Toshiki Komeda

Sensitive detection of circulating hepatocellular carcinoma cells in peripheral venous blood.

Background. This study was performed to develop a sensitive method for the detection of circulating hepatocellular carcinoma (HCC) cells in peripheral blood, in advance of the diagnosis of distant metastasis of HCC by conventional means.

Disruption of the p16/Cyclin D1/Retinoblastoma Protein Pathway in the Majority of Human Hepatocellular Carcinomas

p16, cyclin D1 and retinoblastoma protein (pRB) regulate G1 to S transition and are commonly targeted in various cancers. However, few studies have simultaneously examined all components of the p16/cyclin D1/pRB pathway (RB pathway) in hepatocellular carcinoma (HCC). To clarify the role of the disruption of the RB pathway in HCC, we analyzed p16, pRB and cyclin D1 in 47 HCCs. Inactivation of p16 was detected in 30 of 47 HCCs (64%) by Western blot analysis and significantly correlated with hypermethylation of the promoter of this gene. pRB expression was found to be absent in 13 of 47 HCCs (28%) by immunohistochemistry. We found that 38 of 47 HCCs (81%) contained at least one inactivation in either pRB or p16. Furthermore, there was a significant inverse correlation between p16 and pRB inactivation (p = 0.041). Overexpression of cyclin D1 was detected in 5 of 47 HCCs (11%) by immunohistochemistry. The cases with cyclin D1 overexpression exhibited an advanced clinicopathological appearance and also contained inactivation of pRB and/or p16. These findings suggest that inactivation of pRB and/or p16 is a major event in human hepatocarcinogenesis, while cyclin D1 overexpression may confer additional growth advantages to the tumor in addition to pRB and/or p16 inactivation in HCC.

Infrequent alterations of the p16INK4A gene in liver cancer

We examined the genomic status of the p16INK4A (inhibitor of cyclin‐dependent kinase 4 A) and cyclin‐dependent kinase 4 (CDK4) genes in 62 human hepatocellular carcinomas (HCCs), 5 cholangiocellular carcinomas and 6 cell lines derived from human liver cancers. Although no samples showed the homozygous deletion of the p16INK4A gene, we detected intragenic mutations of the p16INK4A gene in 3 HCCs and one HCC cell line, which led to an amino‐acid substitution or a frameshift. In 2 HCC samples with mis‐sense mutations of the p16INK4A gene, loss of heterozygosity on 9p22 was also detected, suggesting that the loss of function of p16 was induced during hepatocarcinogenesis. On the other hand, amplification or rearrangement of the CDK4 gene was not detected in any samples examined in this study. These results indicated that the mutations or deletions of the p16INK4A gene are not frequent, but may play a role in a sub‐set of human HCC.

Comprehensive allelotyping of well-differentiated human hepatocellular carcinoma with semiquantitative determination of chromosomal gain or loss.

Allelic imbalance (AI), which represents certain chromosomal gains or losses, has been described in human hepatocellular carcinoma (HCC), but the impact of AI on the early stage of hepatocarcinogenesis has not been fully clarified. Moreover, no previous allelotype studies have identified the difference in chromosomal gain and loss that results in AI. To resolve these problems, we examined 18 well‐differentiated HCCs with comprehensive allelotyping by using 400 microsatellite markers with semiquantitative assessment of chromosomal gain or loss. To detect allelic gain effectively, the cutoff value of the allelic imbalance index was set at 0.70. Each allele showing imbalance was subjected to multiplex PCR with use of a retained allele as an internal control to determine whether the imbalance was the result of chromosomal gain or loss. High frequencies of chromosomal gains were detected at 1q (D1S196‐D1S2785, 56%), 5q (D5S647‐D5S2027, 44%), 6p (6pter‐D6S309, 33%), 7 (7pter‐D7S657, 22%), and 8q (D8S514‐qter, 44%), whereas chromosomal losses were frequently observed at 1p (1pter‐D1S234, 22%), 8p (8pter‐D8S549, 44%), and 17p (17pter‐D17S921, 28%). The extent of overall chromosomal aberration was closely related to the maximum tumor diameter (P = 0.002) and the presence of hepatitis B surface antigen (P = 0.03). Recurrent chromosomal losses at 1p and 8p and gains at 1q and 8q, even in HCCs with a minimal extent of aberrant chromosomes, indicate that these alterations were critical in the early stage of hepatocarcinogenesis. On the other hand, deletions of 13q and 16q were infrequent and were seen only in the most aberrant cases, which suggested that these were late events.

Hepatitis B virus strains of subgenotype A2 with an identical sequence spreading rapidly from the capital region to all over Japan in patients with acute hepatitis B

Objective To examine recent trends of acute infection with hepatitis B virus (HBV) in Japan by nationwide surveillance and phylogenetic analyses. Methods During 1991 through 2009, a sentinel surveillance was conducted in 28 national hospitals in a prospective cohort study. Genotypes of HBV were determined in 547 patients with acute hepatitis B. Nucleotide sequences in the preS1/S2/S gene of genotype A and B isolates were determined for phylogenetic analyses. Results HBV genotype A was detected in 137 (25% (accompanied by genotype G in one)) patients, B in 48 (9%), C in 359 (66%), and other genotypes in the remaining three (0.5%). HBV persisted in five with genotype A including the one accompanied by genotype G; another was co-infected with HIV type 1. The genotype was A in 4.8% of patients during 1991–1996, 29.3% during 1997–2002, and 50.0% during 2003–2008 in the capital region, as against 6.5%, 8.5% and 33.1%, respectively, in other regions. Of the 114 genotype A isolates, 13 (11.4%) were subgenotype A1, and 101 (88.6%) were A2, whereas of the 43 genotype B isolates, 10 (23.3%) were subgenotype B1, 28 (65.1%) were B2, two (4.7%) were B3, and three (7.0%) were B4. Sequences of 65 (64%) isolates of A2 were identical, as were three (23%) of A1, and five (18%) of B2, but none of the B1, B3 and B4 isolates shared a sequence. Conclusions Acute infection with HBV of genotype A, subgenotype A2 in particular, appear to be increasing, mainly through sexual contact, and spreading from the capital region to other regions in Japan nationwide. Infection persisted in 4% of the patients with genotype A, and HBV strains with an identical sequence prevailed in subgenotype A2 infections. This study indicates the need for universal vaccination of young people to prevent increases in HBV infection in Japan.

Genetic polymorphisms in CTLA4 and SLC4A2 are differentially associated with the pathogenesis of primary biliary cirrhosis in Japanese patients

BackgroundAnti-gp210 and anti-centromere antibodies are different risk factors for the progression of primary biliary cirrhosis (PBC). In order to dissect the genetic basis for the production of these autoantibodies, as well as the development and progression of PBC in Japanese patients, we examined single nucleotide polymorphisms (SNPs) in cytotoxic T-lymphocyte antigen 4 (CTLA4) and solute carrier family 4 anion exchanger, member 2 (SLC4A2), which have been associated with the pathogenesis of PBC in Caucasian patients.MethodsFour SNPs for both CTLA4 and SLC4A2 were genotyped, using the polymerase chain reaction–restriction fragment length polymorphism method and TaqMan assay, in 450 Japanese PBC patients and 371 sex-matched healthy controls.ResultsThe CTLA4 rs231775, rs3087243, and rs231725 SNPs were significantly associated with PBC susceptibility. The CTLA4 rs231725 SNP was significantly associated with progression to late-stage disease. The CTLA-4 haplotype 1 (rs231775 G, rs231777 C, rs3087243 G, rs231725 A; GCGA) was a risk factor for PBC susceptibility but a protective factor for PBC progression. Conversely, the CTLA-4 haplotype 2 (ACAG) was a protective and risk factor, respectively, for PBC susceptibility and progression. In addition, the CTLA4 rs231777 SNP and haplotype 3 (ATGG) was significantly associated with anti-gp210 antibody production, while SLC4A2 haplotype 4 (rs2069443 A, rs2303933 G, rs2303937 A, rs2303941 T; AGAT) and haplotype 3 (AAGC) were significantly associated with PBC susceptibility and anti-centromere antibody production, respectively.ConclusionsCTLA4 and SLC4A2 genetic polymorphisms are differentially associated with PBC development and progression, as well as anti-gp210 or anti-centromere antibody production, in Japanese PBC patients.

Prognostic Impact of Multiple Allelic Losses on Metastatic Recurrence in Hepatocellular Carcinoma after Curative Resection

Loss of heterozygosity (LOH) on chromosomes 13q, 16q and 17p has been associated with the progression of hepatocellular carcinoma (HCC). To investigate the prognostic impact of such LOH, we examined the metastasis-free survival of curatively resected HCC cases, in whom these LOHs were analyzed. Among the 49 HCC patients examined, the frequency of LOHs was 28% on 13q, 33% on 16q and 40% on 17p. The patients were followed up for metastatic recurrence after surgery and for analysis of the relationship between chromosomal changes and patients’ metastasis-free survival. Univariate survival analysis showed the presence of LOH on 16q, 17p and the number of chromosomes with LOH were significantly and negatively associated with metastasis-free survival, indicating that patients with LOH on multiple chromosomes had a poorer prognosis after surgery than those with LOH on a single chromosome or no LOH. Multivariate Cox survival analysis identified the presence of LOH on 16q and the number of chromosomes with LOH as the most significant independent negatively predictive factors for metastasis-free survival. These findings indicate that accumulation of chromosomal changes is associated with metastatic behavior, and that LOH on 16q was the most useful prognostic indicator for metastasis after curative resection of HCC.

Postoperative detection of α-fetoprotein mRNA in blood as a predictor for metastatic recurrence of hepatocellular carcinoma

Background: We tested for the presence of α‐fetoprotein (AFP) mRNA by using nested RT‐PCR in the peripheral blood of hepatocellular carcinoma (HCC) patients who had undergone curative surgery, and investigated the occurrence of intrahepatic and/or extrahepatic metastasis thereafter, to reveal the optimal timing of blood sampling for the prediction of metastatic recurrence.

Discrete breakpoint mapping and shortest region of overlap of chromosome arm 1q gain and 1p loss in human hepatocellular carcinoma detected by semiquantitative microsatellite analysis.

Recurrent chromosomal gain at 1q is one of the most common features of human hepatocellular carcinoma (HCC), but how the gain at 1q contributes to hepatocarcinogenesis is still unclear. To identify the target genes, precise determination of the shortest region of overlap (SRO) and of breakpoints is necessary. Similarly, the role of loss at 1p, which is also a major cytogenetic aberration in HCC, needs to be determined. Fifty HCCs were examined with the aid of 59 microsatellite markers distributed throughout both arms of chromosome 1. To detect allelic gain effectively, the cutoff value of the allelic imbalance index was set at 0.70. Alleles showing imbalance were subjected to multiplex PCR, using a retained allele as an internal control, to determine whether the imbalance was the result of chromosomal gain or loss. The SRO of the gains was defined as D1S2878–D1S2619 (1q23.–q25.3, 16.9 Mb), which involved 36 cases (72%). Gains in the number of copies of certain oncogenes within this region seemed to be critical for the pathogenesis of HCC. In contrast, the centromeric breakpoints of these gains varied, but they tended to occur mainly in the pericentromeric region (26 of 50 cases, 52%). Rearrangement of specific genes associated with the gains is unlikely. On the other hand, the SRO of deletion was defined as D1S2893–D1S450 (1p36.32–p36.22, 5.1 Mb). Four known putative tumor‐suppressor genes (TP73, RIZ1, NBL1/DAN, and CDKN2C) were outside the SRO, suggesting the presence of other candidate genes with critical roles in hepatocarcinogenesis.

Alteration of the p14ARF gene and p53 status in human hepatocellular carcinomas

BackgroundThe INK4a/ARF locus encodes p16INK4a and p14ARF, both of which are crucial for two tumor suppressor pathways, retinoblastoma (RB)/p16INK4a and p53/ARF. Inactivation of RB/p16INK4a was frequently reported, but alterations of the p14ARF gene in hepatocellular carcinoma (HCC) in the Japanese population have been insufficiently analyzed.MethodsTo determine the role of p53/ARF alteration in hepatocarcinogenesis, we examined 44 HCCs for mRNA expression, deletion, mutation, and promoter hypermethylation of the p14ARF gene; alterations of p53 were also analyzed in the same series of HCCs.ResultsHomozygous deletion, spanning from exon 1 β to exon 2, was found in 1 HCC mutations within exon 2 were found in 2 HCCs, but no promoter hypermethylation was detected. All 3 HCCs with p14ARF alteration were well differentiated. Twelve of the 44 HCCs (27.2%) showed immunohistochemical evidence of p53 alteration; however, only 1 of the tumors with p53 alteration was well differentiated. TaqMan polymarase chain reaction (PCR) indicated that the expression of p14ARF in HCCs was higher than in that in all but three of the corresponding non-tumorous tissues (P < 0.0001), and increased expression of p14ARF seemed to be associated with poorly differentiated phenotype. Absence of p14ARF expression was seen in only one HCC, with homozygous deletion of the p14ARF gene.ConclusionsCompared with p53 alteration, p14ARF alteration does not occur frequently, but may play a role in a subset of Japanese HCCs in the early stage of hepatocarcinogenesis. On the other hand, overexpression of p14ARF was frequently observed in HCC, especially in poorly differentiated tumors, probably reflecting oncogenic stimuli in these tumors.