Quantitative evaluation of genomic instability as a possible predictor for development of hepatocellular carcinoma: comparison of loss of heterozygosity and replication error.

Both loss of heterozygosity (LOH) and replication error (RER) are considered to be phenotypes of genomic instability. To unveil the role of the genomic instability in hepatocarcinogenesis, frequencies of LOH and RER were simultaneously determined in 15 hepatocellular carcinomas (HCCs), surrounding nontumorous liver tissues (SL), and 13 liver tissues with chronic viral hepatitis void of cancer (NC) by referencing peripheral blood leukocytes (PBLs) from the corresponding donor using 18 microsatellite markers spread throughout the genome. LOH was significantly frequent in HCC compared with that in SL or NC (P =.005, P =.0003, respectively) and observed preferentially at particular microsatellite loci, D1S204, D2S123, D8S1106, D9S266, D16S748, and D19S601. Although the higher prevalence of RER was also significant in HCC compared with that in NC (P =.03), in most cases the errors were detected at very low frequencies and random loci. Both LOH and RER tended to appear more prevalently in SL than in NC. The occurrence rate of LOH was higher in the tissues associated with hepatitis B virus (HBV) than with hepatitis C virus (HCV) infection especially in HCC (P =.03). When referencing SL instead of PBLs, the prevalence of LOH and RER in HCC significantly decreased (P =.02 and P =.03, respectively). These results suggest that LOH is closely associated with multistep hepatocarcinogenesis especially under HBV infection, but RER is imperceptibly associated. The quantitative evaluation of the frequency of LOH by referencing PBLs may be a useful predictor for HCC development in chronic liver diseases.

Reduction of telomeric repeats as a possible predictor for development of hepatocellular carcinoma: convenient evaluation by slot-blot analysis.

Hepatocellular carcinoma (HCC) mainly arises from the liver with chronic inflammation. Because telomere reduction reflects replicative history in somatic cells, we analyzed the possibility that liver tissues surrounding HCC consist of the cells carrying substantial reduction of telomere. We studied 20 HCC and surrounding noncancerous liver tissues (SL) obtained by surgical resection, and 10 laparoscopically obtained needle biopsy specimens of the liver with chronic inflammation including no overt HCC (CI). Five liver tissues without chronic liver diseases (ND) were also examined. Extracted genomic DNAs were blotted on a nylon membrane, and probed at first with radio-labeled d(TTAGGG)(3) and reprobed with radio-labeled d(CCT)(7). The intensity caused by d(TTAGGG)(3) was divided by that of d(CCT)(7). The ratio was defined as telomeric repeats content (TC). Dilution experiments reproducibly revealed almost the same TC. The reduction rate of telomere length through aging estimated by regression analysis of TC was 0.62% per year. Concomitant analyses of TC and average telomere length revealed that both values were significantly correlated (r =.45; P =.009). To compare TC in the liver with respect to chronic inflammation, the value was divided by TC in peripheral blood leukocytes (PBL) from the same donor. The ratio was defined as relative TC (RTC). There was a statistically significant decrease of RTC in CI compared with that in ND (P =.03). Furthermore, RTC in SL was significantly lower than that in CI (P =.0001). These observations suggest that RTC value in liver tissues may digitally indicate a replicative history of hepatocytes under chronic inflammation, and a risk of HCC development.

The fucosylation index of alpha-fetoprotein as a possible prognostic indicator for patients with hepatocellular carcinoma.

BACKGROUND: The aim of this study was to elucidate the usefulness of measuring the fucosylation index (FI) of alpha-fetoprotein (AFP) before the initiation of therapy as a new prognostic indicator for patients with hepatocellular carcinoma (HCC). METHODS: One hundred twelve patients with HCC who underwent transcatheter arterial embolization, chemoembolization, and/or percutaneous ethanol injection were examined in the current study. FI was determined by crossed immunoaffino-electrophoresis in the presence of Lens culinaris agglutinin. RESULTS: When the tentative discriminating value of FI was set at 18%, the mean survival rate for the group whose FI was higher than 18% was significantly lower than that for the group whose FI was equal to or less than 18%, according to the generalized Wilcoxon test (P = 0.0117) and the log rank test (P = 0.0183). The survival rate for HCC patients with AFP concentrations of more than 200 ng/mL was also significantly lower than that for patients with AFP in the range of 21-200 ng/mL, according to the generalized Wilcoxon test (P = 0.0017) and the log rank test (P = 0.0018). When FI was combined with AFP concentration, a highly significant difference was observed between the group with FI >18% and AFP >200 ng/mL and another group with FI < or =18% and AFP < or =200 ng/mL, as determined by the generalized Wilcoxon test (P < 0.0001) and the log rank test (P = 0.0003). An analysis of multiple covariates in the prognostic factors with the Cox proportional hazards model showed that FI was one of the independent prognostic factors. CONCLUSIONS: The current study indicates that measuring FI from sera before the initiation of treatment serves as a new prognostic factor and may improve prognostic estimates and appraisal of therapeutic outcomes for patients with HCC.

[Efficacy of granisetron as anti-emetic agent for emesis induced by intra-arterial injection therapy for hepatocellular carcinoma].

The effect of granisetron in preventing nausea and emesis induced by intraarterial chemotherapy was comparatively studied with a historical control group (46 cases) in 50 patients with hepatocellular carcinoma receiving intraarterial anti-tumor drugs such as cisplatin and doxorubicin. Emesis was perfectly controlled in 39 out of 50 patients in the treatment group (78%), in comparison to 33 out of 46 patients (71.7%) in the historical control group. This represented no statistical significance between the two groups. In terms of the severity of nausea, however, the granisetron group demonstrated significant superiority to the control group with 27 out of 50 patients (54%) being free of symptoms compared with 16 out of 46 patients (34.8%) in the control group. A stratified analysis of the data also demonstrated significant superiority of the granisetron group over the historical group in the number of emetic episodes and the severity of nausea in female patients, who are more predisposed to emesis. The above results confirm the usefulness of granisetron as an antiemetic agent used for the prevention of acute nausea and emesis induced by intraarterial chemotherapy.

Quantitation of telomerase activity in hepatocellular carcinoma: a possible aid for a prediction of recurrent diseases in the remnant liver.

Because telomerase activity is necessary for cell immortality and probably associated with tumor progression, we have evaluated a possible aid for quantitation of the activity to predict intrahepatic recurrences after surgery in patients with hepatocellular carcinoma (HCC). HCC tissues obtained by surgical resection from 20 patients were studied. Telomerase activity was expressed as peaks with a periodicity through a fluorescence-based telomeric repeat amplification protocol using an autosequencer, and the quantity of activity was calculated from peak areas. A ratio of fluorescence intensity depending on telomerase to that of an internal standard was used as a value of relative telomerase activity (RTA). RTA in serially diluted S100 extracts from HepG2 cells was well correlated with the amount of the extracts. The mean RTA value of 36.4 +/- 27.8 (mean +/- SD, 3.21 to 105) in 9 patients suffering from early recurrences after surgery was significantly higher than that (9.84 +/- 7.65; mean +/- SD, 3.00 to 29.0) in 11 patients without intrahepatic recurrences during the early period (P = .004). These results indicate that RTA value can be a useful predictor for intrahepatic recurrences during the early period after surgical resection of HCC.

Microheterogeneity of serum transferrin in the diagnosis of hepatocellular carcinoma.

Heterogeneous reactivity of human serum transferrin (Tf) with lectins was analysed using patient sera to determine whether it can be used to distinguish patients with hepatocellular carcinoma (HCC) from those with liver cirrhosis (LC). Microheterogeneity of Tf was analysed by crossed immunoaffinity electrophoresis (CIAE) with concanavalin A (Con A) and Lens culinaris agglutinin (LCA). Sample sera from 58 patients with HCC, 43 patients with LC and 10 normal controls were used in this study and the results were evaluated statistically. The increments of Con A-non-reactive (C1) and -weakly reactive (C2) species of Tf were observed in HCC compared with those of LC and Norm. Significant increase in the combined percentage of Con A- C1 + C2 species was also revealed in HCC (35.5 +/- 8.5%, mean+/-s.d.) compared with those of LC (29.1 +/- 6.8%; P < 0.001) and normal controls (17.1 +/- 2.3%; P < 0.001). The elevation of LCA-reactive (L2) species of Tf was recognized in HCC (8.2 +/- 3.8%) in comparison with those of LC (4.8 +/- 3.1%; P < 0.001) and normal controls (1.3 +/- 1.7%; P < 0.001). The increment of C1 + C2 species and/or L2 species of Tf was observed in 78% (sensitivity) of patients with HCC. The specificity, the positive predictive value and the overall accuracy were 81, 88 and 72%, respectively. Positive ratio of C1 + C2 and/or L2 species was 77 and 70% in alpha-fetoprotein low and -high producing HCC patients, respectively. These results indicate that the microheterogeneity analysis of human serum Tf is useful for distinguishing patients with HCC from those with LC and normal controls.

Alpha-fetoprotein-producing renal cell carcinoma with increased activity of N-acetylglucosaminyl-transferase III.

N-acetylglucosaminyltransferase (GnT) III catalyzes the addition of N-acetylglucosamine through a beta 1-4 linkage to the mannose of the trimannosyl core, resulting in conversion of the concanavalin-A-(ConA)-reactive glycan into the ConA-nonreactive one. In this study, we measured GnT III levels in serum, tumor, and surrounding normal tissues together with a glucosaminylation index of alpha-fetoprotein (AFP), which is defined as the percentage of the ConA-nonreactive species in total AFP, in a case of AFP-producing renal cell carcinoma. The glucosaminylation index was determined by affinoelectrophoresis in the presence of ConA. GnT III was measured by using a pyridylaminated asialoagalactobiantennary sugar chain as a substrate by high-performance liquid chromatography. The glucosaminylation index of serum AFP, the concentration of which was 68 ng/ml, was 60%. This value is much higher than observed in hepatocellular carcinomas. The tumor tissue level of GnT III was 55.34 pmol/mg/h which was about six fold higher (9.50 pmol/mg/h) than in normal surrounding tissues. The serum level of this enzyme before surgery was 27.65 pmol/ml/h and decreased to 5.38 pmol/ml/h thereafter in association with a depression of serum AFP from 68 to 5.4 ng/ml. Thus, an increased level of GnT III in tumor tissues could account for the elevated conversion of a biantennary complex type sugar chain of AFP into a bisecting glucosaminylated biantennary one resulting from the addition of an N-acetylglucosamine residue at the trimannosyl core. This is, to our knowledge, the first report explaining the change in the carbohydrate structure of AFP with different affinity to ConA on the enzymatic basis in a renal cell carcinoma.