Add-on combination therapy with adefovir dipivoxil induces renal impairment in patients with lamivudine-refractory hepatitis B virus.

Combination therapy with adefovir dipivoxil (ADV) and lamivudine (LAM) is recommended for patients infected with LAM-refractory hepatitis B virus (HBV). However, the effects of such therapy on renal function and serum phosphorus levels have not been fully evaluated. Combination therapy with ADV and LAM was given to 37 patients infected with LAM-refractory HBV, including 17 with hepatic cirrhosis. Serum HBV DNA levels decreased to below 2.6 log(10) copies/mL in 23 (62%) of 37 patients at 12 months, 25 (78%) of 32 patients at 24 months, and 16 (84%) of 19 patients at 36 months. Except for one cirrhotic patient, serum alanine aminotransferase levels were below 50 IU/L in all patients during combination therapy. Serum creatinine levels increased in 14 (38%) of 37 patients, and serum phosphate levels decreased to below 2.5 mg/mL in 6 (16%) of 37 patients during combination therapy. Patients who received combination therapy for 36 months or longer had a significantly incidence of elevated serum creatinine levels. Fanconi syndrome occurred in a 57-year-old woman with cirrhosis after ADV was added to LAM. Combination therapy with ADV and LAM can maintain biochemical remission in patients with LAM-refractory HBV. However, the dosing interval of ADV should be adjusted according to renal function and serum phosphate levels in patients receiving long-term treatment.

Development of hepatocellular carcinoma in patients with chronic hepatitis C who had a sustained virological response to interferon therapy: a multicenter, retrospective cohort study of 1124 patients.

BACKGROUND: Interferon (IFN) improves hepatic inflammation/fibrosis and reduces the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C (CH-C). However, HCC develops in some patients who have a sustained virological response (SVR) to IFN therapy. We designed this study to establish a follow-up protocol for patients with CH-C who have SVR to IFN therapy. METHODS: We retrospectively studied 1124 patients with CH-C who received IFN. RESULTS: HCC developed in 3.5% of patients with SVR to IFN. As compared with SVR patients without HCC, SVR patients with HCC were predominantly male (P=0.003), older at the initiation of IFN therapy (P=0.002), and at a more advanced histologic stage of disease (P<0.001). However, three of the 13 SVR HCC patients had mild fibrosis. The mean interval from IFN therapy to the detection of HCC in SVR HCC patients was 5.8 years and did not differ significantly from that in non-SVR HCC patients (P=0.304). Although most patients with HCC received curative therapy, the prognosis of some SVR HCC patients was poor, probably because of insufficient follow-up, resulting in delayed detection of HCC. CONCLUSIONS: SVR patients with CH-C who are elderly, male, or have an advanced histologic stage are at a high risk for the development of HCC after IFN therapy. We recommend that SVR patients should be observed carefully for more than 10 years after the completion of IFN therapy, even if they only have early fibrosis.

Interferon reduces somatic mutation of mitochondrial DNA in liver tissues from chronic viral hepatitis patients.

SUMMARY: We recently reported that the genetic instability resulting in the high rate of mitochondrial DNA (mtDNA) mutation in noncancerous liver tissue is consistent with the multicentric hepatocarcinogenesis detected clinically. Interferon (IFN) has been reported to reduce hepatocarcinogenesis in individuals with hepatitis virus infection. Liver biopsy specimens were obtained from 26 patients with chronic hepatitis C virus (HCV) infection before and after IFN therapy (total dose: 252 million units). The mean (+/-SD) age of the study population was 45 +/- 9 years and 13 (50%) were male [mode of acquisition: blood transfusion (27%), unknown (73%); viral load: 5.2 +/- 1.1 k copies/mL; duration of infection: 17 +/- 9 years (65%), unknown (35%); genotype: I (4%), II (80%), III (8%), IV (8%); alcohol intake: positive (31%), negative (69%)]. DNA samples were extracted from the specimens and subjected to direct sequencing. The mtDNA mutation frequency in the D-loop was increased in liver specimens from individuals with HCV infection compared with 21 controls (2.5 vs 0.6, P < 0.001). IFN therapy decreased the mtDNA mutation (mean difference = 0.7, P < 0.001) and the decreased number of mtDNA mutations was positively correlated with suppression of the total histological activity index score (mean difference = 1.3, P < 0.01). These results clearly indicate that the mutational rate of mtDNA is strongly associated with IFN therapy. Thus, analysis of mtDNA could provide a new criterion for the therapeutic evaluation of the effect of IFN, and may be useful for the prediction of risk of carcinogenesis.

Previous or occult hepatitis B virus infection in hepatitis C virus-associated hepatocellular carcinoma without hepatic fibrosis.

We investigated the role of hepatitis B virus infection in development of hepatocellular carcinoma in hepatitis C virus-infected patients without hepatic fibrosis. Of 253 patients, 8 lacked hepatic fibrosis (group 1); group 2 included the remaining 245 patients. Clinicopathologic findings were compared between the groups. Hepatitis B x gene was sought in cancers and adjoining noncancerous liver. Group 1 showed better liver function parameters and milder active hepatitis than group 2. The proportion of patients with anti-hepatitis B virus antibody tended to be higher in group 1 than in group 2. The proportion of patients with hepatitis B x RNA in cancers was significantly higher in group 1 than in group 2. All group 1 patients had previous or occult hepatitis B virus infection. Previous or occult hepatitis B virus infection may be critical in development of hepatocellular carcinomas in hepatitis C virus-infected patients without hepatic fibrosis.

Accessory spleen in the pelvis diagnosed by Tc-99m phytate scintigraphy.

We report a 58-year-old woman with an accessory spleen in the left side of the pelvis. She visited our outpatient clinic complaining of lower abdominal discomfort. Abdominal ultrasonography revealed a tumor 4 cm in diameter in the left side of the pelvis. Color Doppler ultrasonography demonstrated plentiful pulsating blood flow. Magnetic resonance angiography revealed that the blood supply for the tumor was from a branch of the splenic artery. Scintigraphy with Tc-99m phytate revealed accumulation of radioactivity concordant with a mass in the left side of the pelvis, and the spleen was normally visualized. These findings suggested that this tumor was an accessory spleen, and the patient underwent no further invasive procedures.

Usefulness of positron emission tomography with fluorine-18-fluorodeoxyglucose for predicting outcome in patients with hepatocellular carcinoma.

OBJECTIVES: The present study was designed to assess the usefulness of positron emission tomography with fluorine-18-fluorodeoxyglucose (FDG-PET) for predicting outcome in patients with hepatocellular carcinoma. METHODS: FDG-PET was performed in 48 patients with hepatocellular carcinoma. For quantitative evaluation, a region of interest (ROI) was placed over the area of maximum activity within the lesion. A background ROI was then placed over the nontumor region of the liver. The average activity within each ROI was subsequently corrected for radioactive decay, and the standardized uptake value (SUV) was calculated by dividing the tissue activity by the injected dose of radioactivity per unit body weight. SUV ratio was expressed as the tumor-to-nontumor ratio of the SUV. RESULTS: The tumor-volume doubling time, as index of the growth rate of hepatocellular carcinoma, correlated significantly with SUV ratio but did not correlate with SUV. On the basis of the SUV ratio, the patients were divided into two groups of similar size: group A, SUV ratio of < or = 1.5; and group B, SUV ratio > 1.5. The cumulative survival rate was significantly lower in group B than in group A. On the basis of the SUV, the patients were divided into two groups of roughly equal size: group C, < or = SUV 2.6; and group D, > SUV 2.6. The cumulative survival rate was similar in these groups. On regression analysis with the Cox proportional hazards model, the SUV ratio and tumor number were significantly related to survival. CONCLUSIONS: These results suggest that FDG-PET is useful not only for the evaluation of the malignancy of hepatocellular carcinoma but also for the prediction of outcome in patients with hepatocellular carcinoma.

Effects of long-term postoperative interferon-alpha therapy on intrahepatic recurrence after resection of hepatitis C virus-related hepatocellular carcinoma. A randomized, controlled trial.

BACKGROUND: Interferon therapy decreases the incidence of hepatocellular carcinoma in patients with chronic hepatitis C. OBJECTIVE: To evaluate effects of interferon-alpha on recurrence after resection of hepatitis C virus-related hepatocellular carcinoma. DESIGN: Randomized, controlled trial. SETTING: University hospital, medical center, and affiliated hospital in Osaka, Japan. PATIENTS: 30 men were randomly allocated after resection to the interferon-alpha group (n = 15) or the control group (n = 15). INTERVENTION: Patients in the interferon-alpha group received interferon-alpha, 6 MIU intramuscularly daily for 2 weeks, then three times weekly for 14 weeks, and finally twice weekly for 88 weeks. MEASUREMENTS: Recurrence rates after resection. RESULTS: Recurrent tumors were detected in 5 patients in the interferon-alpha group and in 12 control patients. The recurrence rate was significantly lower in the interferon-alpha group than in the control group (P = 0.037). CONCLUSION: Postoperative interferon-alpha therapy appears to decrease recurrence after resection of hepatitis C virus-related hepatocellular carcinoma.

Tumor-suppressor effect of interferon regulatory factor-1 in human hepatocellular carcinoma.

IFN regulatory factor-1 (IRF-1) regulates the IFN system, inhibits cell growth, and has tumor-suppressor activities. p21 is a universal cyclin-dependent kinase inhibitor, the induction of which depends on both p53 and IRF-1 in mouse embryonic fibroblasts. The expression of p21 in hepatocellular carcinomas (HCCs) is regulated by wild-type p53. We examined the expressions of IRF-1 and p21 in 32 HCCs by quantitative reverse transcription-PCR and the mutation p53 gene in 32 HCCs by single-strand conformation polymorphism and direct sequencing. The expression of IRF-1 mRNA in 15 of 32 HCCs was lower than that in adjacent noncancerous tissue. IRF-1 mRNA expression was reduced in 0 of 3 specimens of well-differentiated HCC, 9 of 21 (42%) specimens of moderately differentiated HCC, and 6 of 8 (75%) specimens of poorly differentiated HCC. IRF-1 mRNA expression was significantly lower in tumors with portal thrombus than in those without portal thrombus (P = 0.003). p53 mutations were detected in 7 of 32 HCCS: p21 expression was reduced in 6 of the 7 (86%) HCCs with p53 mutations. In contrast, p21 expression was reduced in 13 of 25 (52%) HCCs with wild-type p53. IRF-1 expression was reduced in 7 of 13 (53%) HCCs with both wild-type p53 and reduced expression of p21. These results suggest that IRF-1 may be a tumor-suppressor gene for HCC and that IRF-1 is related to p21 expression in HCC with wild-type p53.

Identification of genes differentially expressed by putrescine in HepG2 hepatoblastoma cells.

In order to identify genes differentially expressed by putrescine, a polyamine, which play important roles in the regulation of cell proliferation and the development of cancer, we performed mRNA differential display analysis using total RNA extracted from HepG2 cells (human hepatoblastoma cell line) treated with a specific inhibitor of polyamine biosynthesis, alpha-difluorometylornithine (DFMO). A total of 25 genes were up-regulated and 32 genes down-regulated by putrescine. Of the genes differentially expressed by putrescine, we chose three that were related to the respiratory chain and oxidative phosphorylation and analyzed them by Northern blot analysis. Cytochrome oxidase subunit 1, low molecular mass ubiquinone-binding protein, and NADH dehydrogenase subunit 2 were found to be down-regulated by putrescine. We examined intracellular ATP level in HepG2 cells, and found that ATP level in DFMO-treated cells was increased by exogenous putrescine.

Somatic mutation of mitochondrial DNA in cancerous and noncancerous liver tissue in individuals with hepatocellular carcinoma.

Unlike other types of cancer, hepatocellular carcinoma (HCC) is usually preceded by chronic inflammation caused by viral infection. The mutation of mitochondrial DNA (mtDNA) in hepatocarcinogenesis associated with viral infection was investigated. Compared with control liver tissue, the frequency of mtDNA mutations was markedly increased in both noncancerous and cancerous liver specimens from individuals with HCC. The accumulation of mtDNA mutations in HCC tissue reflected the degree of malignancy. The frequency of mtDNA mutations in HCC tissue was also greater than that described previously for other types of tumors. These observations suggest that the repeated destruction and regeneration of liver tissue associated with chronic viral hepatitis lead to the accumulation of mtDNA mutations. The genetic instability that results in the high rate of mtDNA mutation in cancerous liver tissue is also consistent with the multicentric hepatocarcinogenesis detected clinically.

Prevention of hepatocellular carcinoma in patients with chronic active hepatitis C and cirrhosis.

In a prospective randomised controlled study, 90 patients with chronic active hepatitis C and compensated cirrhosis were assigned symptomatic treatment or interferon alfa (IFN-alpha). We report data on decompensation, detection of hepatocellular carcinoma, and mortality rates. IFN-alpha gave a sustained response in only a small proportion of patients, but worsening of compensated cirrhosis was prevented and development of hepatocellular carcinoma was inhibited, increasing the survival rate. The risk ratio of IFN-alpha versus symptomatic treatment decreased by 0.250 for progression to Child-Pugh grade B, 0.256 for detection of hepatocellular carcinoma, and 0.135 for a fatal outcome.

Clinical evaluation of staging system for nasopharyngeal carcinoma: comparison of fourth and fifth editions of UICC TNM classification.

Staging nasopharyngeal carcinoma (NPC) by the UICC 4th-edition TNM classification system (the old system) did not give an accurate prognosis because of the uneven distribution of patients in each stage. This system was revised in 1997 (the new system). To evaluate the performance of the new system, 35 patients with NPC who had been staged by the old system were restaged according to the new system. Restaging of the patients resulted in an overall "downstaging." Differences in the overall survival rates of the early group (stages I, II, III), stage IVA, stage IVB, and stage IVC patients were statistically significant (75%, 48%, 74%, and 0%. respectively; p = .01). T4 was a significant factor of poor outcome (hazard rate, 2.932; 95% CI, 1.667 to 8.545), whereas N3 was not (hazard rate, 0.858; 95% CI, 0.281 to 2.618). The new staging system is more useful than the old system.

Resected cases of hepatocellular carcinoma detected after interferon therapy for chronic hepatitis C.

BACKGROUND/AIMS: Interferon therapy decreases the incidence of hepatocellular carcinoma in patients infected with hepatitis C virus. However, hepatocellular carcinoma was detected after interferon therapy in some patients. METHODOLOGY: Of the 167 patients who underwent liver resection for hepatitis C virus-related hepatocellular carcinoma between 1993 and September 1998, the carcinoma was detected after interferon therapy in 11 patients. The clinicopathologic findings in these 11 patients were studied. RESULTS: The response to interferon was complete (n = 4), partial (n = 4), or no response (n = 3). Hepatocellular carcinoma was detected 2 months to 3 years 9 months, after interferon therapy. The interval period from the end of interferon therapy to the detection of the carcinoma were significantly correlated with the longest diameter of the main tumor (P = 0.0043), indicating that most carcinomas have already developed before the end of interferon therapy. In one non-responder, multicentric carcinogenesis occurred after liver resection for primary hepatocellular carcinoma. Another patient with advanced hepatocellular carcinoma died of the recurrence. CONCLUSIONS: Surveillance for hepatocellular carcinoma must be performed even in patients successfully treated with interferon because occult carcinoma may have developed before or during the therapy.

TT virus infection in patients with chronic liver disease of unknown etiology.

The role of a novel virus, designated as TT virus (TTV), as a cause of chronic liver disease has not been well defined. We investigated the prevalence of TTV among 69 patients with chronic liver disease of unknown etiology and 50 volunteer blood donors with normal transaminase levels. TTV DNA was amplified by polymerase chain reaction (PCR) by using two different sets of primers: one based on the sequence of the original N22 clone within the open reading frame 1 (set A) and the other derived from the untranslated region (set B). The prevalence of TTV detected by PCR primers set A only, set B only, and in total (by either set A or B) was 11 (31%), 31 (86%), and 31 (86%) of 36 patients with chronic hepatitis; 2 (40%), 4 (80%), and 4 (80%) of 5 with cirrhosis; 11 (39%), 17 (61%), and 22 (79%) of 28 with hepatocellular carcinoma; and 9 (18%), 39 (78%), and 40 (80%) of 50 volunteer blood donors, respectively. Of the interpretable 25 PCR products amplified with primers set A, 9 were classified as genotype 1a, 10 as genotype 1b, 4 as genotype 2, 1 as genotype 3, and 1 as genotype 4. Molecular evolutionary analysis did not suggest any particular strains of TTV that might be associated with chronic liver disease. The nucleotide sequences of the untranslated region on which PCR primers set B were designed were highly conserved, and the interpretable 22 PCR products amplified with primers set B were not clearly divisible into distinct genotypes. Our findings provided no evidence that TTV is a causative agent of chronic liver disease.

Outcomes of dysplastic nodules in human cirrhotic liver: a clinicopathological study.

The number of dysplastic nodules detected clinically has increased since patients with hepatitis virus-associated cirrhosis, who are at increased risk for hepatocellular carcinoma (HCC), began to undergo regular cancer surveillance. Although it is potentially important to determine which type(s) of nodule may be prone to progress to HCC, outcomes of dysplastic nodules have not been fully investigated. This prompted us to examine the outcomes of dysplastic nodules in cirrhotic patients clinicopathologically. We studied 33 dysplastic nodules of <20 mm in maximum diameter, diagnosed by fine needle aspiration biopsy under ultrasonography (US). These nodules were clinically followed, mainly by US examination, for up to 70 months. When the nodules enlarged or exhibited changes on US, they were histologically reexamined by second biopsy. Surprisingly, 15 of the 33 nodules (45.5%) disappeared, 14 nodules (42.4%) remained unchanged, and only 4 nodules (12.1%) progressed to HCC. The latter 4 nodules were all hyperechoic on US and were composed of clear cells with fatty change or small cells with increased nuclear density, and in all 4 patients serum was positive for hepatitis C virus antibody. Univariate analyses revealed that, although not significant, the hyperechoic nodules or nodules with small cell change showed a higher HCC progression rate in comparison with the hypoechoic nodules or the nodules without small cell change. In summary, most of the dysplastic nodules we followed disappeared or remained unchanged, but some progressed to HCC. Hyperechoic nodules in patients with hepatitis C virus-associated cirrhosis, which show small cell change with increased nuclear density, may be prone to progress to HCC.

Ornithine decarboxylase activity in the non-cancerous hepatic tissue of patients with hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma may develop in patients with chronic hepatitis and cirrhosis. Active hepatitis is an important etiologic factor in the development of hepatocellular carcinoma. We measured ornithine decarboxylase activity, an important enzyme during cell proliferation, in non-cancerous hepatic tissue in patients with hepatocellular carcinoma. METHODOLOGY: Thirty-four patients who underwent liver resection for hepatocellular carcinoma were the subjects of this study. Hepatitis B surface antigen was detected in 7 patients (HBV group) and hepatitis C virus antibody was detected in 27 patients (HCV group). Tissue ornithine decarboxylase activity was measured. Histologic severity in active hepatitis (activity score) and degree of fibrosis (staging score) were determined. RESULTS: Ornithine decarboxylase activity was significantly higher in the HCV group than in the HBV and control groups. In all patients, ornithine decarboxylase activity correlated directly with the histologic activity score and the histologic staging score. In the HCV group, ornithine decarboxylase activity correlated with the histologic activity score. CONCLUSIONS: Ornithine decarboxylase activity in non-cancerous hepatic tissue correlated with the severity of active hepatitis and degree of fibrosis. In patients with hepatitis C virus, active hepatitis with increased ornithine decarboxylase activity is an important factor in the development of hepatocellular carcinoma.

HOXA5 regulates expression of the progesterone receptor.

The majority of breast carcinomas show reduced or no expression of the transcription factor, HOXA5. Recently, we have shown that HOXA5 is a potent transactivator of p53 in breast cells and thus may affect the response of breast cancer cells to DNA damage. To determine whether HOXA5 played a role in growth and homeostasis in breast cells, we studied its interaction with the progesterone receptor. The progesterone receptor (PR) belongs to the superfamily of nuclear receptors whose members co-ordinate morphogenesis of the mammary gland in response to binding to their cognate ligands. An increased expression of the endogenous PR gene was seen in MCF-7 cells following induced expression of an exogenously transfected HOXA5 gene. HOXA5, but not HOXB4, -B5, or -B7 activated the PR promoter in two breast cancer cell lines, MCF-7 and Hs578T. Deletion and mutation analysis of the promoter identified a single HOXA5-binding site required for transactivation of the PR gene by HOXA5. HOXA5 binds directly to this site in the PR promoter. Thus, HOXA5 may behave as a transcriptional regulator of multiple target genes, two among which are p53 and the progesterone receptor.

Ultrasonographic evaluation of portal blood flow using transhepatic carbon dioxide injection.

BACKGROUND/AIMS: The evaluation of portal blood flow in hepatic mass is important for the diagnosis of hepatocellular carcinoma. We have designed a new method to easily evaluate portal blood flow in hepatic mass using ultrasonography with injection of carbon dioxide into the intrahepatic portal vein by direct puncture with a fine needle. METHODOLOGY: We evaluated 29 masses in the liver of 20 patients ultrasonically with injection of carbon dioxide into the intrahepatic portal vein. RESULTS: Of 29 space-occupying lesions (SOLs), 13 were found to have portal blood flow and 16 were found to have no portal flow by this method. All 15 SOLs which had no portal flow were histologically confirmed to be hepatocellular carcinoma. Of 13 SOLs with portal flow, 5 were confirmed to be hepatocellular carcinoma. For 7 of 9 SOLs in which both this method and arterial portographic computed tomography were performed, the results were in agreement. CONCLUSIONS: Ultrasonographic evaluation of portal blood flow using transhepatic carbon dioxide injection appears to be useful for the evaluation of portal flow in mass and may aid in the diagnosis and management of mass in patients with liver disease.

Relationship between ornithine decarboxylase gene abnormalities and human hepatocarcinogenesis.

BACKGROUND/AIMS: Ornithine decarboxylase is essential for cell growth. Its activity was high in human hepatocellular carcinoma tissues and was highest in poorly differentiated tumors. METHODOLOGY: To find if there are tumor-specific ornithine decarboxylases, we examined the ornithine decarboxylase cDNA sequences of 91 clones prepared from hepatoma tissue and non-cancerous tissue of resected liver specimens from 15 patients with hepatocellular carcinoma. RESULTS: Ornithine decarboxylase gene mutations were more frequently detected in the hepatoma tissue. The incidence of mutation in hepatoma tissue was related to dedifferentiation. Mutation in regions rich in proline, glutamic acid, serine, and threonine were detected in moderately and poorly differentiated hepatocellular carcinoma only. CONCLUSIONS: Our findings suggested that the sequence of ornithine decarboxylase in hepatocellular carcinoma often is different from that in normal liver and that mutation of its gene is related to the progression of hepatocellular carcinoma.