Hepatocellular pseudotumor in the cirrhotic liver. Report of three cases

Three cases of regenerating hyperplastic nodule in the cirrhotic liver are reported in detail. The patients were all men, ranging in age from 41 to 57 years. They were all heavy drinkers and had chronic liver disease for 6 to 10 years. All patients had slightly elevated alpha-fetoprotein levels and were positive for HBs and HBc antibodies. Radiologic findings were not uniform except for ultrasonography, which demonstrated the lesion in all cases. Preoperative diagnosis was hepatocellular carcinoma (HCC) or highly suspicious of HCC in all of them. At laparotomy, all lesions were embedded in the liver parenchyma and invisible from the hepatic surface. With the aid of intraoperative sonography, partial hepatic resections were performed. Pseudocapsule was seen in two cases. Definitive differential diagnosis of regenerating nodules from HCC in the cirrhotic liver was extremely difficult. The authors propose the name "hepatocellular pseudotumor in the cirrhotic liver" to describe such lesions.     

Galectin-3 expression is induced in cirrhotic liver and hepatocellular carcinoma

Galectins are a family of beta-galactoside-binding animal lectins. In particular, a widely studied member galectin-3, previously designated as epsilonBP, CBP35, Mac-2, L-29 and L-34, has been associated with assorted processes such as cell growth, tumor transformation and metastasis. Galectin-3 is expressed in various tissues and organs but is significantly absent in normal hepatocytes. However, evaluation of patient liver biopsies for galectin-3 expression resulted in the finding that hepatocellular carcinoma (HCC) frequently expressed significant levels of this lectin (76% immunohistochemically positive). Further investigation revealed that galectin-3 expression in HCC is independent of whether the patient had prior hepatitis B virus infection: 14 of 18 HCC cases from HBV- patients, and 5 of 7 cases from HBV patients demonstrated positive galectin-3 immunohistochemistry. However, co-transfection studies using a galectin-3 promoter construct and an HBV-X protein (HBV-X) expression vector demonstrated that galectin-3 expression can occur through transactivation of the lectin promoter by HBV-X. Based on presently known properties of this lectin, it is possible that deregulated expression of galectin-3 can result in tumor transformation and invasiveness, or confer propensity for tumor cell survival. In addition, galectin-3 was abundantly expressed in cirrhotic liver in peripheral distribution within regenerating nodules. Such galectin-3 expression in rapidly proliferating hepatocytes in cirrhotic liver may be a result of the high mitotic index. Alternatively, it is possible that proliferating cells expressing galectin-3 are in the process of being transformed, thus indicating an early neoplastic event.     

肝细胞癌染色体不稳定性的研究进展

肝细胞癌 (hepatocellularcarcinoma ,HCC)的发展是一个多步过程 ,涉及多染色体的改变 ,且与肿瘤的形态和进展相关。在癌前病变 ,由于DNA甲基化的改变、HBV和HCV的侵入、肿瘤抑制基因的点突变或杂合子丢失 (lossofheterozygosity ,LOH )引起基因表达的改变。肿瘤进展期的特征包括许多染色体上肿瘤抑制基因的LOH。不同的HCC结节所观察到的改变常常是不同的 ,表明其在分子水平上的异质性。这些发现表明HCC的发生过程中遗传学的改变也是一个进行性积累的过程。对HCC分子机制的理解可能获得肿瘤分期的新的标记物 ,评价肿瘤形成的相对危险性 ,为肿瘤的治疗提供新的机会     

Expression of intercellular adhesion molecule-1 in hepatocellular carcinoma

The expression of intercellular adhesion molecule-1 (ICAM-1) was investigated in frozen sections obtained from 40 resected liver specimens of patients with hepatocellular carcinoma using immunoperoxidase techniques and immunoelectron microscopy. ICAM-1 was expressed in 80% of the HCC specimens on the membrane of cancer cells. In noncancerous regions characterized by cirrhosis in 28 cases and chronic hepatitis in 12 cases, ICAM-1 was rarely expressed on hepatocytes but was expressed mainly on the endothelium of portal vessels and sinusoidal lining cells. These results suggest that expression of ICAM-1 in hepatocellular carcinoma may be induced by malignant transformation of hepatocytes. © 1993 Wiley-Liss, Inc.     

Hepatitis B virus antigens in human primary hepatocellular carcinoma tissues.

The presence of hepatitis B virus (HBV) antigens was examined in specimens of liver tissue obtained at necropsy from black Senegalese patients suffering from primary hepatocellular carcinoma (PHC). The results were correlated with markers of hepatitis B infection in serum. Hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) were sought for in 15 liver extracts. HBsAg was found in the liver in 10 of 12 cases with HBsAg-positive serum. HBcAg was detected in three livers. The HBsAg was detected in seven of eight livers by immunofluorescence and orcein staining. HBsAg-positive cells were mainly located in the peri-tumoral cirrhotic tissue, although positive hepatocytes were also found in tumour nodules in liver from one of the patients. HBcAg was found in five of seven cases by immunofluorescence in hepatocytes of the cirrhotic areas. HBcAg fluorescence was primarily nuclear but, in some lobules, a patchy cytoplasmic fluorescence was observed. This suggests a cytoplasm-nucleus pathway in the synthesis of the HBV core antigen. Electron microscopy was performed on two HBsAg- and HBcAg-positive cases. Fibrillar and crystalline cytoplasmic inclusions were observed in tumour cells. In the same cells, 20-25 nm virus-like particles were present in swollen cisternae of the endoplasmic reticulum.     

Changes in poly (A) + RNA translational pattern during chemically induced hepatocarcinogenesis

Hepatocarcinoma was induced by administration of diethylnitrosamine to rats. The rats were sacrificed 70 weeks after the administration and the carcinoma nodules were separated from the perinodular parenchymental cells after perfusion of liver with collagenase. The in vitro translational pattern of mRNAs from hepatocellular carcinomas, from perinodular hepatocytes and from regenerating liver after partial hepatectomy were compared by one- and two-dimensional electrophoreses to the pattern obtained with RNA from normal hepatocytes. An increased synthesis of several peptides was observed with RNAs from carcinoma and from regenerating liver and to a lesser extent with RNA from perinodular hepatocytes, which suggests that the increase in synthesis is at least partly related to cell proliferation. A decreased synthesis of several other peptides was observed with RNA from carcinoma nodules and to a lesser extent with RNA from perinodular hepatocytes, but not with RNA from regenerating liver, which suggests that this decrease in synthesis is related to some transformation specific process. These changes are observed as soon as 22 weeks after carcinogen administration. These observations also suggest that at least part of the perinodular hepatocytes have some characteristics of the transformed cells.     

Raf kinase inhibitor protein is downregulated in hepatocellular carcinoma

The Ras/Raf/MEK/ERK signalling cascade is frequently deregulated in tumourigenic diseases and known to be involved in proliferation and transformation of cells. Also in hepatocellular carcinoma (HCC) increased ERK levels are observed and known to correlate with tumour progression, but the underlying molecular mechanism are unknown. We analyzed expression of Raf-1 kinase inhibitory protein (RKIP) in HCC. Expression of RKIP mRNA and protein was downregulated in HCC cell lines and tissue as compared to primary human hepatocytes (PHH) or non-tumorous liver tissue, respectively. Transfection of an HCC cell line with an RKIP expression construct blocked the Raf kinase pathway resulting in decreased activity of ERK1/2 and AP-1. In contrast, downregulation of RKIP by transfection with an antisense RKIP construct led to increased ERK1/2 and AP-1 activity. Since HCC develop in the majority of cases in cirrhotic liver tissue and cirrhosis is the main risk factor for HCC development, we analyzed RKIP expression also in non-cancerous cirrhotic liver tissues by immunohistochemistry. In contrast to normal liver tissue, where the staining was equally distributed within the cytoplasm, hepatocytes in cirrhotic liver revealed an intense RKIP staining of the membrane. It can be speculated that this changed RKIP expression pattern parallels impaired protein function in PHH in cirrhotic livers that may predispose PHH to malignant transformation. In addition, our study demonstrates functional relevance of downregulation of RKIP in HCC that may play an important role in HCC development and progression.     

Clonal state of human hepatocellular carcinoma and non-tumorous hepatocytes

We determined the clonal state in specimens of hepatocellular carcinoma (HCC) and non-tumorous hepatocytes from the integration mode of hepatitis B virus (HBV) DNA. The integration mode of HBV DNA in several parts of the tumors and non-tumorous regions of the same liver, as well as in metastatic tumors, was examined using the Southern blot analysis. In 13 of the 14 cases of HCC, the liver tumors, including metastatic tumors in lymph nodes and the lungs, were monoclonal. In one case, a different HCC clone was found in one part of the liver tumor. The integration of HBV DNA was also observed in non-tumorous tissues in 38 of the 78 cases (49%) of chronic hepatitis with and without HCC; in 16 cases of chronic hepatitis in which HBV DNA was integrated, several clones of the hepatocytes that had HBV DNA integrated into their chromosomal DNA and had proliferated clonally were found in non-tumorous tissues. These clones were different from the tumor clone of the same liver. Thus HCCs were usually monoclonal. The development of different tumor clones appeared to be unusual, but the nontumorous hepatocytes could have proliferated clonally from different multicentric clones before carcinogenesis. The clonal growth of the non-tumorous hepatocytes suggests that the integration of HBV DNA plays an important role in hepatocarcinogenesis.     

肝细胞癌患者外周血甲胎蛋白mRNA的检测及意义

目的：探讨肝细胞癌患者外周血中微小转移及其临床意义。方法：用巢式逆转录聚合酶链反应（Ｎｅｓｔｅｄ－ＲＴ－ＰＣＲ）技术检测６５例肝细胞癌、２１例非肝癌的恶性肿瘤、２２例慢性乙型肝炎或肝硬化患者及２１例健康志愿者外周静脉血中的甲胎蛋白（ＡＦＰ）ｍＲＮＡ。结果： ＡＦＰ－ｍＲＮＡ在健康志愿者、非肝癌恶性肿瘤患者外周血中均为阴性;在肝细胞癌组外周血中ＡＦＰ－ｍＲＮＡ的检出率（６７．７％,４４／６５）明显高于慢性乙型肝炎或肝硬化组（９．１％,２／２２,Ｐ＜０．０１）。ＡＦＰ－ｍＲＮＡ检出率与临床分期、门静脉癌栓、肝外转移显著相关。经１年的随访发现治疗后外周血ＡＦＰ－ｍＲＮＡ阳性与肝癌患者预后有显著相关。在１２例血清ＡＦＰ＜２５μｇ／Ｌ的肝癌病人血中,７例（５８． ３％）可检出ＡＦＰ－ｍＲＮＡ。结论： ＡＦＰ－ｍＲＮＡ可作为血循环中有肝癌细胞或肝细胞的标志物,在肝癌患者,阳性预示有血源性转移的可能,可作为预测肝癌复发、转移的参考指标;并且ＡＦＰ－ｍＲＮＡ对血清ＡＦＰ阴性或低值的肝癌患者能起到补充诊断作用。     

Dominance of functional androgen receptor allele with longer CAG repeat in hepatitis B virus-related female hepatocarcinogenesis

The CAG polymorphism in exon 1 of the androgen receptor (AR) gene has been shown associated with the development of human male hepatocellular carcinoma (HCC) with the shorter AR alleles conferring a higher risk. However, the significance of AR-CAG repeats in female hepatocarcinogenesis remains to be addressed. In this study, seventy-six pairs of female HCCs and corresponding nontumorous tissues were collected, and 180 cirrhotic nodules were microdissected from 7 cirrhotic livers. The clonality status, functional AR alleles, and CAG repeat number of each sample were determined by AR methylation analysis. In a total of 44 monoclonal HCCs, the mean of CAG repeats in the active alleles was significantly longer than that in the inactive alleles (22.0 +/- 2.8 versus 20.7 +/- 3.6; P = 0.047). When we divided HCCs into hepatitis B virus-positive [HBV(+)] and HBV(-) subgroups, the long AR allele dominance was found only in HBV(+) ones (P = 0.006 versus P = 0.923). Notably, the preference of long CAG repeat has also been found in the 100 monoclonal nodules (P = 0.013). For comparison of monoclonal nodules obtained from the same individual, a dominant long AR allele was found in 6 patients. The proportion of monoclonal cirrhotic nodules and HCCs expressing longer AR allele, 69 and 68%, are both significantly higher than 50%, the assumed value in normal liver (P < 0.001 for cirrhotic nodules and P = 0.005 for HCC). The dominance is again only prominent in HBV-infected HCCs [85% for HBV(+) HCC; P < 0.001 but 54% for HBV(-) HCC; P = 0.27]. The results indicated that in female hepatocarcinogenesis, hepatocytes expressing the longer AR allele seem to be favorably selected for autonomous growth and transformation, especially in synergy with HBV infection.     

肝细胞癌多基因甲基化异常及其临床意义

目的 探讨肝细胞癌中多基因甲基化异常的发生率,研究肝细胞癌中多基因甲基化异常的临床意义.方法 收集60例肝细胞癌及相应的癌旁组织、16例肝炎后肝硬化组织、5例慢性肝炎和5例正常肝组织,筛选消化道肿瘤中APC、RASSF1A、p16、GSTP1、MGMT、DAPK、SOCS-1和RIZ18个甲基化异常频率高的肿瘤抑制基因,应用甲基化特异件聚合酶链反应(MSP)检测8个肿瘤抑制基因在所有标本中的甲基化状态.比较不同基因甲基化与非甲基化肝细胞癌患者的临床病理特征和生存情况.结果 肝细胞癌组织中,RASSF1A、APC、GSTP1、p16、RIZ1和MGMT基因的甲基化率分别为95.0%、90.0%、73.3%、65.0%、61.6%和60.0%,均高于相应的癌旁组织(均P＜0.05);癌旁组织中,MGMT、GSTP1和RIZ1基因的甲基化率分别为41.6%、40.0%和25.0%,均高于肝硬化组织(均P＜0.05).p16基因甲基化的肝细胞癌患者的平均年龄大于非甲基化者;巨块性肝细胞癌中,MGMT基因甲基化者的比例高于非甲基化者;MGMT基基甲基化者的无瘤生存期短于非甲基化者.结论 不同基因在肝细胞癌、癌旁和肝硬化组织中的甲皋化率差异显示了肝细胞癌发生中渐进的表观遗传学改变;GSTP1、RIZ1和MGMT基因的甲基化异常具有肿瘤风险评估和早期诊断价值,而MGMT基因的甲基化异常同时具有预后评估价值.     

Prohibitin Expression Is Decreased in the Regenerating Liver but Not in Chemically Induced Hepatic Tumors in Rats

Expression of prohibitin, a growth-regulatory protein, was immunohistochemically investigated in normal rat tissues, regenerating livers, and chemically induced preneoplastic and neoplastic hepatic lesions. Specific cell types including hepatocytes, striated and smooth muscle cells, cardiac cells, squamous epithelial cells, sebaceous gland cells, hair root outer sheath cells, salivary gland duct epithelial cells, chondrocytes, immature spermatocytes and oocytes were found to be positive. In regenerating livers, prohibitin protein disappeared as early as 3 h after two-thirds hepatectomy and returned to near the original level by 24 h, while its mRNA level did not markedly vary. The timing of the disappearance was coincident with the expression of c-myc, suggesting a relation to quiescent hepatocytes entering the cell cycle. However, no pronounced decrease was evident in the most hyperplastic hepatic nodules and hepatocellnlar carcinomas investigated. Examination of 9 rat hepatocellular carcinoma cell lines, 6 hyperplastic hepatic nodules and 5 hepatocellular carcinomas revealed a single case of a base substitution in prohibitin cDNA, identified as a synonymous sense change. The observed abundant expression of prohibitin in quiescent hepatocytes and its rapid loss under conditions of regeneration indicate a growth-regulatory function, but our results do not suggest any critical role in rat hepatocarcinogenesis.     

肝癌发生过程中CD31与VEGF的表达

目的:探讨血管内皮生长因子(VEGF),CD31在人肝癌发生过程中结节性病变组织中的表达。方法:对肝癌28例,高度发育不良结节9例,低度发育不良结节13例,肝硬化结节22例进行了VEGF,CD31免疫组织化学法检测,并进行半定量分析。结果:CD31在肝癌组织中的表达高于发育不良结节和肝硬化结节(P<0.05);肝癌组织VEGF表达水平最高(P<0.05);高度发育不良结节组织中VEGF表达水平高于低度发育不良结节和肝硬化结节组织(P<0.05);肝癌发生过程中VEGF在和微血管密度(MVD)水平呈正相关。结论:VEGF和CD31的阳性表达为肝癌与肝癌发生过程中的结节性病变的鉴别诊断提供了较为可靠的形态学标记。     

Profiles of carbohydrate-metabolizing enzymes in human hepatocellular carcinomas and preneoplastic livers

Activities of key carbohydrate-metabolizing enzymes in biopsied human tissues of hepatocellular carcinoma and related conditions were determined by established methods. Among the enzymes analyzed, fetal-type liver enzymes (low-Km hexokinase, glucose 6-phosphate dehydrogenase, and pyruvate kinase-M2) showed increased activities, and adult-type liver enzymes [glucose 6-phosphatase, fructose 1,6-bisphosphatase, high-Km hexokinase (or glucokinase), and pyruvate kinase-L] showed decreased activities, resulting in undifferentiated enzyme patterns not only in fetal livers and hepatocellular carcinomas but also in livers of acute and chronic hepatitis and liver cirrhosis with or without tumors. Hepatocellular carcinomas showed a general tendency of having greater enzyme deviations than hepatitic and cirrhotic livers. The extent of the enzyme deviation in hepatocellular carcinomas varied considerably from one enzyme to another for each tumor tissue as compared with that in the benign liver diseases. Thus, the phenotypic heterogeneity was important for discriminating between the neoplastic and inflammatory changes in differentiation markers. The enzyme patterns of tumors and their corresponding host cirrhotic livers were unrelated, suggesting that the cirrhotic liver has a significance as preneoplastic state only in terms of having a high incidence of evolving hepatocellular carcinoma.     

Beta-catenin mutations and expression, 249serine p53 tumor suppressor gene mutation, and hepatitis B virus infection in southern African Blacks with hepatocellular carcinoma

BACKGROUND AND OBJECTIVES: To ascertain the prevalence of deregulating mutations of beta-catenin gene, and to correlate this with the occurrence of 249(serine) p53 gene mutation and hepatitis B virus infection in southern African Blacks with hepatocellular carcinoma. METHODS: Paired cancer/non-cancerous liver tissues from 21 and cancer tissues alone from 20 Black Africans with hepatocellular carcinoma were studied. RT-PCR-SSCP and sequencing were used to detect mutations in exon 3 of the beta-catenin gene, and PCR, restriction endonuclease analysis, and sequencing to detect the p53 gene mutation. Immunostaining was used to identify beta-catenin protein expression in hepatocytes. RESULTS: No mutations in exon 3 of the beta-catenin gene were found in tumor or non-tumorous tissues. Immunohistochemical staining showed beta-catenin protein expression in membranes and cytoplasm of hepatocytes but not in the nuclei. The 249serine p53 gene mutation was detected in 27.2% of the hepatocellular carcinoma tissues but not in non-cancerous tissues. No correlation was found between beta-catenin mutation and over-expression and 249serine p53 gene mutations or hepatitis B virus surface antigenemia. CONCLUSIONS: Unlike hepatocellular carcinomas in China, Japan, and Europe, deregulating beta-catenin gene mutations do not appear to occur in southern African Blacks with this tumor and do not therefore interact with either the 249serine p53 gene mutation or hepatitis B virus infection in its pathogenesis.     

p21和IGF-II在肝癌、肝硬化组织中表达的研究

目的　探讨 p2 1、IGF II在肝硬化、肝细胞癌 (HCC)中表达的意义及其与肝细胞不典型增生(LCD)的关系。方法　用免疫组化S P法检测单纯肝硬化、癌旁肝硬化及肝细胞癌 (HCC) p2 1、IGF II表达情况。结果　p2 1、IGF II在癌旁肝硬化和单纯肝硬化组织中的阳性表达率 (92 .86 %和 75 % ;6 8%和 74 % )与HCC(5 4 .5 5 %和 36 .36 % )相比差别有显著性意义 (P <0 .0 5 )。IGF II与p2 1阳性表达存在相关关系。HBsAg阳性或伴LCD改变的肝硬化 ,p2 1、IGF II阳性率均高于HBsAg阴性或不伴有LCD改变的肝硬化 (P <0 .0 5 )。结论　 (1)在肝细胞癌变演化过程中 p2 1、IGF II可能发挥协同作用。 (2 )LCD可能是一群具有肿瘤增殖潜能的癌前细胞群 ,尤其有 p2 1、IGF II共同过度表达者 ,有发生HCC的高度危险。     

Establishment of a human cell line (HCC-T) from a patient with hepatoma bearing no evidence of hepatitis B or A virus infection

A human hepatoma cell line, designated HCC-T, was established. The tumor was surgically obtained from a Japanese male patient with hepatocellular carcinoma (HCC) arising in a cirrhotic liver that had supposedly developed from nonAnonB (NANB) chronic hepatitis. HCC-T exhibited a typical morphology of epithelial cells in culture. Population doubling time was 24 hours and HCC-T cells had characteristics of malignant cells demonstrated by the ability to grow in a soft agar medium and transplantability to nude mice. The histologic condition of the tumor transplanted to a nude mouse showed similarity to the original tumor. A chromosome analysis showed that there were ten identifiable marker chromosomes and sex chromosomes with its modal number of 64. Alpha-fetoprotein (AFP) production was demonstrated by direct immunofluorescence study, but albumin or hepatitis B surface antigen were not detectable. The integration of hepatitis B viral DNA was not demonstrable in the genome of HCC-T cells or the original hepatoma.     

Identification and characterization of a rat hepatic oncofetal membrane glycoprotein.

A major interest of our laboratory is to delineate the pathways leading to experimentally induced liver cancer in the rat. Although the cellular progenitors of primary hepatocellular carcinoma remain controversial, current findings suggest that proliferation of chemically initiated liver epithelial cells gives rise to hepatic nodules, a rare population of which eventually progress to carcinoma. Presently, the availability of cell surface markers that are closely associated with malignant progression is needed for the identification, isolation, and further characterization of these rare malignant cells. In this paper, we describe two new monoclonal antibodies (MAbs), MAb 324.5 and MAb 324.9, that recognize a novel oncofetal membrane glycoprotein, designated TuAg1. MAbs 324.5 and 324.9 were produced using three different transplantable hepatocellular carcinoma cell lines during immunization and screening. MAb 324.5 and MAb 324.9 were shown to be reactive with different epitopes on TuAg1 by competitive immunoprecipitation assays combined with results from immunodepletion analysis and one-dimensional V-8 peptide maps. TuAg1 showed variations in molecular weight from 78,000 to 92,000, and a marked heterogeneity in pI, with charge variants ranging between 4.3 and 6.0. The 324.5-epitope was not expressed at detectable levels in any adult normal tissues or during liver regeneration but was transiently expressed during fetal liver development as shown by indirect immunofluorescence analysis of frozen tissue sections. In contrast, the 324.9-epitope was observed on nerve fibers and ganglia and on sperm tails in the adult rat and also appeared independently of the 324.5-epitope during fetal development. Although normal hepatocytes did not express TuAg1, isolated hepatocytes became positive during the first 24 h of primary culture. Attempts to modulate the in vitro expression of TuAg1 were unsuccessful; however, TuAg1 was lost within 7 days following ectopic transplantation of cultured hepatocytes into the pancreas. During the carcinogenic process, TuAg1 was expressed by a rare population of hepatic nodules, by many primary liver tumors, and by all lung metastases and transplantable hepatocellular carcinomas examined to date. Taken together, these observations suggest that the in vivo constitutive expression of this novel oncofetal membrane antigen is closely associated with acquisition of the malignant phenotype during hepatocarcinogenesis.