Keita Itatsu

Biliary Papillary Tumors Share Pathological Features With Intraductal Papillary Mucinous Neoplasm of the Pancreas

Recently, attention has been drawn to papillary neoplasm of the pancreatobiliary systems. In the pancreas, the disease entity of intraductal papillary mucinous neoplasm (IPMN‐P) is widely recognized. In contrast, the pathological characteristics of biliary papillary tumors, such as biliary papilloma(tosis) and papillary cholangiocarcinoma, have not yet been well documented. In this study, we compared the pathological features and post‐operative prognosis among biliary papillary tumors (10 cases of biliary papilloma(tosis) and 22 cases of papillary cholangiocarcinoma), conventional non‐papillary cholangiocarcinoma (15 cases), and IPMN‐P (31 cases). Macroscopically, all biliary papillary tumors were characterized by the prominent intraductal papillary proliferation, and macroscopic mucin‐hypersecretion was seen in 9 of 32 cases (28%). Histologically, biliary papillary tumors consisted of three types of tumor cells (pancreaticobiliary, intestinal and gastric types), whereas only the pancreaticobiliary type was observed in non‐papillary cholangiocarcinoma. Immunohistochemically, biliary papillary tumors were characterized by the common expression of MUC2, CDX2 and cytokeratin 20. In addition, biliary papillary tumors could be associated with two types of invasive lesions: tubular adenocarcinoma (9 cases) and mucinous carcinoma (5 cases). Patients with tubular adenocarcinoma had a poor prognosis compared to non‐invasive papillary tumor or papillary tumor with mucinous carcinoma. These pathological characteristics and the survival status of biliary papillary tumors were different from those of non‐papillary cholangiocarcinoma, and rather closely resembled those of IPMN‐P. In conclusion, biliary papillary tumors may be the biliary counterpart (intraductal papillary neoplasm of the bile duct) of IPMN‐P. (HEPATOLOGY 2006;44:1333–1343.)

Biliary cystic tumors with bile duct communication: a cystic variant of intraductal papillary neoplasm of the bile duct.

Biliary cystic tumors, which are also called biliary cystadenoma and cystadenocarcinoma, are thought to be a heterogeneous disease entity, and some of them are known to show a luminal communication to the bile duct. In this study, we examined the clinicopathological features of nine cases of biliary cystic tumors with bile duct communication. They were composed of five males and four females with an average age of 67 years (52–84 years). They were multilocular (eight cases) or unilocular (one case), and all cases contained mucinous fluid. A direct luminal communication with the bile ducts was identified in five cases on preoperative or intraoperative cholangiographies. Biliary cystic tumors examined in this study were histologically adenoma (one case), adenocarcinoma in situ (six cases), and adenocarcinoma associated with microinvasive mucinous carcinoma (two cases). One case of adenocarcinoma in situ also had the adenoma component (adenocarcinoma in adenoma). Dysplastic mucinous epithelium proliferated in flat, micropapillary and papillary fashions within the intracystic spaces. Intraepithelial neoplasm was observed within non-dilated adjacent bile ducts, suggesting a direct luminal communication between the cystic tumors and the bile duct. Ovarian-like stroma was not observed in their walls in any cases. Immunohistochemically, seven cases expressed MUC1 or MUC2 in the neoplastic biliary epithelium. All cases except one were alive without any evidences of tumor recurrence after total excision (3–156 months after surgery). These clinicopathological features resembled those of intraductal papillary neoplasm of the bile duct, which had been reported as a biliary counterpart of pancreatic intraductal papillary mucinous neoplasm. In conclusion, biliary cystic tumors with bile duct communication could be regarded as intraductal papillary neoplasm with a prominent cystic dilatation of the bile duct and mucin retention, rather than true biliary cystic neoplasms.

The overexpression of polycomb group proteins Bmi1 and EZH2 is associated with the progression and aggressive biological behavior of hepatocellular carcinoma

Polycomb-group proteins Bmi1 and EZH2 are involved in the malignant transformation and biological aggressiveness of several human carcinomas. We herein examined the significance of the Bmi1 and EZH2 expression in hepatocellular carcinoma (HCC) and its preneoplastic lesions, dysplastic nodules. The expression of Bmi1 and EZH2 were examined immunohistochemically in HCC (n=27) and dysplastic nodules (n=14), and combined hepatocellular and cholangiocarcinoma (HC-CC) (n=14). The effect of Bmi1 and EZH2 knockdown was examined in cultured HCC cells (HuH7 and HepG2) using siRNA. It was determined that Bmi1 was constantly expressed in cholangiocytes, but not in hepatocytes, and EZH2 was detected in neither cholangiocytes nor hepatocytes. Bmi1 and EZH2 were overexpressed in HCC and more extensively in HC–CC (P<0.01). Interestingly, Bmi1 and EZH2 were not overexpressed in the dysplastic nodules. The expression of Bmi1 and EZH2 was heterogeneous and associated with vascular infiltration, the histological grades, and the cell proliferation activity in HCC and HC–CC. In cultured carcinoma cells overexpressing Bmi1 and EZH2, knockdown of Bmi1 and EZH2 resulted in decreased cell proliferation activities. Therefore, the overexpression of polycomb-group proteins Bmi1 and EZH2 is associated with the malignant progression of HCC, thereby reflecting the aggressive biological behavior in HCC and HC–CC.

Over‐expression of polycomb group protein EZH2 relates to decreased expression of p16INK4a in cholangiocarcinogenesis in hepatolithiasis

Polycomb group protein EZH2 and Bmi1 are reportedly involved in the progression of malignant tumours. We examined the participation of EZH2 in multi‐step cholangiocarcinogenesis in hepatolithiasis with respect to tumour suppressor gene p16INK4a. We examined 20 hepatolithiatic livers with intrahepatic cholangiocarcinoma (ICC) and 10 histologically normal livers. Neoplastic biliary lesions were classified into biliary intraepithelial neoplasm (BilIN‐1, 2 and 3) and invasive carcinoma. We selected 15 foci of invasive carcinoma, 8 BilIN‐3 (carcinoma in situ), 12 BilIN‐2 (high‐grade dysplasia), 32 BilIN‐1 (low‐grade dysplasia) and 37 non‐neoplastic biliary epithelia from these livers. Expression of p16INK4a, EZH2 and Bmi1 were surveyed in these foci. P16INK4a promoter methylation was examined in microdissected tissues. Taking advantage of two cell lines of CC (HuCTT‐1 and TFK‐1) and small interfering RNA (siRNA), the effects of the knockdown of EZH2 on p16INK4a methylation of CC cells were examined. Expression of p16INK4a, which was frequent in BilIN1, was decreased in BilIN‐2/3 and invasive carcinoma, while EZH2 expression showed step‐wise increase from BilIN‐1, ‐2 and ‐3 to invasive carcinoma (p < 0.01). P16INK4a promoter hypermethylation was related to aberrant expression of EZH2. The knockdown of EZH2 in cultured CC cells decreased p16INK4a methylation and decreased binding of EZH2 to the p16INK4a gene promoter. The latter suggested that direct binding of EZH2 is involved in the regulation of the p16INK4a gene. Our data suggest that over‐expression of EZH2 may induce hypermethylation of p16INK4a promoter followed by decreased expression of p16INK4a in the multi‐step cholangiocarcinogenesis through intraepithelial neoplasm in hepatolithiasis. Copyright

Innate immune response to double-stranded RNA in biliary epithelial cells is associated with the pathogenesis of biliary atresia†

Infections of Reoviridae consisting of a double‐stranded RNA (dsRNA) genome are a possible cause of biliary atresia (BA). The aim of the present study is to clarify the pathophysiological function of dsRNA viruses in the pathogenesis of BA. The expression of dsRNA pattern‐recognizing receptors, Toll‐like receptor 3 (TLR3), retinoic acid inducible gene I (RIG‐I), melanoma differentiation‐associated gene‐5 (MDA‐5), and dsRNA‐activated protein kinase R (PKR) was constitutively detected in cultured human biliary epithelial cells (BECs). Stimulation with polyinosinic‐polycytidylic acid [poly(I:C), a synthetic analog of viral dsRNA] induced the activation of transcription factors [nuclear factor (NF)‐κB and interferon regulatory factor 3 (IRF3)] and the production of interferon‐β1 (IFN‐β1) and MxA as potent antiviral responses. Moreover, poly(I:C) up‐regulated the expression of tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL), and both poly(I:C) and TRAIL reduced the viability of cultured human BECs by enhancing apoptosis. Experiments in vivo using tissue sections of extrahepatic bile ducts from patients with BA and controls (choledochal cysts and nonbiliary diseases) showed that the activation of NF‐κB, interferon regulatory factor‐3 (IRF‐3), and PKR, and the enhancement of TRAIL and single‐stranded DNA (ssDNA)–positive apoptosis were significant in BA, although extrahepatic bile ducts diffusely and constantly expressed TLR3 in all diseases. Conclusion: dsRNA viruses could directly induce the expression of TRAIL and apoptosis in human biliary epithelial cells as a result of the biliary innate immune response, supporting the notion that Reoviridae infections are directly associated with the pathogenesis of cholangiopathies in cases of BA. (HEPATOLOGY 2007.)

Expression of cell cycle-related molecules in biliary premalignant lesions: biliary intraepithelial neoplasia and biliary intraductal papillary neoplasm

Cholangiocarcinoma of intrahepatic and extrahepatic bile ducts has a multistep carcinogenesis. Two premalignant lesions have been suggested for invasive cholangiocarcinoma: biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct. How the carcinogenetic process differs between biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct is not clear. In this study, we performed a pathological study to reveal the expression of key molecules related to the cell cycle during 2 carcinogenetic lineages. We immunohistochemically examined the expression of p21, p53, cyclin D1, and Dpc4 in a total of 89 cases: nonneoplastic biliary epithelium, biliary intraepithelial neoplasia, intraductal papillary neoplasm of the bile duct, and invasive cholangiocarcinoma. The expression of p21, p53, and cyclin D1 was up-regulated with histological progression in both biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct, whereas Dpc4 expression was down-regulated in these 2 lineages. In biliary intraepithelial neoplasia, p21 expression was significantly up-regulated early on. In contrast, levels of all molecules changed gradually in intraductal papillary neoplasm of the bile duct. Changes in p53 expression during histological progression differed significantly between biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct. p53 expression was dramatically up-regulated at the invasive stage of biliary intraepithelial neoplasia, whereas it was quite low in noninvasive biliary intraepithelial neoplasia. In contrast, p53 expression was already up-regulated in low-grade intraductal papillary neoplasm and reached a plateau in high-grade intraductal papillary neoplasm and invasive cholangiocarcinoma. This study suggested p21, p53, cyclin D1, and Dpc4 to be involved in the carcinogenesis of both biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct. p53 expression was regulated differently in biliary intraepithelial neoplasia compared with intraductal papillary neoplasm of the bile duct.

Epithelial-Mesenchymal Transition Induced by Transforming Growth Factor-β1/Snail Activation Aggravates Invasive Growth of Cholangiocarcinoma

Epithelial-mesenchymal transition is an important mechanism behind initiation of cancer invasion and metastasis. This study was performed to clarify the involvement of epithelial-mesenchymal transition in the progression of cholangiocarcinoma. Cholangiocarcinoma cell lines, CCKS-1 and TFK-1, were treated with transforming growth factor-beta1 (TGF-beta1), and the phenotypic changes and invasive activity were examined. Immunohistochemical analysis was performed using tissue sections of cholangiocarcinoma. In vitro, TGF-beta1 induced mesenchymal features in CCKS-1 and TFK-1 characterized by the reduction of E-cadherin and cytokeratin 19 expression and the induction of mesenchymal markers, such as vimentin and S100A4. TGF-beta1 also induced the nuclear expression of Snail, and the invasive activity was significantly increased in both cell lines. Studies using a mouse xenograft model showed that TGF-beta1 worsened the peritoneal dissemination of CCKS-1. All these changes by TGF-beta1 were inhibited by the simultaneous administration of soluble TGF-beta type II receptor. In vivo, six (16%) of 37 cholangiocarcinoma cases showed marked immunoreactivity of Snail in their nuclei. In these six cases, the immuno-expression of cytokeratin 19 was significantly reduced, and the expression of vimentin was significantly increased. The Snail expression significantly correlated with the lymph node metastasis and a poor survival rate of the patients. These results suggest that epithelial-mesenchymal transition induced by TGF-beta1/Snail activation is closely associated with the aggressive growth of cholangiocarcinoma, resulting in a poor prognosis.

Incidence of and risk factors for incisional hernia after abdominal surgery

Few larger studies have estimated the incidence of incisional hernia (IH) after abdominal surgery.

Immunohistochemical analysis of the progression of flat and papillary preneoplastic lesions in intrahepatic cholangiocarcinogenesis in hepatolithiasis

Background: Two types of precursor lesions, flat‐type ‘biliary intraepithelial neoplasia (BilIN)’ and papillary‐type ‘intraductal papillary neoplasm of the bile duct (IPNB)’, are proposed in the tumorigenesis of intrahepatic cholangiocarcinoma (ICC) in hepatolithiasis.

Expression of matrix metalloproteinase 7 is an unfavorable postoperative prognostic factor in cholangiocarcinoma of the perihilar, hilar, and extrahepatic bile ducts

Cholangiocarcinoma of the perihilar, hilar, and extrahepatic bile ducts (collectively referred to as the large bile ducts) is an intractable disease, and a papillary phenotype and well-differentiated histologic grade have been proposed as indicators of a favorable prognosis after surgical resection. In this study, we examined the significance of matrix metalloproteinases (MMPs) in cholangiocarcinoma with respect to clinicopathologic features. We subjected 66 surgically resected specimens of cholangiocarcinoma of the large bile ducts to clinicopathologic examination, including postoperative survival, papillary phenotype, and immunohistochemical expression of MMP-2,-7, -9, and membrane type 1 MMP (MT1-MP). Nonneoplastic biliary epithelium did not express these 4 MMPs, whereas cholangiocarcinoma frequently expressed MMP-2 (33.9%), -7 (75.8%), -9 (47.5%), and MT1-MMP (54.5%). In particular, conventional (nonpapillary) cholangiocarcinoma expressed MMP-7 and MT1-MMP more frequently than papillary cholangiocarcinoma. The expression of MMP-7 and MT1-MMP significantly correlated with the nonpapillary phenotype, poorly differentiated histologic grade, perineural invasion, and advanced TNM stage. In contrast, the expression of MMP-2 and -9 was not associated with any of the clinicopathologic features. Univariate analysis of disease-specific survival revealed that a papillary phenotype and expression of MMP-7 were prognostic factors of cholangiocarcinoma, in addition to TNM stage, poorly differentiated histologic grade, perineural invasion, and microscopic margin status. Multivariate analysis showed only TNM stage to be an independent prognostic factor. Expression of MMP-7 in cholangiocarcinoma is an unfavorable postoperative prognostic factor of cholangiocarcinoma arising from the large bile ducts. Underexpression of MMPs in papillary cholangiocarcinoma might be associated with a favorable prognosis.