Biliary Epithelial Differentiation Research Articles

Biliary epithelial cell differentiation of bipotent human liver-derived organoids by 2D and 3D culture

Organoid culture is a technology for creating three-dimensional (3D) tissue-like structures in vitro, and is expected to be used in various fields. It was reported that human adult bile duct cells derived from human biopsy can be expanded as organoids in vitro that exhibit stem cell-like properties including high proliferative ability and differentiation ability toward both hepatocytes and biliary epithelial cells (BECs). Although many studies have achieved the efficient differentiation of bipotent human liver-derived organoids (hLOs) toward mature hepatocytes, the differentiation potency toward mature BECs remains unclear. In this study, we attempted to evaluate the differentiation potency of bipotent hLOs, which were generated from primary (cryopreserved) human hepatocytes (PHHs), toward BECs by sequential treatment with epidermal growth factor (EGF), Interleukin-6 (IL-6), and sodium taurocholate hydrate. Along with the differentiation toward bipotent hLOs-derived BECs (Org-BECs), increases in the gene expression levels of BEC markers and formation of the lumen-like structures typical of BECs were observed. In addition, Org-BECs exhibited P-glycoprotein-mediated drug transport capacity. Finally, in order to expand the applicability of Org-BECs, we succeeded in the differentiation of bipotent hLOs toward BECs in a two-dimensional (2D) culture system. Our findings demonstrated that bipotent hLOs can indeed differentiate into mature BECs, meaning that they possess a capacity for differentiation toward both hepatocytes and BECs.

Biliary Epithelial Cell Differentiation of Bipotent Human Liver-Derived Organoids by 2D and 3D Culture

Biliary Epithelial Cell Differentiation of Bipotent Human Liver-Derived Organoids by 2D and 3D Culture

Estrogen Acts Through Estrogen Receptor 2b to Regulate Hepatobiliary Fate During Vertebrate Development.

During liver development, bipotent progenitor cells differentiate into hepatocytes and biliary epithelial cells to ensure a functional liver required to maintain organismal homeostasis. The developmental cues controlling the differentiation of committed progenitors into these cell types, however, are incompletely understood. Here, we discover an essential role for estrogenic regulation in vertebrate liver development to affect hepatobiliary fate decisions. Exposure of zebrafish embryos to 17β-estradiol (E2) during liver development significantly decreased hepatocyte-specific gene expression, liver size, and hepatocyte number. In contrast, pharmacological blockade of estrogen synthesis or nuclear estrogen receptor (ESR) signaling enhanced liver size and hepatocyte marker expression. Transgenic reporter fish demonstrated nuclear ESR activity in the developing liver. Chemical inhibition and morpholino knockdown of nuclear estrogen receptor 2b (esr2b) increased hepatocyte gene expression and blocked the effects of E2 exposure. esr2b-/- mutant zebrafish exhibited significantly increased expression of hepatocyte markers with no impact on liver progenitors, other endodermal lineages, or vasculature. Significantly, E2-stimulated Esr2b activity promoted biliary epithelial differentiation at the expense of hepatocyte fate, whereas loss of esr2b impaired biliary lineage commitment. Chemical and genetic epistasis studies identified bone morphogenetic protein (BMP) signaling as a mediator of the estrogen effects. The divergent impact of estrogen on hepatobiliary fate was confirmed in a human hepatoblast cell line, indicating the relevance of this pathway for human liver development. Our studies identify E2, esr2b, and downstream BMP activity as important regulators of hepatobiliary fate decisions during vertebrate liver development. These results have significant clinical implications for liver development in infants exposed to abnormal estrogen levels or estrogenic compounds during pregnancy.

Tubular bile duct structure mimicking bile duct morphogenesis for prospective in vitro liver metabolite recovery

BackgroundLiver metabolites are used to diagnose disease and examine drugs in clinical pharmacokinetics. Therefore, development of an in vitro assay system that reproduces liver metabolite recovery would provide important benefits to pharmaceutical research. However, liver models have proven challenging to develop because of the lack of an appropriate bile duct structure for the accumulation and transport of metabolites from the liver parenchyma. Currently available bile duct models, such as the bile duct cyst-embedded extracellular matrix (ECM), lack any morphological resemblance to the tubular morphology of the living bile duct. Moreover, these systems cannot overcome metabolite recovery issues because they are established in isolated culture systems. Here, we successfully established a non-continuous tubular bile duct structure model in an open-culture system, which closely resembled an in vivo structure. This system was utilized to effectively collect liver metabolites separately from liver parenchymal cells.ResultsTriple-cell co-culture of primary rat hepatoblasts, rat biliary epithelial cells, and mouse embryonic fibroblasts was grown to mimic the morphogenesis of the bile duct during liver development. Overlaying the cells with ECM containing a Matrigel and collagen type I gel mixture promoted the development of a tubular bile duct structure. In this culture system, the expression of specific markers and signaling molecules related to biliary epithelial cell differentiation was highly upregulated during the ductal formation process. This bile duct structure also enabled the separate accumulation of metabolite analogs from liver parenchymal cells.ConclusionsA morphogenesis-based culture system effectively establishes an advanced bile duct structure and improves the plasticity of liver models feasible for autologous in vitro metabolite-bile collection, which may enhance the performance of high-throughput liver models in cell-based assays.

Longitudinal in vivo bioimaging of hepatocyte transcription factor activity following cholestatic liver injury in mice

Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-κB and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-κB and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes.

Hepatic differentiation of human pluripotent stem cells on human liver progenitor HepaRG-derived acellular matrix

Human hepatocytes are extensively needed in drug discovery and development. Stem cell-derived hepatocytes are expected to be an improved and continuous model of human liver to study drug candidates. Generation of endoderm-derived hepatocytes from human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, is a complex, challenging process requiring specific signals from soluble factors and insoluble matrices at each developmental stage. In this study, we used human liver progenitor HepaRG-derived acellular matrix (ACM) as a hepatic progenitor-specific matrix to induce hepatic commitment of hPSC-derived definitive endoderm (DE) cells. The DE cells showed much better attachment to the HepaRG ACM than other matrices tested and then differentiated towards hepatic cells, which expressed hepatocyte-specific makers. We demonstrate that Matrigel overlay induced hepatocyte phenotype and inhibited biliary epithelial differentiation in two hPSC lines studied. In conclusion, our study demonstrates that the HepaRG ACM, a hepatic progenitor-specific matrix, plays an important role in the hepatic differentiation of hPSCs.

Abstract 3890: Whole-genome mutational landscape of liver cancers displaying biliary phenotype reveals hepatitis impact and molecular diversity

Abstract Primary liver cancer is the third leading cause of cancer death worldwide. Virus infection is the most common and strongest etiological factor for liver cancer development. Pathologically, primary liver cancer can be classified into ∼90% hepatocellular carcinoma (HCC), and 5∼10% intrahepatic cholangiocarcinoma (ICC), and the combined hepatocellular-cholangiocarcinoma (cHCC/CC) representing only a small portion. Clinically, ICC and cHCC/CC show much more aggressive behavior with poorer prognosis than HCC, and no standard treatment currently exists, other than surgical resection. ICC and cHCC/CC show varying degrees of biliary epithelial differentiation, which can be defined as liver cancer displaying a biliary phenotype (LCB). To gain insight into molecular alterations of LCBs, we performed whole genome sequencing analysis on 30 LCBs. Here we show the genome-wide substitution patterns of LCBs developed in chronic hepatitis livers overlapped with those of 60 HCCs, while those of hepatitis-negative LCBs diverged. The subsequent validation study on 68 LCBs identified recurrent mutations in TERT promoter, chromatin regulators (BAP1, PBRM1 and ARID2), a synapse organization gene (PCLO), IDH genes and KRAS. The frequencies of KRAS and IDHs mutations, which are associated with poor disease-free survival, were significantly higher in hepatitis-negative LCBs. This study reveals the strong impact of chronic hepatitis on the mutational landscape in liver cancer and the genetic diversity among LCBs. Citation Format: Hidewaki Nakagawa, Akihiro Fujimoto, Mayuko Furuta, Kunihito Gotoh, Toru Nakamura, Masakazu Yamamoto, Hiroki Yamaue, Kazuaki Chayama, Satoru Miyano, Tatsuhiko Tsunoda. Whole-genome mutational landscape of liver cancers displaying biliary phenotype reveals hepatitis impact and molecular diversity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2015-3890

Intrahepatic Cholangiocarcinoma with Ductal Plate Malformation-like Feature Associated with Bile Duct Adenoma.

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor with biliary epithelial differentiation. Malignant transformation of von Meyenburg complex (VMC) to ICC has been reported [1,2], and a new subtype of ICC with ductal plate malformation (DPM) pattern has been suggested [3]. However, bile duct adenoma (BDA) is a rare entity and is not as well known as DPM. Moreover, it has not been determined whether BDA is a risk factor of ICC. We present a rare case of ICC with DPM-like features associated with BDA.

Whole-genome mutational landscape of liver cancers displaying biliary phenotype reveals hepatitis impact and molecular diversity.

Intrahepatic cholangiocarcinoma and combined hepatocellular cholangiocarcinoma show varying degrees of biliary epithelial differentiation, which can be defined as liver cancer displaying biliary phenotype (LCB). LCB is second in the incidence for liver cancers with and without chronic hepatitis background and more aggressive than hepatocellular carcinoma (HCC). To gain insight into its molecular alterations, we performed whole-genome sequencing analysis on 30 LCBs. Here we show, the genome-wide substitution patterns of LCBs developed in chronic hepatitis livers overlapped with those of 60 HCCs, whereas those of hepatitis-negative LCBs diverged. The subsequent validation study on 68 LCBs identified recurrent mutations in TERT promoter, chromatin regulators (BAP1, PBRM1 and ARID2), a synapse organization gene (PCLO), IDH genes and KRAS. The frequencies of KRAS and IDHs mutations, which are associated with poor disease-free survival, were significantly higher in hepatitis-negative LCBs. This study reveals the strong impact of chronic hepatitis on the mutational landscape in liver cancer and the genetic diversity among LCBs.

Combined Hepatocellular Cholangiocarcinoma: A Case Report and Review of Literature

The two commonly seen subtypes of primary liver cancer are hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). They both have a distinct clinico-pathologic profile. Combined hepatocellular cholangiocarcinoma (cHCC-CC) is a far more uncommon tumor with histologic evidence of both hepatocellular and biliary epithelial differentiation [1, 2]. It accounts for about 1.0–14.2 % of all primary liver cancers with incidence varying in different studies [3, 4]. The reported incidence of cHCC-CC ranges from 1.4 to 6.5 % and varies geographically [5]. In 1949, Allen and Lisa first described this entity in detail and morphologically classified into three subtypes [6]. Since then there have been a few case series and single case reports describing this malignancy. Owing to its rarity, the natural history, clinical characteristics and optimal management of cHCC-CC is not well understood. We report a case of cHCC-CC along with review of literature. Wherever necessary, data from the Surveillance, Epidemiology and End Results (SEER) cancer registry (http://seer.cancer.gov/) was used to supplement the literature review. The results of our review suggests that cHCC-CC is closer to CC than to HCC. Further, surgery appears to be the modality of choice and regional lymph node dissection is recommended, but radiation therapy adds little to the overall survival of these patients.

Calpain Induces N-terminal Truncation of β-Catenin in Normal Murine Liver Development: DIAGNOSTIC IMPLICATIONS IN HEPATOBLASTOMAS

Hepatic competence, specification, and liver bud expansion during development depend on precise temporal modulation of the Wnt/β-catenin signaling. Also, loss- and gain-of-function studies have revealed pleiotropic roles of β-catenin in proliferation and hepatocyte and biliary epithelial cell differentiation, but precise mechanisms remain unknown. Here we utilize livers from different stages of murine development to determine β-catenin signaling and downstream targets. Although during early liver development full-length β-catenin is the predominant form, at late stages, where full-length β-catenin localizes to developing biliary epithelial cells only, a 75-kDa truncated β-catenin species is the principal form localizing at the membrane and in the nucleus of differentiating hepatocytes. The truncated species lacks 95 N-terminal amino acids and is transcriptionally active. Our evidence points to proteolytic cleavage of β-catenin by calpain as the mechanism of truncation in cell-free and cell-based assays. Intraperitoneal injection of a short term calpain inhibitor to timed pregnant female mice abrogated β-catenin truncation in the embryonic livers. RNA-seq revealed a unique set of targets transcribed in cells expressing truncated versus full-length β-catenin, consistent with different functionalities. A further investigation using N- and C-terminal-specific β-catenin antibodies on human hepatoblastomas revealed a correlation between full-length versus truncated β-catenin and differentiation status, with embryonal hepatoblastomas expressing full-length β-catenin and fetal hepatoblastomas expressing β-catenin lacking its N terminus. Thus we conclude that calpain-mediated cleavage of β-catenin plays a role in regulating hepatoblast differentiation in mouse and human liver, and the presence of the β-catenin N terminus correlates with differentiation status in hepatoblastomas.

Combined hepatocellular-cholangiocarcinoma in a lesser flamingo (Phoenicopterus minor)

A case of combined hepatocellular-cholangiocarcinoma (CHCC) in an adult male lesser flamingo (Phoenicopterus minor) that was part of a breeding programme at a private facility is reported. Grossly, the liver was markedly enlarged with multifocal, well-circumscribed, pinpoint to 2 cm diameter pale tan nodular masses. Histologically, the hepatic parenchyma was replaced by neoplastic cells that demonstrated hepatocellular and, less frequently, biliary epithelial cell differentiation. Positive pan-cytokeratin (AE1/AE3/PCK26) immunolabelling of the neoplastic cells forming bile ducts with the scattered immunoreactivity of cells forming glandular structures within the areas of hepatocellular differentiation supported the diagnosis. No metastases were detected. CHCC is a rare neoplasm in mammals and birds. This is the first report where gross, histological, and immunohistochemical characteristics of CHCC in a bird are described, and the first report of CHCC in a lesser flamingo.

A Case of Combined Hepatocellular-Cholangiocarcinoma with Underlying Schistosomiasis

Combined hepatocellular-cholangiocarcinoma is a rare form of primary liver cancer showing features of both hepatocellular and biliary epithelial differentiation. We report here on a case with collision tumor, which apparently was the coincidental occurrence of both hepatocellular carcinoma and cholangiocarcinoma underlying schistosomiasis. A 39-year-old-Philippine female was transferred to our hospital for evaluation of a liver mass that was found on ultrasonography at a local hospital. HBsAg and Anti-HCV were negative and serum alpha-fetoprotein (AFP) level was normal. The tumor mass was histologically diagnosed as adenocarcinoma by sono-guided biopsy before the operation. Partial lobectomy was performed and we histologically identified the concurrent occurrence of hepatocellular carcinoma and cholangiocarcinoma, (a "collision type carcinoma").

Clinical and molecular analysis of combined hepatocellular-cholangiocarcinomas

Background/Aims Combined hepatocellular-cholangiocarcinoma (HCC-CC) show dual hepatocellular and biliary epithelial differentiation. To better understand the relations between cholangiocarcinoma (CC), HCC-CC and hepatocellular carcinoma (HCC), we screened for genetic alterations. Methods A series of nine CC, 15 HCC-CC and three separated HCC and CC lesions (‘collision tumors’) were screened for loss of heterozygosity (LOH) using 400 microsatellite markers and for p53 and β-catenin mutations. A comparison with a previously characterized series of 137 HCC was performed. Results In six cases of CC and HCC-CC, we identified TP53 gene mutations. A CTNNB1/β-catenin was identified in two patients presenting collision tumors, but no mutations were found in CC or in HCC-CC. A high level of chromosome instability in both CC and HCC-CC was found. Recurrent specific LOH were identified at 3p and 14q in more than 50% of the CC and the HCC-CC cases, whereas these chromosomal regions were deleted in less than 10% of the HCC cases ( P<10 −5). Minimal common regions of deletion (MCRD) were defined at 3p24-p14 and 14q24-q32, respectively. Conclusions These results suggest that combined HCC-CC are genetically closer to CC than HCC and common carcinogenesis pathways may be altered in HCC-CC and CC.

Clinical and molecular analysis of combined hepatocellular-cholangiocarcinomas

<h2>Abstract</h2><h3>Background/Aims</h3> Combined hepatocellular-cholangiocarcinoma (HCC-CC) show dual hepatocellular and biliary epithelial differentiation. To better understand the relations between cholangiocarcinoma (CC), HCC-CC and hepatocellular carcinoma (HCC), we screened for genetic alterations. <h3>Methods</h3> A series of nine CC, 15 HCC-CC and three separated HCC and CC lesions (‘collision tumors') were screened for loss of heterozygosity (LOH) using 400 microsatellite markers and for p53 and β-catenin mutations. A comparison with a previously characterized series of 137 HCC was performed. <h3>Results</h3> In six cases of CC and HCC-CC, we identified <i>TP53</i> gene mutations. A CTNNB1/β-catenin was identified in two patients presenting collision tumors, but no mutations were found in CC or in HCC-CC. A high level of chromosome instability in both CC and HCC-CC was found. Recurrent specific LOH were identified at 3p and 14q in more than 50% of the CC and the HCC-CC cases, whereas these chromosomal regions were deleted in less than 10% of the HCC cases (<i>P</i><10⁻⁵). Minimal common regions of deletion (MCRD) were defined at 3p24-p14 and 14q24-q32, respectively. <h3>Conclusions</h3> These results suggest that combined HCC-CC are genetically closer to CC than HCC and common carcinogenesis pathways may be altered in HCC-CC and CC.

Hepatic Resection for Combined Hepatocellular and Cholangiocarcinoma

Combined hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) is an uncommon form of primary liver cancer with features of hepatocellular and biliary epithelial differentiation. The clinical significance of this disease entity is poorly understood. The aim of the present study was to determine the operative and survival outcomes of patients with HCC-CC who underwent hepatic resection. Retrospective study. Tertiary referral center. The study comprised 12 patients (aged 28-72 years) with HCC-CC (HCC-CC group) who underwent hepatic resection between January 1, 1991, and December 31, 2000. The diagnosis was based on a combination of histological, immunohistochemical, and, if necessary, electron microscopic examinations of the resected specimen. Clinicopathological data and operative and survival outcomes of the HCC-CC group were compared with those of 476 patients with HCC (HCC group) and 25 patients with intrahepatic CC (CC group) who underwent hepatic resection during the study period. Ten patients (83%) in the HCC-CC group underwent major hepatic resection. The operative morbidity and mortality were 17% (2 of 12 patients) and 0%, respectively. The clinicopathological variables of the HCC-CC group resembled those of the HCC group more closely than those of the CC group. The incidence of positive hepatitis B serologic test results in the HCC-CC group (7 [58%] of 12 patients) was intermediate between that of the HCC group (392 [82%] of 476 patients) and that of the CC group (5 [20%] of 25 patients) (P<.001). Underlying chronic liver disease was significantly less common in the CC group (5 [20%] of 25 patients) than in either the HCC-CC group (9 [75%] of 12 patients) or the HCC group (421 [88%] of 476 patients) (P<.001). After hepatic resection, the median disease-free survival of the HCC-CC, HCC, and CC groups was 10, 18, and 24 months, respectively (P =.44). The overall median survival was shortest in the HCC-CC group (17 months) and was not significantly different from that of the CC group (26 months) (P =.38), but was significantly worse than that of the HCC group (52 months) (P =.02). Although the clinicopathological variables of patients with HCC-CC were most similar to those of patients with HCC, patients with HCC-CC had a significantly worse survival outcome after hepatic resection when compared with patients with HCC. Further studies on postoperative adjuvant therapy and multimodality treatment for recurrent disease are required to prolong the survival of these patients.

Telomerase activity is repressed during differentiation along the hepatocytic and biliary epithelial lineages: verification on immortal cell lines from the same origin.

Recent investigations indicate that telomerase activity regulates the life span of cells by compensating for telomere shortening during DNA replication. In addition, as differentiation progresses, telomerase activity is reduced in several different cell lineages. These findings lend support to the theory that more immature cells have greater remaining proliferative capacity and longer life span. However, it has not been directly demonstrated that the differentiation along a hepatocytic or a bile ductal lineage is accompanied by reduction of telomerase activity. In this study, we present direct evidence that telomerase activity is reduced during hepatocytic and biliary epithelial differentiation by using our unique cell lines including a stem-like cell line, ETK-1. When hepatocytic differentiation was induced in ETK-1 by 5-azacytidine, telomerase activity decreased significantly. Similarly, when we compared the telomerase activity on SSP-25 and RBE cell lines from the same origin but representing different maturation stages of cholangiocarcinoma, more mature cells were found to possess significantly lower activity. These results indicate that the generally accepted relationship between telomerase activity and differentiation stage also applies in the hepatocytic and biliary epithelial lineages.

Combined hepatocellular-cholangiocarcinoma: a clinicopathological study.

Combined hepatocellular-cholangiocarcinoma (HCC-CC) is an uncommon form of primary liver cancer having features of both hepatocellular and biliary epithelial differentiation. We reviewed 21 cases of this tumour diagnosed between 1972 and 1996 (patient age range 16-79 years; mean patient age 49.7 years; 18 male and three female patients). Histologically, the majority (n = 18) of tumours were 'mixed' tumours, in which areas of hepatocellular and biliary epithelial differentiation were intimately mixed within the same tumours. Two patients had separate tumours in which discrete nodules of HCC and CC occurred in the same livers. One patient had a 'fibrolamellar' tumour that histologically simulated the fibrolamellar variant of HCC, but some of the tumour cells were mucin-producing cells. Of the 21 cases, mucin was demonstrable in 16 and, in the few mucin-negative tumours, electron microscopic studies confirmed the presence of the dual differentiation. The tumours frequently exhibited an invasive character with frequent venous permeation, direct invasion into adjacent liver parenchyma and tumour microsatellite formation, similar to that of ordinary HCC. Histological evidence of cirrhosis or chronic hepatitis was present in 77.8% of patients and 75% of patients were hepatitis B surface antigen positive. Raised serum alpha-fetoprotein (AFP) levels (above 300 ng/mL) were present in 61.5% of patients and AFP was detected immunohistochemically in 55% of tumours. The overall survival times of patients with HCC-CC were short. In conclusion, HCC-CC showed clinical and pathological features more akin to those of ordinary HCC than to CC.

Cholangiocarcinomas in Japanese and Thai patients: difference in etiology and incidence of point mutation of the c-Ki-ras proto-oncogene.

Point-mutational activation of the c-Ki-ras proto-oncogene has been shown to be rare in human hepatocellular carcinoma, the most common primary liver cancer and one usually associated with chronic viral infection. To reveal the association of c-Ki-ras activation with cholangiocarcinogenesis under different etiological backgrounds, the incidence of point mutation at codons 12 and 13 of the c-Ki-ras proto-oncogene was examined in three groups of human liver cancers with differentiation to biliary epithelial cells: Group 1, cholangiocellular carcinoma in Japanese with normal livers; Group 2, cholangiocellular carcinoma in Thais who had lived in an area where the liver fluke Opisthorchis viverrini is endemic; and Group 3, combined hepatocellular-cholangiocellular carcinoma, a rare type showing features of both hepatocellular and biliary epithelial differentiation, in Japanese with chronic viral hepatitis with or without cirrhosis. The polymerase chain reaction and direct sequencing of its product were used to detect the mutation. Point mutation at codon 12 of the c-Ki-ras gene was detected in five (56%) of nine cases in Group 1. In contrast, the mutation was not detected in any of the cases in Groups 2 and 3. Therefore, point-mutational activation of c-Ki-ras did not seem to be involved in the development of primary liver cancers associated with apparent chronic irritation of liver cells or biliary epithelial cells caused by exogenous liver-fluke or viral infection. On the other hand, point-mutational activation of the c-Ki-ras proto-oncogene may be involved in cholangiocarcinogenesis in liver without preexisting liver-fluke or viral infection.

Combined hepatocellular-cholangiocarcinoma. A histologic and immunohistochemical study.

Combined hepatocellular-cholangiocarcinoma is a rare form of primary liver cancer showing features of both hepatocellular and biliary epithelial differentiation. In a review of 24 cases of this tumor, three histologic types were encountered. Four cases were Type I or "collision tumors," apparently a coincidental occurrence of both hepatocellular carcinoma and cholangiocarcinoma in the same patient. Twelve cases were Type II or "transitional tumors," in which there were areas of intermediate differentiation and an identifiable transition between hepatocellular carcinoma and cholangiocarcinoma. Eight cases were Type III or "fibrolamellar tumors" which resembled the fibrolamellar variant of hepatocellular carcinoma but which also contained mucin-producing pseudoglands. Type III tumors differ from other combined tumors, occurring at a younger age, in the absence of cirrhosis, and having a slightly longer survival. Immunohistochemical (immunoperoxidase) staining for intracellular antigens showed that alpha-fetoprotein is a fairly specific, although insensitive, marker of hepatocellular differentiation in primary liver cancers, being present in 50% of typical hepatocellular carcinomas and in hepatocellular areas in 29% of combined tumors, but in no cholangiocarcinomas or cholangiocellular areas of combined tumors. Keratin is a good marker of biliary epithelial differentiation, being found in 90% of cholangiocarcinomas and in 52% of combined hepatocellular cholangiocarcinomas, but in no hepatocellular carcinomas. Alpha-1-antitrypsin, fibrinogen, IgG, and carcinoembryonic antigen may be found in both hepatocellular carcinoma, cholangiocarcinoma, and in combined tumors; these antigens are therefore of limited use in differential diagnosis.