recurrence rate at 5 years is 60% to 100% (10-13). Several studies have evaluated the factors that contribute to the recurrence of hepatocellular carcinoma (12, 13). Occasionally, early recurrence develops adjacent to the treated lesion (local recurrence, 6% to 33% depending on tumor size) (14), but most tumors (80% to 90%) recur at different sites (15). Because hepatocellular carcinoma recurrence and decompensation of underlying liver disease are major problems after medical or surgical treatment, liver transplantation is another option for treating small, unresectable hepatocellular carcinomas in patients with cirrhosis. Studies report 5-year survival rates as high as 75% with liver transplantation (16-18). Interferon therapy has beneficial effects in chronic HCV infection (19, 20). In long-term follow-up studies, sustained virologic responders have remained in remission with normal liver function and improved histologic features of inflammation; in some of these responders, fibrosis even regresses (21, 22). Recently, the frequency of hepatocellular carcinoma in patients receiving interferon therapy has substantially decreased, especially in patients with sustained virologic and biochemical responses (23-25). This decreased frequency has occurred even in patients with cirrhosis (25, 26). Our study evaluated whether complete ablation of neoplastic nodules and administration of antiviral therapy could increase survival rates. Methods Study Design Our prospective study was designed by an eight-member committee in December 1992. The Ethics Committee of the University of Tokyo approved the study. We obtained informed consent from each patient in accordance with the Helsinki declaration. Patients with compensated cirrhosis, three or fewer nodules of hepatocellular carcinoma, and low HCV RNA loads were recruited after complete ablation of the lesions. Eligibility Criteria Inclusion Criteria Hepatitis C virus infection was diagnosed on the basis of identification of anti-HCV antibody using the passive hemagglutination test (Dinabbot, Tokyo, Japan) or enzyme-linked immunosorbent assay (ELISA; Ortho Diagnostic Systems, Tokyo, Japan). Hepatitis C virus RNA was identified by reverse transcriptase polymerase chain reaction (RT-PCR). The serum HCV RNA level was measured by competitive reverse transcriptase (CRT)-PCR according to the method of Kato and colleagues (27); HCV genotype was determined by the method of Okamoto and colleagues (28). Hepatocellular carcinoma was suspected on the basis of several imaging methods, including abdominal ultrasonography, dynamic computed tomography (CT), magnetic resonance imaging (MRI), and arteriography. We confirmed the diagnosis by histologic examination of tumor biopsy specimens obtained from all patients. Evaluation was based on the criteria of the International Working Party (29). In addition, we obtained and evaluated biopsy specimens from non-neoplastic lesions according to the methods of Desmet and colleagues (30). Hepatocellular carcinoma was treated with percutaneous ethanol injection therapy (7, 8, 10). Real-time linear-array scanners were used with 3.5-MHz transducers for the sonographic guidance of needles [21-gauge with a 15-cm or 20-cm needle; Hanako, Tokyo, Japan] into the tumors. Two to 10 mL of 99.5% ethanol was injected into each lesion. Ethanol injection was repeated several times at different sessions. Complete destruction of the nodules was confirmed on dynamic CT 1 month after ethanol injection according to the following criteria: 1) The destructive area was larger than the area of the tumor nodule shown on pretreatment dynamic CT and 2) dynamic CT showed no early-phase contrast enhancement of nodules (7, 8, 10). Inclusion criteria were as follows: 1) hepatocellular carcinoma with three or fewer lesions [verified by histologic examination] and dynamic CTconfirmed complete ablation of hepatocellular carcinoma lesions by percutaneous ethanol injection therapy, 2) detection of HCV RNA by RT-PCR and an HCV RNA load of 2 106 copies/mL or less by CRT-PCR (the cutoff value was based on unpublished data indicating that interferon treatment was effective in patients with HCV RNA loads of 105 copies/50 L of serum by CRT-PCR [27]], 3) platelet count of 50 109 cells/L, 4) leukocyte count of 3 109 cells/L or greater, 5) compensated cirrhosis in ChildPugh stage A, 6) age younger than 70 years, 7) no previous treatment with interferon, and 8) submission of informed consent. Exclusion Criteria Exclusion criteria were as follows: 1) liver diseases due to other causes, such as hepatitis B or primary biliary cirrhosis; 2) HCV RNA load of 2 106 copies/mL or greater by CRT-PCR; 3) severe comorbid diseases, such as heart disease, lung disease, or diabetes mellitus; 4) decompensated cirrhosis in ChildPugh stage B or C; and 5) failure to obtain informed consent. Randomization Patients who enrolled in the study were randomly assigned in a 2:1 ratio to the interferon group or the control group by the controller. We assigned patients to the treatment group or control group by using a randomization list. Interferon Therapy and Follow-up of Patients Interferon Therapy We started interferon therapy with natural interferon- (Sumitomo Pharmaceuticals, Tokyo, Japan) 2 to 3 months after tumor ablation was confirmed. Patients received 6 million U of interferon by intramuscular injection three times weekly for 48 weeks as outpatients. If patients could not tolerate this dose, the interferon dose was reduced to 3 million U. If HCV RNA in serum was still detected by RT-PCR (detection limit, 102 copies/mL) after 24 weeks of interferon therapy and serum alanine aminotransferase (ALT) levels were higher than pretreatment ALT levels, therapy was discontinued. Criteria for Interferon Response We defined the efficacy of interferon therapy virologically and biochemically. Patients who were negative for HCV RNA (as determined by RT-PCR; detection limit, 102 copies/mL) more than 6 months after the completion of interferon therapy were classified as showing a sustained virologic response. Patients with persistently normal ALT levels after the completion of interferon therapy were classified as showing a sustained biochemical response; patients with abnormal ALT levels were classified as showing a nonsustained biochemical response. Follow-up Patients attended a monthly medical consultation at the University of Tokyo Hospital outpatient clinic. Blood biochemical measures, including -fetoprotein (AFP) tumor markers, were measured every 1 to 2 months; ultrasonography was performed every 2 to 3 months; and dynamic CT was performed every 6 months. Recurrence of hepatocellular carcinoma was detected by the finding of abnormal nodules with low or high echogenic appearance on abdominal ultrasonography or by the finding of abnormal density on dynamic CT. The diagnosis was confirmed histologically through ultrasonography-guided fine-needle biopsy of the tumor. Recurrent nodules were divided into two categories [14, 15]: 1) local recurrence, in which the nodule appeared adjacent to the previously treated nodules, suggesting that residual tumor cells had not been completely ablated by percutaneous ethanol injection therapy, or 2) new foci developing at a distant site. New foci of hepatocellular carcinoma, as well as local recurrent nodules at tumor, node, metastasis (TNM) stage I, II, and III, were mainly treated by a second course of percutaneous ethanol injection therapy; local recurrent nodules at TNM stage IV were treated with transarterial chemoembolization or chemotherapy. New development of hepatocellular carcinoma and survival of the patients (tumor recurrence rate and survival rate) were analyzed in relation to the time interval after initial treatment. Statistical Analysis When estimating the sample size, we assumed that 5-year survival in the control group would be 40% according to the data of our previous unpublished study. We predicted that 5-year survival would be increased by 35% as a result of treatment

Des‐γ‐carboxy prothrombin as a useful predisposing factor for the development of portal venous invasion in patients with hepatocellular carcinoma

Portal venous invasion (PVI) in patients with hepatocellular carcinoma (HCC) is an important factor affecting prognosis. The objective of this study was to elucidate predisposing factors for the development of PVI.

The rs8099917 Polymorphism, When Determined by a Suitable Genotyping Method, Is a Better Predictor for Response to Pegylated Alpha Interferon/Ribavirin Therapy in Japanese Patients than Other Single Nucleotide Polymorphisms Associated with Interleukin-28B

ABSTRACT We focused on determining the most accurate and convenient genotyping methods and most appropriate single nucleotide polymorphism (SNP) among four such polymorphisms associated with interleukin-28B (IL-28B) in order to design tailor-made therapy for patients with chronic hepatitis C virus (HCV) patients. First, five different methods (direct sequencing, high-resolution melting analysis [HRM], hybridization probe [HP], the InvaderPlus assay [Invader], and the TaqMan SNP genotyping assay [TaqMan]) were developed for genotyping four SNPs (rs11881222, rs8103142, rs8099917, and rs12979860) associated with IL-28B, and their accuracies were compared for 292 Japanese patients. Next, the four SNPs associated with IL-28B were genotyped by Invader for 416 additional Japanese patients, and the response to pegylated interferon/ribavirin (PEG-IFN/RBV) treatment was evaluated when the four SNPs were not in linkage disequilibrium (LD). HRM failed to genotype one of the four SNPs in five patients. In 2 of 287 patients, the results of genotyping rs8099917 by direct sequencing differed from the results of the other three methods. The HP, TaqMan, and Invader methods were accurate for determination of the SNPs associated with IL-28B. In 10 of the 708 (1.4%) patients, the four SNPs were not in LD. Eight of nine (88.9%) patients whose rs8099917 was homozygous for the major allele were virological responders, even though one or more of the other SNPs were heterozygous. The HP, TaqMan, and Invader methods were suitable to determine the SNPs associated with IL-28B. The rs8099917 polymorphism should be the best predictor for the response to the PEG-IFN/RBV treatment among Japanese chronic hepatitis C patients.

Does dual infection by hepatitis B and C viruses play an important role in the pathogenesis of hepatocellular carcinoma in Japan

There are contradictory data concerning the synergistic effect of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection on the progression from chronic hepatitis to hepatocellular carcinoma (HCC).

Unique clinical characteristics of patients with hepatocellular carcinoma who present with high plasma des‐γ‐carboxy prothrombin and low serum α‐fetoprotein

Although the importance of α‐fetoprotein (AFP) and des‐γ‐carboxy prothrombin (DCP) in the clinical treatment of hepatocellular carcinoma (HCC) has been studied extensively, the authors examined the clinical picture of HCC with regard to the state of these two tumor markers.

Quantitation of α‐fetoprotein and albumin messenger RNA in human hepatocellular carcinoma

To analyze gene expression of α‐fetoprotein (AFP) and albumin in hepatocellular carcinoma (HCC), messenger RNAs (mRNAs) of these proteins in six human hepatoma cell lines and in 30 cases of HCC were quantitatively analyzed by competitive reverse transcription (RT) followed by polymerase chain reaction (PCR). The transcriptional levels of both AFP and albumin genes in HepG2 and Huh 7 cell lines were 1010 copies/μg RNA, in contrast to approximately 105 copies/μg RNA in HLE and HLF cell lines. AFP and albumin mRNA levels in three normal livers were 105 and 1010 transcripts/μg RNA, respectively. In 30 cases with HCC AFP mRNA level in neoplasm was 10 to 105‐fold enhanced as compared with that of nonneoplastic portion, and correlated with serum AFP level and tumor size (P < .01). In contrast, albumin mRNA level was not reduced in the neoplasms presenting enhanced AFP mRNA levels, indicating that AFP and albumin gene expression in situ is not necessarily mutually exclusive. Prospective analysis revealed that an increased serum AFP was shown at the time of recurrence among patients with enhanced AFP mRNA levels in neoplasm only, indicating that AFP mRNA levels in neoplasm could be a clinically predictable tool.

Evaluation of transcatheter arterial embolization prior to percutaneous tumor ablation in patients with hepatocellular carcinoma: a randomized controlled trial.

Abstract: Background: Transcatheter arterial embolization (TAE) may reduce the risk of hepatocellular carcinoma (HCC) recurrence when performed before percutaneous tumor ablation (PTA), either percutaneous ethanol injection therapy (PEIT) or radiofrequency ablation (RFA). We conducted a randomized, controlled trial comparing the use of TAE combined with percutaneous ethanol injection therapy (TAE/PEIT) to the use of PEIT only to assess the effects on HCC recurrence and survival. We continued the study after the introduction of RFA and compared TAE combined with RFA (TAE/RFA) with RFA only.

Presence of α-fetoprotein mRNA in blood correlates with outcome in patients with hepatocellular carcinoma

Abstract Background/Aims: Since hematogenous spread of tumor cells may adversely affect the prognosis of patients with hepatocellular carcinoma, we prospectively analyzed whether the presence of alpha-fetoprotein (AFP) messenger RNA (mRNA) in blood, used as a marker of circulating hepatocellular carcinoma cells, correlates with outcome. Methods: Eighty-eight patients were enrolled between December 1993 and August 1995, and 81 were followed until the end of 1997. All patients were treated with percutaneous ethanol injection therapy and/or transarterial embolization during follow-up. The status of AFP mRNA in blood was serially determined. Cumulative metastasis-free survival and overall survival were analyzed in relation to AFP mRNA and other clinical and laboratory variables. Results: Among 81 patients followed, 54 were positive for AFP mRNA at entry and 27 were negative. Extrahepatic metastasis developed more frequently among the AFP mRNA-positive patients (13 of 54) than among the AFP mRNA-negative patients (2 of 27) ( p =0.0296). After treatment, AFP mRNA became negative in 24 of 54 patients (44%). Cumulative metastasis-free survival and overall survival were significantly better in the 24 patients whose AFP mRNA became negative after treatment than in the 30 patients with persistently positive AFP mRNA ( p =0.0001 and p Conclusions: The presence or absence of AFP mRNA in blood is a predictor of outcome in patients with hepatocellular carcinoma.

Percutaneous ethanol injection for hepatocellular carcinoma: 20‐year outcome and prognostic factors

Ethanol injection is the best‐known image‐guided percutaneous ablation for hepatocellular carcinoma (HCC) and a well‐tolerated, inexpensive procedure with few adverse effects. However, there have been few reports on its long‐term results.