Yoshihiro Fukuda

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.

Impaired adrenocorticotropic hormone response to bacterial endotoxin in mice deficient in prostaglandin E receptor EP1 and EP3 subtypes

Sickness evokes various neural responses, one of which is activation of the hypothalamo–pituitary–adrenal (HPA) axis. This response can be induced experimentally by injection of bacterial lipopolysaccharide (LPS) or inflammatory cytokines such as IL-1. Although prostaglandins (PGs) long have been implicated in LPS-induced HPA axis activation, the mechanism downstream of PGs remained unsettled. By using mice lacking each of the four PGE receptors (EP1–EP4) and an EP1-selective antagonist, ONO-8713, we showed that both EP1 and EP3 are required for adrenocorticotropic hormone release in response to LPS. Analysis of c-Fos expression as a marker for neuronal activity indicated that both EP1 and EP3 contribute to activation of neurons in the paraventricular nucleus of the hypothalamus (PVN). This analysis also revealed that EP1, but not EP3, is involved in LPS-induced activation of the central nucleus of the amygdala. EP1 immunostaining in the PVN revealed its localization at synapses on corticotropin-releasing hormone-containing neurons. These findings suggest that EP1- and EP3-mediated neuronal pathways converge at corticotropin-releasing hormone-containing neurons in the PVN to induce HPA axis activation upon sickness.

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.

Clinical results of radiofrequency hyperthermia for malignant liver tumors

PURPOSE To evaluate thermometry and the clinical results of radiofrequency (RF) hyperthermia for advanced malignant liver tumors. METHODS AND MATERIALS One hundred seventy-three patients with malignant liver tumors treated between 1983 and 1995 underwent hyperthermia. The 173 tumors consisted of 114 hepatocellular carcinomas (HCCs) and 59 non-HCCs (47 metastatic liver tumors and 12 cholangiocarcinomas). Eight-megahertz RF capacitive heating equipment was used for the hyperthermia. Two opposing 25-cm electrodes were generally used for heating the liver tumors. Our standard protocol was to administer hyperthermia 40-50 min twice a week for a total of eight sessions. The liver tumor temperature was measured by microthermocouples when possible. Transcatheter arterial embolization, radiotherapy, immunotherapy, and chemotherapy were combined with hyperthermia treatment in accordance with each patients liver function. RESULTS One hundred forty (81%) of the 173 patients who underwent more than four sessions of hyperthermia were evaluated in this study. Thermometry was performed in 77 (55%) of these 140 patients. The maximum tumor temperature, average tumor temperature, and minimum tumor temperature in the HCC were (mean +/- standard error) 41.2 +/- 0.2 degrees C, 40.3 +/- 1.3 degrees C, and 40.1 +/- 0.2 degrees C, respectively. The same thermometry results for non-HCC were 42.3 +/- 0.2 degrees C, 41.2 +/- 0.2 degrees C, and 40.9 +/- 0.2 degrees C, respectively. The maximum and minimum temperatures (41.8 +/- 0.2 degrees C and 40.3 +/- 0.4 degrees C) in the patients with a complete or partial response (CR or PR) were higher than those in the patients with no response or progressive disease (NR or PD) (41.3 +/- 0.5 degrees C and 39.8 +/- 0.4 degrees C), but the difference was not significant. Of the 73 cases with HCC who were evaluated by computed tomography (CT), CR was achieved in 7 (10%), PR in 15 (21%), NR in 37 (51%), and PD in 14 (19%). Of the 45 cases involving liver metastases evaluated by CT, CR was achieved in 3 (7%), PR in 17 (38%), NR in 12 (27%), and PD in 13 (29%). The 1-year cumulative survival rate for HCC patients was 30.0%, and the 5-year survival rate was 17.5%. The 1-year survival of non-HCC patients was 32.5%, and the longest survival was 30 months. The sequelae of hyperthermia included focal fat necrosis in 20 patients (12%), gastric ulceration in 4 (2%), and liver necrosis in 1 (1%). The sequelae of thermometry were severe peritoneal pain in seven patients (11%), intraperitoneal hematoma in one (1%), and pneumothorax in one (1%). CONCLUSION Even though the thermometry results for liver tumors were not satisfactory, the treatment results are promising. Further clinical trials of RF capacitive hyperthermia for the treatment of advanced liver tumors should be encouraged.

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.

Treatment of ruptured hepatocellular carcinoma.

AbstractBackground. The problem of whether surgical or conservative treatment is indicated for ruptured hepatocellular carcinoma (HCC) has not been analyzed from the viewpoint of long-term development of hepatitis viral infection from liver fibrosis to liver cirrhosis. Although transcatheter arterial embolization (TAE) for hemostasis followed by two-stage hepatectomy has been established as the best treatment for ruptured HCC, there still remain difficulties in the treatment of some patients. Methods. Twelve patients with ruptured HCC who were surgically or conservatively treated were retrospectively analyzed in terms of modality of treatment, liver function, extension of HCC, complications, survival rate, and cause of death. Results. Tumor rupture can occur either in the early phase or in the terminal phase during the development from liver fibrosis to liver cirrhosis, while tumor rupture occurs at the advanced stage in terms of HCC extension. TAE for emergent hemostasis or prevention of re-bleeding was performed in ten patients, while TAE was contraindicated in one patient and emergent laparotomy for hemostasis was performed in one patient. In four patients, elective extended surgical resection was performed, because liver function was evaluated as clinical stage 1 according to the General rules for the clinical and pathological study of primary liver cancer of the Liver Cancer Study Group of Japan. In seven patients, conservative or medical treat-ment was selected, because liver function was evaluated as poor. The surgically treated group, who could tolerate extensive operation, survived longer than the conservatively treated group. Conclusions. While TAE remains the best method to employ for hemostasis, it still has limitations. Hence, we should be mindful of other possible modalities for hemostasis and their outcomes. Rupture of HCC at an early phase in the development of liver fibrosis is a good indication for elective surgical treatment and should be distinguished from rupture in the terminal phase of liver cirrhosis, which should be treated conservatively. Although elective surgical treatment can be performed in selected patients, tumor size and location of HCC, in addition to liver function, should be taken into consideration.

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.

Liver damage in patients with colony-stimulating factor-producing tumors

PURPOSE We have demonstrated that colony-stimulating factor (CSF)-producing tumor cell lines produce not only CSF but also interleukin-1 (IL-1) and interleukin-6 (IL-6). Clinically, we have observed that patients bearing such tumors present with liver dysfunction and fever in addition to marked leukocytosis. The purpose of this study was to determine whether or not the liver damage was specifically related to CSF-producing tumors. PATIENTS AND METHODS Clinicopathologic examinations were performed in six autopsied patients with CSF-producing tumors. We also transplanted two tumor cell lines (KHC287 and CHU-2), which produce granulocyte (G)-CSF, IL-1, and IL-6, to nude mice. RESULTS Of the six patients, five had G-CSF- and one had granulocyte/macrophage (GM)-CSF-producing tumors. IL-1 and IL-6 concentrations in plasma or culture supernatant were elevated in these patients. Biochemical examinations revealed high serum enzyme levels of the biliary system in contrast to normal or slight increases in transaminase levels in all patients studied. Serum direct bilirubin was elevated in five of the six patients. Three common pathologic changes of the liver were found: (1) focal necrosis associated with neutrophil infiltration in the centrilobular zones, (2) fibrous change and enlargement of the portal area associated with neutrophil infiltration, and (3) intrahepatic cholestasis. The same pathologic changes, except for cholestasis, were reproduced in the liver of mice transplanted with KHC287 or CHU-2. CONCLUSION These results indicate that patients with CSF-producing tumors have characteristic liver damage, and suggest a new paraneoplastic syndrome of leukocytosis and liver damage.

A useful ELISA system for human liver-type arginase, and its utility in diagnosis of liver diseases.

OBJECTIVES To develop a new ELISA system for liver-type arginase using monoclonal antibodies against the enzyme, and to verify the utility of the arginase in diagnosis of hepatic disorders. DESIGN AND METHODS We have developed an enzyme-linked immunosorbent assay (ELISA), using two kinds of monoclonal antibodies (Mo6G3 and Mo9C5) for human liver-type arginase as the first and second antibodies respectively. We have also developed a new method to eliminate the influence of erythrocyte-derived arginase contamination in hemolytic samples. This ELISA was applied to specimens received from patients with acute and chronic hepatic disease and also patients who had undergone partial hepatectomy. RESULTS This assay is sensitive and reproducible for the measurement of liver-type arginase in the sera of patients with liver dysfunction, and enabled us to detect enzyme concentrations as low as 27 pmol/L without any processing of the samples. The assay showed within-run coefficients of variation (CV) ranging from 1.9 to 4.1% and between-day CV from 3.6 to 5.1% for arginase concentrations varying from 57.1 to 1200 pmol/L. The recovery was 113% (mean) with a range of 96 to 129%. These antibodies reacted strongly with both recombinant and native liver-type arginases, while, to some extent, with erythrocyte-derived arginase. Correction for erythrocyte-derived arginase contamination in hemolytic samples was, however, easily made by assaying peroxidase-like activity of hemoglobin. From the view of a limited localization of arginase in the liver, the marked increase of the enzyme in serum reflects initiation of liver injury, while the rapid decrease reflects termination of the damage. Such quick normalization in circulating liver-type arginase indicated another merit of the enzyme in diagnosis of liver diseases. CONCLUSIONS The changes in circulating liver-type arginase level could be helpful not only in the diagnosis of liver diseases but also subsequent treatment of the patients with liver damage.

Liver-type arginase in serum during and after liver transplantation: a novel index in monitoring conditions of the liver graft and its clinical significance.

We quantified liver-type arginase in sera of 47 patients undergoing partial liver transplantation with use of an ELISA method. The level of liver-type arginase fluctuated slightly beyond the normal range in successful liver recipients, while it changed more drastically or precipitously in unsuccessful ones, accompanying or unaccompanying elevation of AST and ALT levels. A higher elevation pattern of the arginase level (above 100 ng ml-1) was observed in each of the unsuccessful recipients with critical condition, except for one patient. Other hepatic markers (LDH, ALP, and T-BIL) remained relatively unchanged until the terminal stage of deceasing patients. The finding that the liver-type arginase emerged in large quantity in the blood stream immediately after reperfusion of the liver graft indicates that the enzyme leaks out of hepatocytes damaged, presumably, by storage in the absence of circulation. A half-life of the liver-type arginase in the human blood was estimated to be 1 h, that is clearly shorter than that of AST. The short half-life of the arginase appears to be ascribable, at least partly, to formation of an immune complex with circulating autoantibody which appears in many liver recipients. These results suggest that liver-type arginase behaves uniquely in the serum among many hepatic enzymes, and could serve as a distinct marker of hepatic lesions, particularly during and after liver transplantation.