Saya Igarashi

Significance of immunoglobulin G4 (IgG4)‐positive cells in extrahepatic cholangiocarcinoma: Molecular mechanism of IgG4 reaction in cancer tissue

IgG4 reactions consisting of marked infiltration by immunoglobulin G4 (IgG4)‐positive plasma cells in affected organs is found in cancer patients as well as patients with IgG4‐related diseases. Notably, extrahepatic cholangiocarcinomas accompanying marked IgG4 reactions clinicopathologically mimic IgG4‐related sclerosing cholangitis. The regulatory cytokine interleukin (IL)‐10 is thought to induce the differentiation of IgG4‐positive cells. In this study, to clarify the mechanism of the IgG4 reaction in extrahepatic cholangiocarcinoma, we investigated nonprofessional antigen‐presenting cells (APCs) generating IL‐10–producing regulatory T cells (anergy T cells) and Foxp3‐positive regulatory cells producing IL‐10. Immunohistochemistry targeting IgG4, HLA‐DR, CD80, CD86, and Foxp3 was performed using 54 cholangiocarcinoma specimens from 24 patients with gallbladder cancer, 22 patients with common bile duct cancer, and eight patients with cancer of the Papilla of Vater. Moreover, a molecular analysis of Foxp3 and IL‐10 was performed using a cultured human cholangiocarcinoma cell line. Consequently, 43% of the cholangiocarcinomas were found to be abundant in IgG4. Those expressing HLA‐DR but lacking costimulatory molecules (CD80 and CD86) and those expressing Foxp3 detected by an antibody recognizing the N terminus accounted for 54% and 39% of cases, respectively. Moreover, the number of IgG4‐positive cells was larger in these cases than in other groups. In cultured cells, the presence of a splicing variant of Foxp3 messenger RNA and the expression of IL‐10 were demonstrated. Conclusion: Extrahepatic cholangiocarcinoma is often accompanied by significant infiltration of IgG4‐positive cells. Cholangiocarcinoma cells could play the role of nonprofessional APCs and Foxp3‐positive regulatory cells, inducing IgG4 reactions via the production of IL‐10 indirectly and directly, respectively. (HEPATOLOGY 2012;56:157–164)

KRAS and GNAS mutations and p53 overexpression in biliary intraepithelial neoplasia and intrahepatic cholangiocarcinomas

Similar to the pancreatic intraepithelial neoplasia (PanIN)‐pancreatic carcinoma sequence model, intrahepatic cholangiocarcinoma (ICC) also reportedly follows a stepwise carcinogenesis process through the a precursor lesion: biliary intraepithelial neoplasia (BilIN). For this study, the authors investigated the status of v‐Ki‐ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and GNAS complex locus (GNAS) mutations and tumor protein 53 (p53) overexpression in the stepwise process of cholangiocarcinogenesis.

Intrahepatic cholangiocarcinomas in cirrhosis are hypervascular in comparison with those in normal livers.

Intrahepatic cholangiocarcinomas (ICCs) are usually adenocarcinomas with fibrotic and hypovascular stroma. Intrahepatic cholangiocarcinomas in cirrhosis and precirrhotic liver (ICC‐cirrhosis) are increasingly being diagnosed, and can display hypervascluar enhancement resembling a hepatocellular carcinoma on dynamic imaging.

Clinicopathologic study of mixed adenoneuroendocrine carcinomas of hepatobiliary organs

Neuroendocrine neoplasms in hepatobiliary organs are very rare, but several cases of mixed adenoneuroendocrine carcinoma (MANEC) have been reported. In this study, we characterized the neuroendocrine component of biliary MANEC. A total of 274 cases of biliary cancer including 17 intrahepatic cholangiocarcinomas (CCs), 15 hepatic hilar CCs without preceding hepatobiliary disease, 55 hepatic hilar CCs with hepatolithiasis, 49 gallbladder cancers, 53 extrahepatic CCs, and 85 hepatocellular carcinomas were examined for a neuroendocrine component using immunohistochemistry with neuroendocrine markers (chromogranin A and synaptophysin). In the MANEC cases, in addition to a close histological examination, the proliferative activity and the expression of somatostatin receptor 2A were also evaluated. In addition to an ordinary adenocarcinoma, a neuroendocrine component occupying more than 30% of the entire tumor was also found in 4% (2/55 cases) of hepatic hilar cholangiocarcinomas with hepatolithiasis, 10% (5/49 cases) of gallbladder cancers, and 4% (2/53 cases) of extrahepatic cholangiocarcinomas, but not in the intrahepatic cholangiocarcinomas, hilar cholangiocarcinomas without preceding hepatobiliary disease, and hepatocellular carcinomas. Two cases were positive for somatostatin receptor 2A. The adenocarcinoma components were predominately located at the surface of the tumors, and the majority of stromal and vascular invasion and lymph node metastasis involved neuroendocrine components, showing the features of neuroendocrine tumor G2 or neuroendocrine carcinomas (NECs). NEC components showed higher proliferative activity on Ki67 immunostaining, compared to the adenocarcinoma components. Biliary MANECs are found in hepatic hilar cholangiocarcinomas with hepatolithiasis, gallbladder cancers, and extrahepatic cholangiocarcinomas and show a characteristic histology. Since the neuroendocrine component in biliary MANEC defines the prognosis, it is important to identify it and consider the indications for adjunctive therapy with somatostatin analogues.

Hilar cholangiocarcinoma and pancreatic ductal adenocarcinoma share similar histopathologies, immunophenotypes, and development-related molecules

Embryologically, intrahepatic small bile ducts arise from hepatic progenitor cells via ductal plates, whereas the pancreato-extrahepatic biliary progenitor cells expressing the transcription factors PDX1 and HES1 are reportedly involved in the development of the extrahepatic biliary tract and ventral pancreas. The expression of cellular markers characteristic of the different anatomical levels of the biliary tree and pancreas, as well as PDX1 and HES1, was examined in cholangiocarcinoma components of combined hepatocellular cholangiocarcinoma (12 cases), intrahepatic cholangiocarcinoma (21 cases), hilar cholangiocarcinoma (25 cases), and pancreatic ductal adenocarcinoma (18 cases). Anterior gradient protein-2 and S100P were frequently expressed in hilar cholangiocarcinoma and pancreatic ductal adenocarcinoma, whereas neural cell adhesion molecule and luminal expression of epithelial membrane antigen were common in cholangiocarcinoma components of combined hepatocellular cholangiocarcinoma. PDX1 and HES1 were frequently and markedly expressed in pancreatic ductal adenocarcinoma and, to a lesser degree, in hilar cholangiocarcinoma, although their expression was rare and mild in cholangiocarcinoma components in combined hepatocellular cholangiocarcinoma. The expression patterns of these molecules in intrahepatic cholangiocarcinoma were intermediate between those in hilar cholangiocarcinoma and cholangiocarcinoma components of combined hepatocellular cholangiocarcinoma. Pancreatic ductal adenocarcinoma and hilar cholangiocarcinoma had a similar expression of mucin, immunophenotypes, as well as transcription factors. Pancreatic ductal adenocarcinoma and hilar cholangiocarcinoma showed similar postoperative prognosis. In conclusion, the similar expression of phenotypes related to pancreatobiliary anatomy and embryology may in part explain why these 2 types of carcinoma present similar clinicopathologic features. Further studies on the carcinogenesis of these carcinomas based on their similarities are warranted.

Monocyte chemoattractant protein-1 derived from biliary innate immunity contributes to hepatic fibrogenesis

Aims Monocyte chemoattractant protein-1 (MCP-1) is a major chemotactic factor for hepatic stellate cells (HSCs) associated with hepatic fibrosis. In this study, among several fibrogenetic factors derived from biliary epithelial cells (BECs), MCP-1 produced by the biliary innate immune system was found to be most critical in the histogenesis of hepatic fibrogenesis. Methods Using cultured human BECs, the expression of five fibrogenetic factors including MCP-1 on stimulation with Toll-like receptor ligands, inflammatory cytokines or bile acids was examined. Moreover, in situ detection of MCP-1 and α-smooth muscle actin proteins was performed using sections from normal and diseased livers by immunohistochemistry. Results All fibrogenetic factors were detected in BECs, but only MCP-1 expression was upregulated, by all the Toll-like receptor ligands, IL-1β, and tumour necrosis factor-alpha. Proliferating bile ductules in interface areas expressed MCP-1 in diseased livers accompanying α-smooth muscle actin-positive activated HSCs. Conclusions Bile ductules proliferate in various hepatobiliary diseases, and its significance is still unknown. This study demonstrated that BECs in bile ductules could produce MCP-1, particularly, via biliary innate immunity, suggesting that MCP-1 derived from BECs plays an important role in the recruitment of HSCs to interface areas and the activation of HSCs resulting in the progression of periportal fibrosis.

Chemoembolization for the Treatment of Large Hepatocellular Carcinoma

PURPOSE To retrospectively evaluate the efficacy of chemoembolization for inoperable hepatocellular carcinoma (HCC) tumors larger than 5 cm in diameter. MATERIALS AND METHODS Chemoembolization was performed in 30 patients with HCCs with a largest diameter of more than 5 cm with three or fewer lesions and no portal vein tumor thrombus. The mean maximum tumor diameter was 7.7 cm +/- 2.4. When the tumor was extremely large and had multiple feeding arteries, stepwise chemoembolization sessions at intervals of 3-10 weeks were performed. In addition, extrahepatic collateral supply was identified and embolized. Local therapeutic effects, survival rates, and complications were analyzed. RESULTS The mean follow-up period was 33.8 months +/- 24.1. One to 13 chemoembolization sessions (mean, 4.0 sessions +/- 3.0) were performed in each patient. Additionally, 62 collateral vessels were embolized in 21 patients, including 22 vessels in 14 patients at the initial procedure. Early tumor response rate 2-3 months after treatment was 43.3% by Response Evaluation Criteria In Solid Tumors. Complete radiologic response was achieved in 19 patients. Eleven patients died between 4 and 61 months after treatment (mean, 27.2 months +/- 21.8), including four deaths unrelated to hepatic causes. Nineteen patients have survived for 6-103 months (mean, 37.5 months +/- 25.2). Overall and progression free-survival rates at 1, 3, and 6 years were 82.3% and 66.0%, 73.9% and 57.6%, and 32.9% and 34.2%, respectively. Three infectious complications developed and were managed by interventions. CONCLUSIONS Chemoembolization was effective for large HCCs, although there is a risk of infectious complications after the procedure.

Participation of peribiliary glands in biliary tract pathophysiologies.

AIM To investigate the roles of peribiliary glands around the bile ducts in the pathophysiology of the biliary tract. METHODS The expression of fetal pancreatic markers, pancreatic duodenal homeobox factor 1 (PDX1) and hairy and enhancer of split 1 (HES1) and endodermal stem/progenitor (S/P) cell markers [CD44s, chemokine receptor type 4 (CXCR4), SOX9 and epithelial cell adhesion molecule (EpCAM)] were examined immunohistochemically in 32 normal adult livers (autopsy livers) and 22 hepatolithiatic livers (surgically resected livers). The latter was characterized by the proliferation of the peribiliary glands. Immunohistochemistry was performed using formalin-fixed, paraffin-embedded tissue sections after deparaffinization. Although PDX1 and HES1 were expressed in both the nucleus and cytoplasm of epithelial cells, only nuclear staining was evaluated. SOX9 was expressed in the nucleus, while CD44s, CXCR4 and EpCAM were expressed in the cell membranes. The frequency and extent of the expression of these molecules in the lining epithelia and peribiliary glands were evaluated semi-quantitatively based on the percentage of positive cells: 0, 1+ (focal), 2+ (moderate) and 3+ (extensive). RESULTS In normal livers, PDX1 was infrequently expressed in the lining epithelia, but was frequently expressed in the peribiliary glands. In contrast, HES1 was frequently expressed in the lining epithelia, but its expression in the peribiliary glands was focal, suggesting that the peribiliary glands retain the potential of differentiation toward the pancreas and the lining epithelia exhibit properties to inhibit such differentiation. This unique combination was also seen in hepatolithiatic livers. The expression of endodermal S/P cell markers varied in the peribiliary glands in normal livers: SOX9 and EpCAM were frequently expressed, CD44s infrequently, and CXCR4 almost not at all. The expression of these markers, particularly CD44s and CXCR4, increased in the peribiliary glands and lining epithelia in hepatolithiatic livers. This increased expression of endodermal S/P cell markers may be related to the increased production of intestinal and gastric mucin and also to the biliary neoplasia associated with the gastric and intestinal phenotypes reported in hepatolithiasis. CONCLUSION The unique expression pattern of PDX1 and HES1 and increased expression of endodermal S/P cell markers in the peribiliary glands may be involved in biliary pathophysiologies.

Notch1-Hes1 signalling axis in the tumourigenesis of biliary neuroendocrine tumours

Aims Biliary neuroendocrine tumours (NETs) are rare and mostly exist as a component of mixed adenoneuroendocrine carcinomas (MANECs). Although the NET component in biliary MANECs is generally more malignant and clinically more important to the prognosis than the ordinary adenocarcinomatous component, the histogenesis of biliary NET has not been clarified. In this study, the role of the Notch1-Hes1 signalling axis in the histogenesis of biliary NETs was examined. Methods Immunohistochemistry for Notch1, its ligand Jagged1 and Hes1 was performed using surgical specimens from 11 patients with biliary MANEC. Moreover, after the knock-down of Notch1 mRNA expression in a cholangiocarcinoma cell line, the expression of chromogranin A (a neuroendocrine marker) and Ascl1 (a neuroendocrine-inducing molecule inhibited by activated Hes1) was examined by quantitative PCR. Results Histological examination revealed that the adenocarcinomatous components were predominately located at the luminal surface of the MANEC and the majority of stromal invasion involved NET components. Ordinary adenocarcinomas and non-neoplastic biliary epithelium constantly expressed Notch1, Jagged1 and Hes1, but the expression of Notch1 and Hes1 was decreased or absent in NET components, suggesting interference with the Notch1-Hes1 signalling axis in biliary NET. Moreover, in the cholangiocarcinoma cell line in which the expression of Notch1 mRNA was knocked down, the mRNA expression of Ascl1 and chromogranin A was increased. Conclusions The Notch1-Hes1 signalling axis suppresses neuroendocrine differentiation and maintains tubular/acinar features in adenocarcinoma and non-neoplastic epithelium in the biliary tree. Moreover, a disruption of this signalling axis may be associated with the tumourigenesis of NETs in biliary MANEC.

Bile duct expression of pancreatic and duodenal homeobox 1 in perihilar cholangiocarcinogenesis

Igarashi S, Matsubara T, Harada K, Ikeda H, Sato Y, Sasaki M, Matsui O & Nakanuma Y 
(2012) Histopathology 61, 266–276