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An expression of ras p21 proteins on cholangiocarcinoma (CC) (intrahepatic bile duct carcinoma) cells was examined by an immunoperoxidase method using an appropriate dilution of mouse monoclonal antibody RAP-5, with which no positive staining was obtained in livers with normal histology. Of 44 CCs examined 39 were positive for the antigens; well-differentiated adenocarcinoma usually showed a diffuse weak, cytoplasmic staining in nearly all tumor cells with the same staining intensity, while in moderately and poorly differentiated adenocarcinoma the expression of p21 varied markedly in intensity from cell to cell in the same cell nest. The number of positive cells decreased with the grade of tumor, and no or little staining was observed in undifferentiated areas. These findings indicate that the expression of ras p21 antigens was lost with increasing dedifferentiation of tumor cells. Carcinoembryonic antigens (CEA) were positive in 42 of 44 CCS. Well-differentiated adenocarcinoma expressed CEA along the apical surfaces of the tumor glands. With the dedifferentiation of tumor cells, the expression of CEA became prominent not only at the apical surfaces but also on the basolateral surfaces and in the cytoplasms, and further in the surrounding stromal tissue. There was no clear-cut correlation between the expression of p21 antigens and the production of CEA in CCs.

Proton-induced x-ray fluorescence has been used to determine the concentration of trace elements in human prostate and liver tissue samples. Ca, Fe, Cu, and Zn were observed in all specimens. Small amounts of Br were detected in most of the prostates, and in nearly half of the livers. The data indicate that the Fe content decreases in malignant liver tissues and the quantities of Cu and Zn are also decreased, but to a lesser extent.

Histochemical and immunohistochemical studies on 47 consecutive specimens excised for choledochal cyst were performed to clarify possible metaplastic changes of the biliary duct in relation to carcinogenesis. An anomalous arrangement of the pancreaticobiliary ductal system was observed in all 39 cases examined. Among the 47 patients, 5 (10.6 per cent) had biliary carcinoma. 27.3 per cent mucous gland, 13.0 per cent goblet cell and 9.5 per cent argyrophil cell in 23 children. On the other hand, 81.8 per cent exhibited mucous gland, 41.7 per cent goblet cell and 27.3 per cent argyrophil cell in 24 adults. These metaplastic changes seemed to be an intestinal metaplasia and increased with age. Immunoreactive-gastrin or -somatostatin were evident immunohistochemically in 4 adults. These findings confirmed that intestinal metaplasia may develop in the biliary duct in cases of choledochal cyst. Although direct evidence between intestinal metaplasia and the development of biliary carcinoma was not found, reflux and stasis of pancreatic enzymes in the biliary duct may relate to the development of intestinal metaplasia and be an important factor related to the carcinogenesis of choledochal cyst.

Primary liver carcinoma (PLC) may express a certain number of markers. Here we communicate results of an analysis of five such markers (alpha-1-antitrypsin--AAT--, carcino-embryonic antigen --CEA--, alpha-fetoprotein --AFP--, and superficial --HBsAg-- and core --HBcAg-- antigens of hepatitis B virus) by means of PAP techniques in 130 cases of PLC, comparing the neoplastic tissue and the non-tumorous liver. Three variants of PLC are distinguished: hepatocarcinoma (HC) (108 cases); cholangiocarcinoma (CC) (19 cases); and three cases of hepatocholangiocarcinoma (HCC). AAT was positive in 29 HC, 2 HCC, and negative in all 19 CC. CEA appeared positive in 16 HC, 16 CC and only one HCC. AFP was positive in two HC, and negative in all CC and HCC. HBsAg displayed positivity in 15 HC and one HCC, being negative in all 19 CC. HBcAg was positive in 4 HC, and negative in all CC and HCC. HBsAg was also positive in two neoplastic emboli associated with HC. On the non-tumorous liver tissue the immunohistochemical results showed positivity for AAT and CEA, but not for AFP. Therefore the present results confirm that in the geographical area from which these tumors proceed, PLC is closely correlated with HBsAg positivity and with cirrhosis.

A series of 10 cases of biliary obstruction due to primary cholangiocarcinoma has been studied with histological and immunocytochemical means. The total duration of cholestasis (as manifested by jaundice) was between 2 and 11 weeks with variable period of preoperative drainage. Liver biopsy specimens taken during surgery for cholangiocarcinoma were investigated for the presence of ductular proliferation and the development of fibrosis, as demonstrated by Sirius Red F3BA collagen staining. The differentiation of epithelial components was evaluated by AEC-immunostaining with chain-specific monoclonal antibodies specifically directed against human keratins type 7, 18 and 19. Keratin 7, normally occurring only in the ductular system, was expressed in hepatocytes at the periphery of the hepatic lobule (zone I) following about 4 weeks' cholestasis, when an increase of ductular profiles in the enlarged portal areas had become manifest. Such keratin 7 positive cells, however, still retained all morphological aspects of hepatocytes. Keratin 19, normally also restricted to the ductular system in liver, is not expressed by zone I hepatocytes even after longer duration (up to 11 weeks) of cholestasis. It is concluded that the increase in ductular profiles during the first week is mainly due do proliferation of pre-existing ductules, while ductular metaplasia occurs in more chronic cholestasis. Development of fibrosis, not always strictly paralleling the multiplication of ductular profiles in sections through a portal tract, represents an early change, and is clearly apparent after 2 weeks of obstruction.

The studies described were designed to examine the question of cross-reactivity between basic protein of myelin and a basic protein common to many human tumours. The lymphocytes from guinea-pigs injected with acid extracts of human cancer tissue showed significant (P less than 0-01) tranformation on exposure to basic protein of human myelin. Conversely lymphocytes from guinea-pigs injected with basic protein of human myelin showed significant (P less than 0-01) transformation on exposure to the acid extracts of human cancer tissue. Lymphocytes from control non-injected guinea-pigs did not transform on exposure to either antigen. These findings demonstrate immunological cross-reactivity in guinea-pigs between basic proteins of human myelin and of human cancer tissue using an assay system other than the macrophage electrophoretic migration hitherto used to show this effect in man.

A method is proposed for the analysis of glycosaminoglycans that were isolated from human liver, combining cellulose acetate electrophoresis and enzymatic digestion with mucopolysaccharidases. The major constituent of glycossaminoglycans in the healthy liver is heparin sulfate and/or heparin (about 65%), with approximately equal quantities of dermatan sulfate and hyalauronic acid (about 13.5 and 13%, respectively) and a small amount of chondroitin sulfate. These components, especially chondroitin sulfate and hyaluronic acid, are markedly increased in hepatic carcinomas.

Since the relationship between tissue ligandin and liver tumors has not been studied yet, we investigated the changes of Y protein and ligandin in human hepatoma and cholangioma by gel filtration, BSP-affinity chromatography, and SDS-gel electrophoresis. The concentration of Y protein was markedly increased in both hepatoma and cholangioma, 2.8 and 4.8 times that of control, respectively. The content of ligandin was also increased in both conditions. SDS polyacrylamide gel electrophoresis of the increased ligandin confirmed the increment of 2.3 K dalton protein, which coincided with the MW of the ligandin subunit. Although the mechanism of the ligandin increase in hepatoma tissue is not clear, one possible reason might be due to the degree of differentiation of the tumor cells. In our case, the pathological examination revealed that the tumor cell was classified as Edmondoson Type II.

The receptors of peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA) and Ulex europaeus agglutinin I (UEA-I) were localized in intrahepatic cholangiocellular carcinoma, hepatocellular carcinoma, intrahepatic bile ducts and normal, cirrhotic and pericarcinomatous liver using the avidin-biotin-peroxidase complex method. It was found that epithelial cells of normal bile ducts had many UEA-I receptors, fewer DBA receptors and no PNA receptors. The positive rates of PNA, UEA-I and DBA receptors in 18 cases of intrahepatic cholangiocellular carcinoma were 88.9%, 61.1% and 33.3% respectively, which were significantly higher than those in hepatocellular carcinoma (16.0%, 4.0% and 4.0% respectively). Hepatocytes in normal, cirrhotic and pericarcinomatous liver had no receptors for these three lectins. It is suggested that lectin receptor distribution in intrahepatic cholangiocellular carcinoma is obviously different from that in normal bile duct cells and in hepatocellular carcinoma, and might be used as an auxiliary index in its clinical diagnosis.

Localization of alpha-fetoprotein (alpha-FP) has been followed in hepatal tissue and tumors during induction of primary hepatomas with the aid of 0.12% 3'-Me-DAB (3'-methyl-4-dimethylammoazobenzene) in Wistar rats. The indirect immunofluorescence method was used for the localization of alpha-FP positive cells. During the course of carcinogenesis, alpha-FP in serum was detected by means of the crossing over immunoelectrophoresis. This study has yielded the following results: Alpha FP positive cells resembling small hepatocytes occurred dispersed and in groups beginning with the 5th week of a carcinogenic diet until the appearance of tumors. No alpha-FP positive oval cells have been found. Alpha-FP positive cells were always found in rats with alpha-FP positive serum, but they were rarely present in rats with alpha-FP negative serum. From the 10th week, tumors of the cholangiohepatoma type began to be formed in which variously scattered alpha-FP positive cells of the type of small hepatocytes were present, with the serum being negative. Between week 14 and 21 hepatoma nodules began to be formed. At week 21 frequent alpha-FP positive cells close to normal hepatocytes were observed both singly and in groups. These are considered to be the sites of developing tumor nodules. In all the hepatoma nodules, the number of positive tumorous cells and the intensity of fluorescence proved to be directly proportional to alpha-FP concentration in serum.

Cholangiocarcinoma is one of the main liver diseases in northeast Thailand. Associations with exposure to liver fluke and N-nitrosodimethylamine in formation of the tumour have been demonstrated in animals. This study was carried out to compare possible endogenous formation of N-nitroso compounds in inhabitants of areas with low and high incidences of cholangiocarcinoma by examining the levels of nitrate and nitrite in their saliva and urine. Thirty-two subjects (16 males and 16 females) living in the north-east (high incidence) and 12 volunteers (6 males and 6 females) in Bangkok (low incidence) were allowed to take regular meals, and their saliva and urine were collected before, and 30, 60 and 120 min after each meal. Nitrate and nitrite concentrations in saliva of the group in the high-incidence area were significantly higher than those of the group in Bangkok: salivary nitrate was 2-2.8 times higher and nitrite 2-5.6 times higher in the north-eastern group when compared with levels at each corresponding time interval in the low-incidence group. Nitrate levels in urine were also significantly higher in the north-eastern group at some time intervals, but urinary nitrite levels were similar and very low in both groups throughout the day. This finding may indicate a greater possibility of in-vivo formation of N-nitroso compounds in the north-east area than in Bangkok and might be associated with the occurrence of cholangiocarcinoma in north-east Thailand.

The immunohistochemical localization of human chorionic gonadotropin (HCG) and alpha-fetoprotein (AFP) was studied in 44 cases with cholangiocarcinoma (CC) to determine the correlation to the expression of ras oncogene product p21 on tumor cells. HCG-immunoreactivity was found in 10 of 44 cases (23%) and AFP in only one (2.3%), whereas the expression of ras p21 was demonstrated in 39 (88.6%). The incidence of HCG-positive cells within the tumor was less than 1% in 8 of 10. The incidence of AFP-positive cells was less than 0.01%. All were histologically classified as adenocarcinoma and none of them had histologic features of trophoblastic tumors, yolk sac tumor or hepatocellular carcinoma. Nine of 10 HCG-positive and one AFP-positive CC expressed ras p21 on their tumor cells. However, one HCG-positive CC was negative for ras p21, though the incidence of HCG-positive cells within the tumor was 25%. HCG- and AFP-immunoreactivity was more frequently observed in poorly or undifferentiated tumor cells than in moderately or well-differentiated areas, whereas the expression of ras p21 was more diffuse in well-differentiated tumor and stronger in moderately differentiated areas, but rarely found in poorly and undifferentiated tumor. These results suggest that HCG production by CC of the usual adenocarcinoma variety is not rare, when compared to AFP production, and is preferentially localized in a small number of poorly differentiated and undifferentiated carcinoma cells, and there is no correlation between the production of HCG or AFP and the expression of ras p21 in CCs.

Ricinus communis agglutinin I(RCAI) receptors in 25 cases of hepatocellular carcinoma and 6 cases of intrahepatic cholangiocellular carcinoma were immunohistochemically localized by avidin-biotin-peroxidase complex (ABC) method. In the meantime, RCAI receptors in normal and cirrhotic liver tissues were also observed as controls. The results showed that there were many irregularly distributed RCAI receptors in HCC in forms of dispersed dots, even or localized lumpy stainings. The receptors in most intrahepatic cholangiocellular carcinomas were distributed in a polar form. However, the distribution of RCAI receptors in hepatocytes of normal, cirrhotic and precancerous liver tissues was band-like. It is suggested that the distribution of RCAI receptors in the cells might be helpful to the diagnosis of hepatoma and to the differentiation of benign from malignant hyperplasia.

The distribution of laminin, a basement membrane glycoprotein, was studied by an immunohistochemical technique in six samples of benign sclerosing lesions of extrahepatic bile ducts and in 11 sclerosing cholangiocarcinomas. The expression of laminin showed that benign glandular structures were surrounded by a mainly intact basement membrane. In sclerosing cholangiocarcinomas laminin was irregularly distributed, and in large areas totally absent. The findings suggest that deposition of basement membrane material is defective. Sclerosing cholangitis may be extremely difficult to distinguish from well differentiated sclerosing cholangiocarcinoma. This sample was small, but the diseases studied were relatively rare. Staining for laminin might be of additional use to surgical pathologists in the differential diagnosis of benign sclerosing lesions and sclerosing cholangiocarcinomas.

Using 109 hepatocellular carcinomas (HCG), 34 cholangiocellular carcinomas (CCC), 4 mixed hepatocellular-cholangiocellular carcinomas (MHC) and 24 metastatic adenocarcinomas in the liver (MA), an immunohistochemical study on primary carcinoma of the liver was performed by means of the ABC method for carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), tissue polypeptide antigen (TPA) and keratin. The material consisted of surgical specimens of Kosin Medical College including 50 HCC, 17 CCC and 1 MHC, surgical specimens of 20 HCC from the University of Occupational and Environmental Health, Japan (UOEH) and autopsied specimens from UOEH that included 39 HCC, 17 CCC, 3 MHC and 24 MA. All the specimens were fixed with 10-15% formalin and embedded in paraplast manually at Kosin Medical College and by utilizing an automatic embedding machine with a decompressing procedure at UOEH. The antigenicity of TPA and keratin was preserved better in the specimens of Kosin Medical College than in those from UOEH. It is therefore assumed that manually embedded specimens are superior to specimens embedded by using an embedding machine with regard to the preservation of some antigenicities. The immunoreactivity of the 4 antigens in CCC cells was significantly higher than that in HCC cells, and the intracellular localization of antigens generally showed several characteristics in HCC and CCC. However, as the same localization of antigens is also seen in both HCC cells and CCC cells, it is considered that the immunohistochemical examination using plural antibodies is not always useful for a differential diagnosis between HCC and CCC, which is difficult in conventional sections. That TPA in HCC may be an oncodevelopmental antigen is suggested by the facts that the higher the grade of HCC, the higher the immunoreactivity of HCC cells, that hepatocytes with possible higher activity sometimes showed a positive reaction in the present study and that TPA is expressed in fetal hepatocytes in a fetus up to 20 weeks in the literature.

A polyclonal anti-cytokeratin antibody has been used to examine the expression of this intermediate filament both during normal development in the rat and in a variety of pathological states in the rat and mouse. Bile duct proliferation induced by the administration of alpha-naphthylisothiocyanate (ANIT) as well as the oval cell proliferation induced by 3'-methyl-4-dimethylaminoazobenzene (3-MeDAB) have been used to examine the expression of the rodent cytokeratins in the proliferating cells regarded as being of bile duct origin. Examples of cholangiofibrosis and cholangiocarcinomas were also examined for evidence of cytokeratin expression using this antibody, as well as proliferations of a morphological intermediate type between epithelial and mesenchymal. In all cases we have been able to demonstrate continuity of phenotypic expression of the cytokeratins recognized by this antibody in cells which are recognized as bile duct in origin, even where their morphological appearance does not resemble an epithelial cell type. Because this antibody can be used on formalin-fixed, paraffin-processed tissues, after trypsin treatment, it is proposed that it can be used routinely in the toxicological evaluation (even retrospectively) of bile duct related proliferations and tumours.

The case of a 61-year-old woman with a surgically resected solitary cholangiocarcinoma of the liver is reported, where many discrete multiple bile duct hamartoma (MBDH) were also seen. The latter is a congenital lesion of the liver that potentially may be confused with widespread metastatic disease. The relationship between cholangiocarcinoma and MBDH was studied histologically by the use of an immunoperoxidase technique for cytokeratin. MBDH was strongly positive for cytokeratin, while the neoplasm showed this to a lesser extent, but a clear continuity between the MBDH epithelial cells and those of the neoplasm was demonstrated by the use of this technic. The potential use for the various cytokeratins in the differentiation of primary from secondary liver tumors, is discussed. This differentiation is a significant problem to the pathologist. Although cholangiocarcinoma may, on occasion, be associated with various congenital lesions of the bile ducts, the association with MBDH is extremely rare, this being only the third reported case.

Tissue copper contents in 38 primary and 45 metastatic hepatic malignancies and 15 control livers were analyzed by atomic absorption spectrophotometry. The average copper content of 15 control livers was 23.1 +/- 13.0 micrograms/g dry weight (microgram/gdw). The copper content of five cholangiocellular carcinomas (CCCs) and 45 metastatic cancers was almost equal to the control level. Thirty three hepatocellular carcinomas (HCCs) contained a larger amount of copper (61.5 +/- 76.8 micrograms/gdw) than the control livers (p less than 0.05), but the copper content of HCCs showed a considerably wide variation. The average copper content of nine minute HCCs (126 +/- 112 micrograms/gdw) was significantly (p less than 0.05) higher than that of 24 large HCCs (37.2 +/- 39.9 micrograms/gdw). Histologically, orcein and paramethylaminobenzylidene rhodamine positive granules were seen in eight and four of nine minute HCCs, respectively. These granules were also found in some large HCCs, but were never found in CCCs and metastatic cancers. It was concluded that these excessive accumulations of copper and copper-binding proteins might present a helpful finding to distinguish some cases of HCC, especially small HCC, from CCCs, metastatic cancers and hypertrophic regenerative nodules of cirrhotic livers. The significance and possible pathogenesis of these copper accumulations in HCCs require further studies.

To reveal the cell-biological character of biliary tract cancer, localization and distribution of three cancer-associated carbohydrate antigens (CA19-9, sialyl SSEA-1, NCC-ST-439) and carcinoembryonic antigen (CEA) were studied immunohistochemically in 35 cases of gallbladder carcinoma, 21 of bile duct carcinoma, 16 of chronic cholecystitis, and 3 of normal gallbladder. 1) All carbohydrate antigens and CEA were present in 70-90% of the cases of gallbladder and bile duct carcinoma. In particular, NCC-ST-439 had the highest incidence of positive staining (95.2%) in bile duct carcinoma. 2) The mode of localization was diverse and was not fixed by the kind of antigen. Antigens flowing out to the surrounding stroma were affected by the rate of positive cells. 3) No significant correlation was observed between the histological type or degree of differentiation and tissue positivity. 4) The positivity of tissue CEA was higher in the cases with serous membrane invasion, gamma INF pattern, and neuro-, vascular-, and lymphatic invasion. 5) In chronic cholecystitis, CA19-9, NCC-ST-439, and CEA were stained in mucosal cells and/or metaplastic cells, while sialyl SSEA-1 was stained only in one case in the goblet cells and the cells with pseudopyloric metaplasia. None of the antigens were stained in normal gallbladders. These results suggest that these antigens may be useful in the diagnosis and therapeutic treatments in patients with biliary tract cancer.

Aflatoxin is localized by fluorescence microscopy in primary liver cancer in man from Zaïre.

Production of human chorionic gonadotropin (hCG) by extragonadal tumours is not a rare phenomenon. In the liver, similar results have been reported in hepatoblastomas. The present study was attempted to survey hCG level in serum and hCG-immunoreactivity in primary liver carcinoma in adults. Although hCG was elevated in serum in 2 (22.2%) of 9 autopsied cases with hepatocellular carcinoma (HCC), the hCG-reactivity of carcinoma cells was found in 2 (2.1%) of 95 HCC cases. Carcinoma cells positive for immunoreactive hCG was found in 2 (15.4%) of 13 cases with cholangiocarcinoma (CC). The patients with hCG-immunoreactivity in carcinoma and/or elevated serum level of hCG failed to reveal distinct clinical and endocrinological disturbance due to excess hCG. The hCG-positive cells were focal within the carcinoma and showed poor histological differentiation in both HCC and CC, and there were no trophoblastic cells. It is suggested that hCG is one of the hormones produced by primary liver carcinoma in adults and can be localised immunohistochemically in a small number of poorly differentiated carcinoma cells.

A combined hepatocellular-cholangiocarcinoma (CHC) of transitional subtype and the surrounding cirrhotic liver tissue were investigated immunocytochemically by monoclonal antibodies specific for each of the keratin polypeptides 7, 8, 18 and 19. Different keratin subsets were found in different parts of the tumour. The hepatocellular component reveals keratins 8 and 18, with the bordering cells of trabecular formations additionally expressing keratins 7 and 19. The same keratins i.e. 7, 8, 18, 19 were found in normal bile duct epithelium as well as in cholangiocarcinomatous and transitional areas of hepatocellular and cholangiocellular differentiation. Normal hepatocytes express only keratin 8 and 18. In cirrhotic liver some modified hepatocytes additionally express keratin 7. When ductal transformation is observed in the marginal parts of portal tracts and fibrous septa the keratin polypeptide pattern mimics that of bile duct epithelium. The cholangiocellular metaplasia of hepatocytes observed here correlates well with findings in hepato-organogenesis and hepatocarcinogenesis and suggests that the transitional subtype of combined hepatocellular-cholangiocarcinoma is a variant of hepatocellular carcinoma.