Toshihiko Koji

Early recognition of hepatocellular carcinoma based on altered profiles of alpha-fetoprotein.

BACKGROUND The sugar-chain structures of circulating alpha-fetoprotein in patients with hepatocellular carcinomas differ from those in patients with cirrhosis. We studied the reactivity of alpha-fetoprotein with two lectins, Lens culinaris agglutinin A and erythroagglutinating phytohemagglutinin, to monitor the evolution of hepatocellular carcinoma in patients with cirrhosis. METHODS Among 361 patients with cirrhosis caused mainly by chronic hepatitis B or hepatitis C virus infection, 33 with base-line serum alpha-fetoprotein concentrations > or = 30 ng per milliliter or more were found to have hepatocellular carcinomas during a mean follow-up of 35 months. The lectin-reactive profiles of the alpha-fetoprotein in the serum of these 33 patients were analyzed and compared with those in the serum of 32 patients with cirrhosis who had increased base-line serum alpha-fetoprotein concentrations and were followed for at least 24 months but in whom hepatocellular carcinoma did not develop. RESULTS At the time of tumor detection, 24 (73 percent) of the 33 patients with cirrhosis and hepatocellular carcinoma had higher percentages of L. culinaris agglutinin A-reactive alpha-fetoprotein (alpha-fetoprotein L3), erythroagglutinating phytohemagglutinin-reactive alpha-fetoprotein (alpha-fetoprotein P4+P5), or both than the 32 patients with cirrhosis but no hepatocellular carcinoma. Among the 24 patients, one or both of the markers were first elevated 3 to 18 months before the hepatocellular carcinoma was detected by imaging techniques. CONCLUSIONS Measurements of the alpha-fetoprotein L3 and alpha-fetoprotein P4+P5 fractions of serum alpha-fetoprotein allow the differentiation of hepatocellular carcinoma from cirrhosis in some cases and serve as predictive markers for the development of hepatocellular carcinoma during the follow-up of patients with cirrhosis.

Evaluation of Nontumorous Tissue Damage by Transcatheter Arterial Embolization for Hepatocellular Carcinoma

The serial changes in serum hepatic enzyme activities by transcatheter arterial embolization (TAE) were analyzed in 17 patients with hepatocellular carcinoma to estimate the contribution to the value by the damage of tumor or nontumorous hepatic cells. The serum levels of relatively tumor-specific fructose 1,6-diphosphate (FDP) aldolase were elevated after TAE in the cases of both superselective and nonsuperselective TAE that were performed from the segmental and the nonsegmental hepatic artery, respectively, but we found the marked elevation of FDP aldolase in the cases of the superselective TAE. In contrast, the non-tumor-specific fructose 1-phosphate (F1P) aldolase was markedly elevated only in the cases of nonsuperselective TAE. The total amount of FDP aldolase released by TAE correlated significantly with the integrated tumor tissue volume (P less than 0.005), whereas the total amount of F1P aldolase output correlated significantly with the integrated nontumorous tissue volume (P less than 0.005) as defined by lipiodol accumulation on computerized tomography scan. The consequent changes in the total nontumorous liver volumes after TAE were also analyzed by the follow-up computerized tomography scan. The nonsuperselective TAE caused the significant total nontumorous liver atrophy when compared with the superselective TAE. The progression of the total nontumorous liver atrophy correlated significantly with F1P aldolase output by TAE (P less than 0.001) but not with FDP aldolase output. These results suggest that the outputs of FDP and F1P aldolase are useful to estimate the degree of the tumorous and nontumorous tissue damage by TAE, respectively, and F1P aldolase output can be used to predict the progression of liver atrophy caused by TAE.

Analysis of alpha-fetoprotein gene expression in hepatocellular carcinoma and liver cirrhosis by in situ hybridization.

The expression of the alpha‐fetoprotein (AFP) gene in hepatocellular carcinoma (HCC) and liver cirrhosis by in situ hybridization analysis of AFP mRNA in cryostat and paraffin‐embedded tissue sections was studied. In HCC sections the majority of tumor cells showed positive hybridization with a 3H‐labeled AFP complementary DNA probe. The number of radioactive hybrid grains detected in HCC sections generally paralleled the level of serum AFP in the patient. In liver cirrhosis sections a small number of cells showed positive hybridization. These cells were dispersed in the tissue and morphologically indistinguishable from surrounding hepatocytes. Each of these cells contained a high level of hybridization signals to permit easy identification. In situ hybridization analysis of AFP mRNA may be of use in detecting preneoplastic cells in liver cirrhosis that cannot be defined on the morphologic and immunohistochemical basis.

Transforming growth factor β1 differentially regulates α-fetoprotein and albumin in HuH-7 human hepatoma cells

Abstract Transforming growth factor β1 (TGF-β1) is known to inhibit hepatocyte growth in vitro and in vivo. In this study, we analyzed the effect of TGF-β1 on α-fetoprotein (AFP) and albumin gene expression in HuH-7 human hepatoma cells. TGF-β1 inhibited cell growth in a dose dependent manner. The cellular secretion rate of AFP but not albumin was suppressed significantly by TGF-β1. TGF-β1 caused a significant reduction in the level of AFP mRNA. In contrast, the levels of albumin mRNA or β-actin mRNA were not changed by TGF-β1. In transient transfection experiments, TGF-β1 resulted in selective repression of AFP promoter activity. These results suggest that TGF-β1 is one of the key factors involved in the differential regulation of the AFP gene and the albumin gene.

Pancreatic tissue damage by transcatheter arterial embolization for hepatoma

We analyzed the serial changes in serum pancreatic enzyme activities by transcatheter arterial embolization (TAE) in 20 hepatoma patients with liver cirrhosis in an attempt to evaluate the incidence of the pancreatic tissue damage by TAE. Serum amylase activities increased in two (10%) cases, elastase 1 levels in six (30%) cases, and trypsin and pancreatic secretory trypsin inhibitor (PSTI) levels in each of five (25%) cases. Consequently, TAE resulted in the elevation of at least more than one serum pancreatic enzyme in eitht (40%) of 20 cases, although none had clinical symptoms related to pancreatitis When the adverse effect on the pancreatic tissue was compared among 6 cases of the superselective TAE and 14 cases of the nonsuperselective TAE, which were perfomed from the segmental and the nonsegmental hepatic arteries, respectively, the elevation of serum pancreatic enzymes was caused only by nonsuperselective TAE, not by superselective TAE. The volumes of Spongel and lipiodol used or the injected doses of the anticancer agent mitomycin C were not different between the two groups. These results indicate that TAE for the treatment of hepatoma frequently causes pancreatic tissue damage, and the position of the inserted catheter tip is very important to avoid the pancreatic tissue damage by TAE.

RADIOIMMUNODETECTION OF CANCER USING ANTIBODIES TO α-FETOPROTEIN AND CARCINOEMBRYONIC ANTIGEN

This study reports the use of radiolabeled antibody to AFP and CEA for the detection and localization of AFP- or CEA-producing tumors. Thirty-one patients received 131I-labeled anti-AFP or anti-CEA antibodies. Photoscans were taken at 24 and 48 hours after injection of radioantibodies. In three of six patients with CEA-producing tumors, radioimmunodetection with anti-CEA antibody showed positive scans. In AFP-producing tumors, 7 of 15 patients had positive findings on immunoscintigraphy using polyclonal anti-AFP antibody, and two of nine patients had positive findings when monoclonal antibodies were used. Analysis of radioantibody in the blood after injection showed both complex and free antibody with immunoreactivity in the circulation, and smaller complexes were seen to form after administration of monoclonal antibodies.

Purification of specific antibody to α-foetoprotein and its immunological effect on cancer cells

Abstract Human or rat α-foetoprotein (AFP) was highly purified from ascitic fluid or serum of hepatoma bearers. The purification was carried out mainly by means of immunoadsorbent chromatography using Sepharose coupled to specific anti-AFP antibody with BrCN activation, and by Sephadex gel filtration. Horses were immunized with the purified AFP and the specific antibody was isolated from the antiserum by means of an immunoadsorbent coupled to purified AFP. The specific antibody was found to bind specifically with AFP-producing tumour cells. The antibody was applied for radio immunodetection of the tumour. 125 I-labelled antibody was administered to patients or rats with hepatoma, and radioactivity localized in the tumours was scintiscanned with a scintillation camera. In this way, the location of the tumour was detected in about 50% of the hepatoma bearers. The cytotoxicity of the antibody was clearly demonstrated both vitro and in vivo in animal experiments. The antibody was administered to patients with advanced hepatoma. Although no improvement of the disease was demonstrated, serum AFP levels decreased greatly and in some cases low AFP levels were maintained for long periods suggesting that the antibody suppressed AFP-producing hepatoma cells. No significant side effects were observed in patients who had been administered with the horse antibody.

Attempts of Treatment of Hepatoma with Antibody to Alpha-Fetoprotein

Abstract Highly purified polyclonal or monoclonal antibodies to human or rat α-fetoprotein (AFP) were proved to be cytotoxic to AFP-producing hepatoma cells. Rat with transplanted hepatoma lived longer by the antibody treatment. The polyclonal horse antibody to human AFP was administered to 25 cases of patients mostly with hepatoma. In 7 out of 25 cases, serum AFP levels were markedly suppressed for a long period. Daunomycin, an antitumor drug, was conjugated to the antibodies according to the method of E. Hurwitz and M. Sela. The conjugate revealed a striking antitumor effect compared to antibody or drug alone. Toxic effect of daunomycin upon the host animals was markedly decreased by the conjugation with antibody protein. Any serious side effect of administration of horse antibody was observed in the patients.

Radioimmunodetection of Cancer Using Radiolabeled Antibodies to α-Fetoprotein

Abstract The tumor imaging using radiolabeled antobodies to α-Fetoprotein (AFP) was undertaken in 27 patients with various AFP producing tumors. In 7 of 15 patients (47%), positive images were obtained using polyclonal antibody and 4 positive and 3 doubtful images were obtained in 12 patients using 4 monoclonal antibodies. The tumor/serum ratios of AFP showed high levels in positive cases, and all monoclonal antibodies could bind to AFP in 7 patients with various primary tumor sites in vitro.

Enhanced DNA synthesis in rat hepatoma cells by conditioned media from Kupffer cells incubated with supernatants of tumor necrosis factor-α-pretreated hepatocytes

The effects of tumor necrosis factor-alpha (TNF-alpha) on DNA synthesis in AH66 rat hepatoma cells and rat hepatocytes were analysed by means of [3H]thymidine incorporation. DNA synthesis in AH66 cells was suppressed when AH66 cells were directly incubated with TNF-alpha. When primary culture of rat Kupffer cells was incubated with hepatocyte conditioned media pretreated with TNF-alpha (0-200 U/ml), and AH66 cells were then treated with these hepatocyte/Kupffer cell-conditioned media, TNF-alpha used in the pretreatment caused a dose-dependent increase in DNA synthesis in AH66 cells with a maximum effect amounting to a more than 10-fold increase. In contrast, DNA synthesis in primary culture of rat hepatocytes was not stimulated by the TNF-alpha-pretreated hepatocyte/Kupffer cell conditioned media. These results suggest that TNF-alpha-mediated hepatocyte-Kupffer cell interaction selectively promotes proliferation of rat hepatoma cells.