Naomi Tanaka

Proton Beam Therapy for Hepatocellular Carcinoma: A Retrospective Review of 162 Patients

Purpose: We present results of patients with hepatocellular carcinoma (HCC) treated with proton beam therapy. Experimental Design: We reviewed 162 patients having 192 HCCs treated from November 1985 to July 1998 by proton beam therapy with or without transarterial embolization and percutaneous ethanol injection. The patients in the present series were considered unsuitable for surgery for various reasons, including hepatic dysfunction, multiple tumors, recurrence after surgical resection, and concomitant illnesses. The median total dose of proton irradiation was 72 Gy in 16 fractions over 29 days. Results: The overall survival rate for all of the 162 patients was 23.5% at 5 years. The local control rate at 5 years was 86.9% for all 192 tumors among the 162 patients. The degree of impairment of hepatic functions attributable to coexisting liver cirrhosis and the number of tumors in the liver significantly affected patient survival. For 50 patients having least impaired hepatic functions and a solitary tumor, the survival rate at 5 years was 53.5%. The patients had very few acute reactions to treatments and a few late sequelae during and after the treatments. Conclusions: Proton beam therapy for patients with HCC is effective, safe, well tolerable, and repeatable. It is the useful treatment mode for either cure or palliation for patients with HCC irrespective of tumor size, tumor location in the liver, insufficient feeding of the tumor with arteries, presence of vascular invasion, impaired hepatic functions, and coexisting intercurrent diseases.

Detection of GST1 gene deletion by the polymerase chain reaction and its possible correlation with stomach cancer in Japanese

SummaryA homozygous gene deletion at the GST1 locus of genomic DNA isolated from peripheral blood was investigated for its relationship with several types of cancer using the polymerase chain reaction (PCR) technique. DNA samples were prepared from blood obtained from 128 healthy blood donors and 150 patients with cancer or chronic hepatitis. PCR primers were prepared based on the human cDNA sequence and the intron/exon sequences of the rat Yb2 gene. The amplified sequence between exons 5 and 6 including intron 5 showed very clearly the presence of absence of the GST1 gene, after electrophoresis in a 2% agarose gel. Segregation of the presence and absence of PCR product from samples of twins and their parents indicated that presence involves homozygous or heterozygous normal GST1 genotypes while absence invovles only homozygous gene deletion. The patients with stomach cancer had a significantly higher frequency of gene deletion than did the healthy controls (P< 0.005). Thus, GST1 deletion may be a possible genetic marker for early detection of a group at high risk of stomach cancer.

The expression levels of plasma membrane transporters in the cholestatic liver of patients undergoing biliary drainage and their association with the impairment of biliary secretory function

OBJECTIVES:Percutaneous transhepatic biliary drainage (PTBD) has been believed to reduce hyperbilirubinemia in patients with obstructive cholestasis and to lessen liver injury through bile acid retention. The efficacy may be closely related to the capability of cholestatic liver to produce and secrete bile, which in turn depends on the expressions and functional activities of plasma membrane transporters in the liver. The aim of the present study was to determine the expression levels of these transporters in the cholestatic liver of patients undergoing PTBD.METHODS:A total of 24 patients who had experienced obstructive cholestasis and had undergone preoperative PTBD were included in the study. Liver biopsy specimens were analyzed to determine the expression levels of the multidrug resistance-associated proteins (MRP) MRP2 and MRP3 and the canalicular bile salt export pump BSEP in the liver.RESULTS:The messenger RNA (mRNA) levels of MRP2, the canalicular bilirubin conjugate export pump, and bile salt export pump (BSEP) were unchanged in liver specimens from the 14 patients well drained by PTBD but were reduced in specimens from the 10 patients poorly drained, compared to the levels of control subjects. Immunostainings of MRP2 and BSEP outlined the canalicular membrane domain but seemed fuzzy to varying degrees in specimens obtained from cholestatic liver, especially in specimens from liver that had been poorly drained, in contrast to the linear and intense localization in the liver of control subjects, correlating with the impaired bilirubin conjugate and bile acid secretion. The mRNA of MRP3, functioning as an inducible export pump for bilirubin conjugate and bile acid, was expressed not only in the cholestatic liver but also in the liver of control subjects, and the mRNA level was increased in specimens from both the cholestatic liver that had been well drained and from the liver that had been poorly drained. Immunostaining of MRP3 was observed in the epithelia of intrahepatic bile ducts in the liver of both control subjects and cholestatic patients, and in the epithelia of proliferated bile ductules and the hepatocytes surrounding the portal tracts in the cholestatic liver.CONCLUSIONS:From the results of the present study, it is concluded that 1) the mRNA and immunohistochemical expression levels of MRP2 and BSEP may be altered in the cholestatic liver of patients undergoing PTBD; 2) both the decreased mRNA levels and the diminished canalicular membrane localization may be associated with the impairment of bile formation and secretion, i.e., the efficacy of PTBD; and 3) upregulated MRP3 in the cholangiocytes and hepatocytes may play a significant role in bile acid transport in the cholestatic hepatobiliary system.

Serum des-gamma-carboxy prothrombin levels determined by a new generation of sensitive immunoassays in patients with small-sized hepatocellular carcinoma.

OBJECTIVE: Des-gamma-carboxy prothrombin (DCP), also called protein induced by vitamin K absence or antagonist II (PIVKA-II), is a tumor marker complementary to AFP for the diagnosis of hepatocellular carcinoma (HCC). Currently available immunoassays for DCP are not sensitive enough to detect HCC at an early stage. Recently, two new immunoassays with enhanced sensitivity were developed. The aim of this study was to assess the diagnostic values of the new methods in patients with small-sized HCC. METHODS: Coded serum samples obtained from 36 patients with small-sized and single-nodular HCC (≤3 cm in diameter) and 49 patients with posthepatitic cirrhosis not carrying HCC were analyzed. DCP levels were determined in three different ways: 1) conventional EIA; 2) a new immunoassay using the electrochemiluminescence (ECLIA) detection system; and 3) a new immunoradiometric assay (IRMA). Lectin-reactive profiles of AFP (AFP-L3) were also determined. RESULTS: In 36 patients with small-sized HCC, the rates of abnormal values obtained by the conventional, ECLIA, and IRMA methods were 2.7%, 27.8%, and 16.7%, respectively. An ROC analysis of the two new methods (ECLIA vs IRMA) revealed a better performance by the ECLIA method (p < 0.05). The true positive rate of AFP-L3 was 22.2%, whereas a combination assay of ECLIA for DCP and AFP-L3 resulted in a 41.7% sensitivity with a specificity of 90%. CONCLUSIONS: Compared with the conventional method, the sensitivity in detecting small-sized HCC was increased in the two new DCP immunoassays (ECLIA and IRMA). The overall performance as evaluated by an ROC analysis was significantly better in ECLIA than in IRMA.

A new, effective and safe therapeutic option using proton irradiation for hepatocellular carcinoma

BACKGROUND/AIMS Conventional radiation is almost useless for hepatocellular carcinoma (HCC) because of the severe adverse effects of the irradiation to the accompanying liver cirrhosis. In contrast, the proton beam has Bragg peak, which limits distribution of the beam. The aim of this study was to prove the usefulness of proton irradiation for HCC. METHODS The proton irradiation was performed in 32 nodular lesions in 24 patients with HCC who had unresectable tumors or serious complications; the proton irradiation was performed either as monotherapy (15 lesions) or as combination therapy to insufficient Lipiodol-targeted chemotherapy (Kodama Co. Ltd., Tokyo, Japan) (17 lesions). The energy was 250 MeV, and 50-87 Gy (76.5 +/- 9.5, mean +/- SD) in total was irradiated for a time period of 17-69 days. RESULTS After 1 year, size reduction was seen in 12 out of 13 lesions (92%) in the monotherapy group and 9 out of 9 lesions (100%) in the combination therapy group; after 2 years, size reduction was seen 4 out of 5 lesions (80%) in the monotherapy group and 5 out of 5 lesions (100%) in the combination therapy group. Local tumor control has being assured for 2 years of the observation, which is continuing for another 2 years. None of the patients have experienced any serious adverse effects. CONCLUSIONS These results show that proton irradiation is a new, safe, and effective therapeutic option in cases of HCC, even in patients with unresectable tumors or those with serious complications.

Genipin enhances Mrp2 (Abcc2)-mediated bile formation and organic anion transport in rat liver

Inchin‐ko‐to (ICKT), an herbal medicine, and its ingredients exert potent choleretic effects by a “bile acid‐independent” mechanism. The current study was designed to determine whether ICKT or its ingredients potentiate multidrug resistance‐associated protein 2 (Mrp2; Abcc2)‐mediated choleresis in vivo. Biliary secretion of Mrp2 substrates and the protein mass, subcellular localization, and messenger RNA (mRNA) level of Mrp2 were assessed in rat liver after infusion of genipin, an intestinal bacterial metabolite of geniposide, a major ingredient of ICKT. The function of Mrp2 was also assessed by the adenosine triphosphate (ATP)‐dependent uptake of Mrp2‐specific substrates using canalicular membrane vesicles (CMVs) from the liver. Infusion of genipin increased bile flow by 230%. It also increased biliary secretion of bilirubin conjugates and reduced glutathione (GSH) by 513% and 336%, respectively, but did not increase bile acid secretion. The ATP‐dependent uptake of estradiol 17‐β‐D‐glucuronide (E217βG; by 265%), leukotriene C4 (LTC4; by 161%), taurolithocholate‐3‐sulfate (TLC‐3S; by 266%), and methotrexate (MTX; by 234%) was significantly stimulated in the CMVs from the liver. These effects were not observed in Mrp2‐deficient rats. Under these conditions, genipin treatment increased the protein mass of Mrp2 in the CMVs but not the mRNA level. In immunoelectron microscopic studies, a marked increase in Mrp2 density in the canalicular membrane (CM) and microvilli was observed in the genipin‐treated liver tissue sections when compared with the vehicle‐treated liver tissue sections. In conclusion, genipin may enhance the bile acid‐independent secretory capacity of hepatocytes, mainly by stimulation of exocytosis and insertion of Mrp2 in the bile canaliculi. ICKT may be a potent therapeutic agent for a number of cholestatic liver diseases. (HEPATOLOGY 2004;39:167–178.)

A novel β1,3-N-acetylglucosaminyltransferase (β3Gn-T8), which synthesizes poly-N-acetyllactosamine, is dramatically upregulated in colon cancer

A new member of the UDP‐N‐acetylglucosamine: β‐galactose β1,3‐N‐acetylglucosaminyltransferase (β3Gn‐T) family having the β3‐glycosyltransferase motifs was identified using an in silico method. This novel β3Gn‐T was cloned from a human colon cancer cell line and named β3Gn‐T8 based on its position in a phylogenetic tree and enzymatic activity. β3Gn‐T8 transfers GlcNAc to the non‐reducing terminus of the Galβ1–4GlcNAc of tetraantennary N‐glycan in vitro. HCT15 cells transfected with β3Gn‐T8 cDNA showed an increase in reactivity to both LEA and PHA‐L4 in a flow cytometric analysis. These results indicated that β3Gn‐T8 is involved in the biosynthesis of poly‐N‐acetyllactosamine chains on tetraantennary (β1,6‐branched) N‐glycan. In most of the colorectal cancer tissues examined, the level of β3Gn‐T8 transcript was significantly higher than in normal tissue. β3Gn‐T8 could be an enzyme involved in the synthesis of poly‐N‐acetyllactosamine on β1–6 branched N‐glycans in colon cancer.

Multivariate analysis of risk factors for hepatocellular carcinoma in patients with hepatitis C virus-related liver cirrhosis.

To elucidate the risk factors for hepatocellular carcinoma (HCC) in hepatitis C virus (HCV)-related liver cirrhosis (LC), we examined 204 cirrhotic patients negative for hepatitis B surface antigen and positive for HCV antibodies. The independent influence of various clinical characteristics in these patients was analyzed by multiple logistic regression, and the risk factors for HCC were identified. Multiple logistic regression analysis identified and ranked the following four risk factors: male sex (P<0.001), habitual heavy drinking (P<0.005), hepatitis B virus antibody positivity (anti-HBs and/or anti-HBc,P<0.05), and age greater than 60 years (P<0.05). The odds ratio of HCC was 4.20 (95% confidence interval; CI, 1.80–9.78) in male patients, 3.27 (95% CI, 1.46–7.30) in habitual heavy drinkers, 2.01 (95% CI, 1.01–3.99) in patients positive for hepatitis B virus antibodies, and 2.06 (95% CI, 1.00–4.23) in patients older than 60 years. The cumulative occurrence rates of HCC after blood transfusion were significantly higher in habitual heavy drinkers (4.8%, 49.4%, and 74.7% at 10, 20, and 30 years, respectively) than in non-drinkers (0%, 21.0%, and 23.3% at 10, 20, and 30 years, respectively,P<0.0003). The mean interval for progression to LC after blood transfusion was significantly shorter in the habitual heavy drinkers than in the non-drinkers (22.4±4.4 years vs 28.4±3.9 years;P<0.0003). This multivariate analysis revealed that habitual heavy drinking and hepatitis B virus antibody positivity are significant risk factors for HCC in HCV-related liver cirrhosis.

The chemopreventive role of ursodeoxycholic acid in azoxymethane-treated rats: suppressive effects on enhanced group II phospholipase A2 expression in colonic tissue

Great interest has been focused on the chemoprevention of colonic carcinogenesis by oral administration of ursodeoxycholic acid (UDCA) because its administration reportedly reduces the incidence of colon cancer in animal experiments. To elucidate the precise role of UDCA in the chemoprevention of azoxymethane-induced colon carcinogenesis, we examined the expression levels of group II phospholipase A2 in the colonic tissue of UDCA-treated and untreated rats and correlated the levels with the findings of aberrant crypt foci, putative preneoplastic lesions. Twelve weeks after azoxymethane exposure, the total number of aberrant crypt foci in 0.4% UDCA-fed rats and 1% UDCA-fed rats was significantly decreased compared to the untreated animals. The mucosal concentrations of PGE2 and 6-keto PGF1alpha were significantly lower in the UDCA-treated rats than in untreated rats. In correlation with lowering, the enhanced activity, protein mass and mRNA levels of group II phospholipase A2 were significantly attenuated in the UDCA-treated animals. The chemopreventive role of UDCA in colon carcinogenesis may lie in its modulation of the arachidonate metabolism in colonic mucosa.

Liver glutathione S-transferase polymorphism in Japanese and its pharmacogenetic importance

SummaryA total of 168 autopsy liver extracts from Japanese individuals were examined for the glutathione S-transferase (GST) isozymes by means of starch gel electrophoresis. The gene frequencies of GST1*1, GST1*2, and GST1*0 in Japanese were 0.252, 0.057, and 0.691, respectively. GST1*3 was detected as a rare variant allele. The incidence of GST1 0 in 41 liver biopsy samples from patients suffering from various liver diseases was investigated using polyacrylamide gel isoelectric focusing. The GST1 0 phenotype was found more frequently in livers with hepatitis and carcinoma than in control livers. The isozymes coded by different GST loci were partially purified and characterized to study their biochemical properties. The apparent Km values with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate for the isozymes at the GST1, GST2, GST3, and GST4 loci were 604, 1345, 776 and 591 μM, respectively.