BRAF V600E mutation in biliary proliferations associated with α1 -antitrypsin deficiency.

AIMS: Both homozygous and heterozygous α1 -antitrypsin (AAT) deficiency patients are at risk of developing hepatocellular carcinoma (HCC), but also of developing cholangiocarcinoma and combined HCC and cholangiocarcinoma. The aim of our study is to report a series of bile duct adenomas (BDAs) and intrahepatic cholangiocarcinoma (ICCs) in adult AAT deficiency patients, observed in our institution over a 5-year period. Our observational study includes a detailed investigation of their immunohistochemical profile and BRAF V600E mutation status. METHODS AND RESULTS: Eleven biliary lesions from five AAT deficiency patients (six BDAs from three cirrhotic patients with other concurrent liver diseases; three BDAs and two ICCs from two non-cirrhotic patients) were identified between 2010 and 2015 during routine histological investigation. Most BDAs expressed CD56, EpCAM, CD133, and CA19-9, similarly to hepatic progenitor cells (HPCs), and carried the BRAF V600E mutation (87.5%). One ICC showed a similar immunohistochemical profile but no evidence of the BRAF V600E mutation. CONCLUSIONS: Most of the biliary proliferations in AAT deficiency patients have an appearance of BDA with an HPC-related immunohistochemical profile. Their frequent BRAF V600E mutations support their neoplastic nature, but not necessarily their progression to ICC. We believe that this may depend on the patient genotype, or require a different pathway or a second mutational hit for malignant transformation. We postulate that BDA represents a heterogeneous group of biliary lesions, and that those associated with AAT deficiency may constitute a group of their own.

BRAF V600E mutational status in bile duct adenomas and hamartomas.

AIMS: Bile duct adenomas (BDA) and bile duct hamartomas (BDH) are benign bile duct lesions considered neoplastic or secondary to ductal plate malformation, respectively. We have reported previously a high prevalence of BRAF V600E mutations detected by allele-specific polymerase chain reaction assay in BDA, and suggested that BDA may be precursors to a subset of intrahepatic cholangiocarcinomas harbouring V600E mutations. The aim of the present study was to assess the existence of BRAF V600E mutations, using immunohistochemical methods, in additional BDA as well as in BDH. METHODS AND RESULTS: Fifteen BDA and 35 BDH were retrieved from the archives of the pathology departments of two French university hospitals. All cases were reviewed by two pathologists specialized in liver diseases. BRAF V600E mutational status was investigated by immunohistochemistry. Mutated BRAF mutant protein was detected in 53% of the BDA and in none of the cases of BDH. CONCLUSION: Our findings suggest that BDA and BDH are different processes, and that BDA represent true benign neoplasms. They also support the hypothesis that mutated BDA might precede the development of the subset of intrahepatic cholangiocarcinomas harbouring BRAF V600E mutations.

Hepatocellular and cholangiolar carcinoma-derived cell lines reveal distinct sets of chromosomal imbalances.

OBJECTIVES: Hepatocellular carcinoma (HCC) and cholangiolar carcinoma (CC) cell lines are used to analyze the basic mechanisms of carcinogenesis and target therapies. However, it is not yet clear which chromosomal aberrations are to be typically expected in such cell lines. It is also not clear whether there are prerequisites for in vitro growth on the genomic and/or expression level. We therefore analyzed HCC and CC cell lines for typical genetic settings. METHODS: The HCC cell lines HLE, HLF, Huh7, HepG2 and Hep3b and the CC cell lines EGI1, MzCha1 and TFK-1 were analyzed using high-density arrays for comparative genomic hybridization (aCGH; 244,000 oligonucleotides). Additional fluorescence in situ hybridization analyses were done to confirm the aCGH results and to add information regarding the aneuploidy of cell lines. RESULTS: The gain of 1q, in particular q21-22, was detected in all HCC cell lines also as a partial loss of 13q. In contrast, a loss of 8p in combination with a relative gain of 8q was seen in all CC but no HCC cell lines. Interestingly, a gain of 17q was seen in all cell lines. These aberrations are also well documented for surgical tumor specimens. Besides these imbalances, the cell lines revealed imbalances for 11p, 12p, 14q, 16p, 16q, 21q and 22q, respectively, only rarely seen in surgical tumor specimens. These aberrations could be of importance for the in vitro cultivation of tumor cells. Structural aberrations were accompanied by aneuploidy in 3 of 5 HCC cell lines and 2 of 3 CC cell lines. Ploidy status was not correlated to any of the imbalances mentioned above. CONCLUSIONS: HCC and CC cell lines revealed characteristic chromosomal imbalances similar to those seen in surgical tumor specimens including chromosomes 1, 8, 13 and 17, respectively. These aberrations are characteristic of the histogenetic origin of the tumor cells. However, the chromosomal imbalances that occurred probably led to the ability of tumor cells to grow in vitro.

[Precursor lesions of pancreatobiliary cancer]. / Vorläuferläsionen pankreatobiliärer Karzinome.

Precursor lesions of pancreatobiliary cancer can be divided into cystic and flat lesions. Mucinous cystic neoplasm and intraductal papillary mucinous neoplasm (IPMN) comprise the cystic precursors in the pancreas, while intraductal papillary neoplasm (IPN) represents their counterpart in the bile duct system. There is an adenoma-carcinoma sequence in the cystic precursors arising from four different types of epithelia: pancreatobiliary, oncocytic, intestinal and gastric. These subtypes of IPMN/IPN are morphologically and immunohistochemically well characterised and show clinical and prognostic relevance: the gastric subtype is associated with the best prognosis, followed by the oncocytic and intestinal subtypes, while the pancreatobiliary subtype is characterized by adverse clinical behaviour. Pancreatic intraepithelial neoplasia (PanIN) and biliary intraepithelial neoplasia (BilIN) represent the flat precursors. PanIN are morphologically and biologically well defined. PanIN with lobulocentric atrophy has recently been described as a putative precursor of pancreatic cancer. Despite well defined morphological features in BilIN, the molecular alterations seen during early tumor progression in the biliary tract are poorly understood.

Frequent p16ink4a inactivation is an early and frequent event of intraductal papillary neoplasm of the liver arising in hepatolithiasis.

Intraductal papillary neoplasm of the liver (IPNL) is a precursor lesion of intrahepatic cholangiocarcinoma (ICC) arising in hepatolithiasis. In this study, 98 foci of IPNL identified in 39 surgically resected hepatolithiatic livers were investigated for expression of p16INK4a, cyclin D1, p21WAF1/CIP1, p53, mouse double-minute 2 (MDM2), and pRb. In addition, methylation-specific polymerase chain reaction (MSP) for p16 INK4a promoter region was performed in these foci. Nonneoplastic bile ducts from 11 hepatolithiatic livers, 5 histologically normal livers, and 9 cases of nonpapillary conventional ICC were used as controls. Decreased expression of p16INK4A was seen in IPNL group 1 with mild dysplasia and continued along the progression of IPNL to ICC. The expression of cyclin D1, p21WAF1/CIP1,and pRb gradually increased along the progression of IPNL to ICC and became significantly high in IPNL of group 3 (carcinoma in situ). The expression of p53 and MDM2 was increased in IPNL group 3 and group 4 with evident invasive carcinoma. MSP revealed that 54.6% of 44 IPNL foci harbored p16INK4a promoter hypermethylation, and such foci were significantly correlated with decreased expression of p16INK4a protein. Ki-67 labeling index exhibited a stepwise increase from IPNL group 1 to group 4. We conclude that p16INK4a inactivation, due mainly to its promoter hypermethylation, is a frequent and early event of IPNL and may be responsible for genetic and epigenetic alterations of other cell cycle regulators in IPNL.

Primary liver carcinoma in genetic hemochromatosis reveals a broad histologic spectrum.

Hepatocellular carcinoma (HCC) is a well-known complication of genetic hemochromatosis (GH). However, the frequency of primary liver carcinoma (PLC) with biliary differentiation, such as cholangiocarcinoma (CC) and combined hepatocholangiocarcinoma (CHCC), in GH remains unclear We analyzed the histologic type of 20 PLCs occurring in the background of GH; all patients were homozygotic for the C282Y mutation. Ten were depleted of iron by successive phlebotomies, while the remaining 10 were untreated. Histologically, 13 cases were classified as HCC, 3 as CC, and 4 as CHCC. Immunohistochemical detection of Hep Par 1, cytokeratin 19 (CK19), and MUC1 supported this classification; PLC with biliary differentiation was immunoreactive for MUC1 in 86% (6/7) of cases and for CK19 in 100% (7/7) of cases. The nontumoral liver exhibited no cirrhosis or extensive fibrosis in 6 cases. Von Meyenburg complexes were present in 11 cases and intraparenchymal bile duct adenomas in 3. These data suggest that PLCs in patients with GH present a wide histologic spectrum, with tumors showing frequent biliary differentiation; may arise on a nonfibrotic or a cirrhotic liver; and often are associated with Von Meyenburg complexes and to a lesser extent with bile duct adenomas.

Mammaglobin B gene as a novel marker for lymph node micrometastasis in patients with abdominal cancers.

Mammaglobin B is a recently-isolated gene speculated to belong to the uteroglobin gene family and is overexpressed in primary breast cancers. We investigated mammaglobin B mRNA expression in various cancers of the digestive system. Given the absence of mammaglobin B expression in normal lymph nodes, we also assessed the usefulness of mammaglobin B as a marker for lymph node micrometastases in cancer patients. Mammaglobin B gene transcripts were frequently detected by reverse transcriptase-polymerase chain reaction (RT-PCR) assay in primary tumors of the esophagus (2/3), stomach (7/7), colon (15/15), pancreas (4/6), common bile duct (6/6), cholangioma (2/2) and gall bladder (1/1). Mammaglobin B overexpression was observed in three of 15 cases (20%) of colon cancer, suggesting its possible contribution to colon carcinogenesis. Down-regulated mammaglobin B expression was observed in hepatoma cells in comparison with corresponding non-cancerous livers (3/3). RT-PCR assay of mammaglobin B detected 14 of 15 histologically positive lymph nodes from patients with gastric cancer, colon cancer and cholangioma. Seven of 32 (22%), three of nine (33%), and three of seven (43%) histologically negative nodes from patients with gastric, colon and cholangiocellular carcinoma, respectively, were found to express mammaglobin B mRNA. Our results showed that expression of mammaglobin B was frequently detected in cancers originating in digestive organs, especially adenocarcinomas, and that mammaglobin B gene detected by RT-PCR may be a potentially useful molecular marker for lymph node micrometastases of various digestive organ cancers.

p53 protein expression and prognosis in gallbladder carcinoma and premalignant lesions.

BACKGROUND/AIMS: p53 protein expression in gallbladder carcinoma has recently been detected by immunohistochemical techniques, but the relationship between p53 expression and prognosis or clinico-pathological factors is still obscure. MATERIALS AND METHODS: We investigated 48 gallbladder carcinoma, 7 adenoma and 11 dysplasia cases for p53 expression by immunohistochemical techniques. RESULTS: p53 expression was positive in 39.6% of gallbladder cancer cases, but in no adenoma or dysplasia cases. No significant correlation was found between p53 overexpression and prognosis or recurrence in 20 patients with carcinoma involvement up to the subserosal layer. p53 overexpression was correlated with DNA aneuploidy pattern and the absence of stones, but was not correlated with clinical staging or lymph node metastasis. CONCLUSION: These results suggest that p53 gene mutation is related to the transition from premalignancy to malignancy in gallbladder carcinogenesis, as well as DNA ploidy alterations and carcinogenesis unassociated with gallstones, but has no bearing on the prognosis.

Mutagenesis of ras proto-oncogenes in rat liver tumors induced by vinyl chloride.

Vinyl chloride is a DNA-damaging carcinogen which induces liver angiosarcomas in humans and animals. Activation of the Ki-ras 2 gene by a GC-->AT transition at the second base of codon 13 in human liver angiosarcomas associated with occupational exposure to vinyl chloride has been reported recently. In order to compare the molecular pathways of carcinogenesis in humans and animals, Sprague-Dawley rats were exposed to vinyl chloride and hepatic tumors, including two hepatocellular carcinomas and five liver angiosarcomas, were investigated for mutations at codons 12, 13 and 61 of the Ha-ras, Ki-ras and N-ras genes. High molecular weight DNA was amplified by the polymerase chain reaction and point mutations were analyzed by allele specific oligonucleotide hybridization, direct sequencing of polymerase chain reaction products and sequencing after cloning. None of the tumors exhibited a mutation in codons 12, 13 and 61 of the Ki-ras gene, nor in codons 12 of the Ha-ras gene or 61 of the N-ras gene. However, an activating AT-->TA transversion at base 2 of codon 61 of the Ha-ras gene was detected in the two hepatocellular carcinomas. Mutations involving codon 13 (GGC-->GAC) and codon 36 (ATA-->CTA) of the N-ras A gene were detected in two liver angiosarcomas, suggesting that the nature of the ras gene affected by a given carcinogen depends on host factors specific to cell types. Several additional base pair substitutions were found in exon 1 of the N-ras B and C sequences. NIH 3T3 transfection assays and Southern blot analysis of DNA from transformed NIH 3T3 cells confirmed the presence of a dominant activated N-ras gene. These results emphasize the differences in the molecular pathways leading to tumors in humans and rats and within a given species between different cell types.

The expression of p53 antigen in primary malignant epithelial tumors of the liver: an immunohistochemical study.

We examined the expression of mutant p53 gene products in primary malignant epithelial tumors of the liver. Fourteen of 68 hepatocellular carcinomas, one of seven hepatoblastomas and one of nine intrahepatic cholangiocarcinomas showed nuclear staining for p53 proteins. None of the surrounding non-tumorous tissues expressed nuclear staining. The detection of p53 proteins in tumor cells was significantly higher in hepatocellular carcinomas of Oriental patients (31.6%) compared to non-Orientals (6.7%, p < 0.015). No significant differences were seen in p53 antigen expression between hepatitis B and non-hepatitis B associated hepatocellular carcinomas in Oriental patients. These results suggest a role for other environmental factors, such as aflatoxin, in the etiology of p53 mutation in hepatocellular carcinoma in Oriental patients.

Loss of constitutional heterozygosity on chromosomes 5 and 17 in cholangiocarcinoma.

It has been established that loss of tumour suppressor genes is crucial in carcinogenesis. There has been no reported study on searching for tumour suppressor genes in cholangiocarcinomas as yet. In order to investigate the loss of heterozygosity (LOH), which may represent such gene loss, in cholangiocarcinoma, we studied 14 patients with this tumour using restriction fragment length polymorphism analysis. Twenty-two probes assigned to chromosomes 1, 5, 7, 9, 11, 12, 13, 14, 16, 17 and 18 were used. Allelic losses were found in chromosomal regions 5q35-qter and 17p13. Loss of genetic material in these regions in cholangiocarcinoma was shared with hepatocellular carcinoma. Probes for other chromosomes have as yet shown no consistent LOH. In conclusion, this study for the first time showed LOH on chromosomes 5 and 17 in cholangiocarcinoma.

Cell kinetic and morphological studies of human cholangiocellular carcinoma.

We investigated the cell kinetics and morphologies of cholangiocellular carcinoma (CCC) using 48 autopsied or surgically resected cases (47 were adenocarcinoma and the remaining adenosquamous cell carcinoma), all of which were formalin-fixed and paraffin-embedded. Cell kinetics were analyzed by counting the number of argyrophilic nucleolar organizer regions (AgNOR) using immunostaining of proliferating cell nuclear antigens (PCNA) and flow cytometric DNA analysis. Dedifferentiation of CCC was positively correlated with AgNOR number (2.22 +/- 0.21 in well differentiated, 3.66 +/- 0.85 in moderately differentiated and 4.17 +/- 0.49 in poorly differentiated adenocarcinomas, respectively). In 22 cases, the labeling index (LI) of PCNA was higher in moderately and poorly differentiated adenocarcinomas (24.0 +/- 2.35 and 26.0 +/- 4.89, respectively) than in well differentiated ones (10.8 +/- 2.14). A majority of well differentiated ones were diploid, while aneuploidy prevailed in moderately to poorly differentiated ones. These data suggest that cell proliferative indices and nuclear DNA analysis of CCC accurately reflect their histological grading. The anatomical location of CCC along the biliary tree had no relation to either of the cell kinetic data. In autopsy cases, the patients with organ and lymph node metastases tended to show a higher DNA index and aneuploidy. This study implies that a combination of several cell kinetic data is valuable for the evaluation of the biological behaviors of CCC, and also supports further studies of cell kinetics of CCC using small-sized biopsy specimens, as a prognostic indicator.

[Study of combined hepatocellular and cholangiocarcinoma].

Four patients with combined hepatocellular and cholangiocarcinoma were studied. One case received hepatic arterial infusion therapy (HAIT), 2 hepatectomy and the case with extrahepatic cholangiocarcinoma underwent enucleation. According to Allen's classification, two were combined type and the others mixed type. Tumor ploidy patterns on flow cytometric DNA analysis were aneuploid in 2 cases with curative resection, of which one is still alive 6 years and 3 months after surgery. It suggested that the patient's survival was affected by the pathological factors more than the ploidy patterns of the tumor. A case treated by HAIT improved considerably in terms of symptoms and evidenced reduction in the size of the deposits. Selection of anticancer agents sensitive to both hepatocellular and cholangiocarcinoma is required.

Two new human cholangiocarcinoma cell lines and their cytogenetics and responses to growth factors, hormones, cytokines or immunologic effector cells.

Two new human cholangiocarcinoma (CC) cell lines (CC-SW-I and CC-LP-I) were established and maintained in culture for 2 years. Histologically, both original liver tumors were adenocarcinomas, and the cell lines exhibited morphologic features of moderately differentiated adenocarcinoma. Immunohistochemistry showed that both cell lines were strongly positive for cytokeratin AEI but negative for carbohydrate tumor-associated antigen, CA19-9. Ultrastructural analysis of both cell lines showed the presence of tight junctional complexes and focally formed microvilli. Both CC cell lines were tumorigenic in nude mice. Cytogenetic analysis showed that both cell lines expressed highly aneuploid karyotypes with numerous structural and numerical deviations. CC-SW-I was hypodiploid with numerous chromosome losses and structural rearrangements, while CC-LP-I was hyperdiploid and displayed multiple additional chromosomes. Doubling times for the CC-SW-I and CC-LP-I cell lines in the presence of 15% fetal bovine serum were 72 hr and 180 hr, respectively. Growth of the CC-SW-I cell line was significantly stimulated in the presence of insulin, while that of the CC-LP-I cell line was significantly augmented by epidermal growth factor (EGF). In contrast, dexamethasone strongly inhibited proliferation of both cell lines in a dose-dependent manner. Among various recombinant cytokines examined for effects on growth or surface antigen expression on CC cell lines, only interleukin I-beta (ILI-beta) strongly inhibited growth of the CC-LP-I cell line, while interferons (IFNs) or tumor necrosis factor-alpha (TNF-alpha) were mildly inhibitory. Both tumor cell lines were resistant to natural killer (NK) cells but sensitive to lymphokine-activated killer (LAK) cells. Preincubation of tumor cells with IFN-gamma, IFN-alpha or TNF-alpha significantly decreased the susceptibility of each tumor cell line to lysis by LAK cells, and the change in sensitivity did not correlate with the expression of HLA antigens or intercellular adhesion molecule-I (ICAM-I) on the surface of tumor cells. These 2 CC cell lines are expected to provide valuable information about cell biology of human CC.

A new human cholangiocellular carcinoma cell line (KMC-1).

We have recently established a cholangiocellular carcinoma (CCC) cell line, designated KMC-1, from a nude mouse subcutaneous tumor which developed after inoculation of a surgically resected peripheral type CCC from a 62-year-old Japanese male patient. KMC-1 cells grew over a 26-month period and passaged 57 times. These cells retained the morphologic characteristics of both the original tumor and the subcutaneous tumor in the nude mouse, which mainly consisted of irregular tubules and invaded surrounding interstitial tissue in part with an indurate pattern. KMC-1 cells grew in a monolayer pavement-like cell arrangement with tubular formation in part. Some cells and/or glands had a mucin-like substance inside. The doubling time of KMC-1 cells growing in serum-containing medium was 54 h at passage 31. Cell growth in serum-free medium was slow but steady. The number of chromosomes was distributed in range from 73 to 83 with modes of 76 and 78. KMC-1 cells secreted some tumor markers such as DUPAN-2, CA125, TPA, hCG, CA19-9 and ferritin, however, the secretion of DUPAN-2, and CA19-9 and ferritin were only detectable in serum-containing and serum-free medium, respectively. These findings suggest that KMC-1 cells will provide a variety of experimental models for research on CCC and the mechanisms of tumor marker secretion.

Establishment and characterization of a new human extrahepatic bile duct carcinoma cell line (KMBC).

A new human extrahepatic bile duct carcinoma cell line (KMBC) was established from a serially transplanted tumor in nude mice that originated from a surgically resected tumor from a 73-year-old Japanese man; the cell line has been maintained for 5 five years. KMBC cells proliferate in a monolayered sheet with a population doubling time of 30 hours. Chromosome number was distributed in a range from 37 to 44, with modal numbers of 40 and 41. KMBC cells and the reconstituted tumor in a nude mouse showed moderately to poorly differentiated adenocarcinoma and possessed various functional characteristics of extrahepatic bile duct carcinoma. KMBC cells secreted carbohydrate antigen 19-9, tissue polypeptide antigen, carcinoembryonic antigen, ferritin, beta 2-microglobulin, fibronectin, and alpha 2-macroglobulin and produced glutamic oxaloacetic transaminase and alkaline phosphatase. KMBC is the second established cell line that originated from a human extrahepatic bile duct carcinoma in the world literature, and it will be applicable to various experiments.

Lack of increase in heterozygous alpha 1-antitrypsin deficiency phenotypes among patients with hepatocellular and bile duct carcinoma.

Homozygous alpha 1-antitrypsin deficiency (PiZZ phenotype) is known to be associated with increased risk of cirrhosis and primary liver cancer. Although a relationship between heterozygous alpha 1-antitrypsin deficiency and chronic liver disease was suggested recently, it is still a matter of controversy whether such patients are at increased risk of liver cancer. The goal of this study was to determine the prevalence of heterozygous alpha 1-antitrypsin deficiency of different phenotypes among patients with primary hepatobiliary cancers. We studied 82 patients with primary hepatobiliary cancer; 59 had hepatocellular carcinoma and 23 had bile duct carcinoma. alpha 1-Antitrypsin quantitation and phenotyping were performed in each patient using standard methods. The distribution of the various Pi phenotypes was compared with that found in a normal population and reported elsewhere. Odds-ratio and chi 2 tests were used to measure the relative risk and the significance of association, respectively, between primary hepatobiliary cancers and heterozygous alpha 1-antitrypsin deficiency. Four patients in each of the cancer groups were heterozygous. Among the hepatocellular carcinoma patients, three had the PiMS phenotype and one had the PiMZ phenotype. Of these four heterozygous patients, only two had cirrhosis; one had cryptogenic cirrhosis and the other had hepatitis B virus-related cirrhosis. One noncirrhotic patient with a PiMZ phenotype had a fibrolamellar carcinoma. Of the four patients with bile duct carcinoma, three had the PiMS phenotype and one had the PiMZ phenotype. Of the four heterozygous patients, two had primary sclerosing cholangitis without associated inflammatory bowel disease and one patient had had previous biliary operations.(ABSTRACT TRUNCATED AT 250 WORDS)

c-erbB-2 oncogene expression in hepatocellular carcinoma and cholangiocarcinoma.

The c-erbB-2 proto-oncogene encodes a transmembrane protein which is homologous to the epidermal growth factor receptor. This protein can be localized immunohistochemically in formalin-fixed paraffin-embedded material using a monoclonal antibody NCL-CB11; positive membrane staining correlates with gene amplification and protein overexpression in breast cancer. Using this technique we have shown that only 2/26 (8%) of hepatocellular carcinomas, 0/10 (0%) of cholangiocarcinomas and 0/2 (0%) hepatoblastomas overexpressed c-erbB-2 as evidenced by membrane staining. Moreover c-erbB-2 mRNA was not detected in seven hepatocellular carcinomas examined by Northern blot analysis. c-erbB-2 overexpression is, therefore, unlikely to be contributing to the malignant phenotype in hepatocellular carcinoma and cholangiocarcinoma.