Identification of Novel Biomarker Candidates for the Immunohistochemical Diagnosis of Cholangiocellular Carcinoma

Abstract

The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell-type-specific expression profile via immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma, and stress-induced phosphoprotein 1 in tumorous cholangiocytes relative to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Because stress-induced phosphoprotein 1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as a potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases, as well as metastatic malignancies in the liver. The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell-type-specific expression profile via immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma, and stress-induced phosphoprotein 1 in tumorous cholangiocytes relative to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Because stress-induced phosphoprotein 1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as a potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases, as well as metastatic malignancies in the liver. Cholangiocellular carcinoma (CCC) 1The abbreviations used are:CCCcholangiocellular carcinoma2D-DIGEtwo-dimensional differential in-gel electrophoresisAUCarea under the curveABAT4-aminobutyrate aminotransferase, mitochondrialAPOA4apolipoprotein A-IVBHMTbetaine-homocysteine S-methyltransferase 1CIconfidence intervalFABP1fatty acid–binding protein 1SFN14-3-3 protein σSTIP1stress-induced phosphoprotein 1. 1The abbreviations used are:CCCcholangiocellular carcinoma2D-DIGEtwo-dimensional differential in-gel electrophoresisAUCarea under the curveABAT4-aminobutyrate aminotransferase, mitochondrialAPOA4apolipoprotein A-IVBHMTbetaine-homocysteine S-methyltransferase 1CIconfidence intervalFABP1fatty acid–binding protein 1SFN14-3-3 protein σSTIP1stress-induced phosphoprotein 1. is a malignant neoplasm that arises from the cholangiocytes, the epithelial cells lining the bile ducts. The tumors, consisting of a significant amount of fibrous stroma, are classified as intrahepatic, extrahepatic, or hilar according to their anatomic location. Most common are the Klatskin tumors, originating from the confluence of the right and left hepatic ducts (1.Khan S.A. Davidson B.R. Goldin R. Pereira S.P. Rosenberg W.M. Taylor-Robinson S.D. Thillainayagam A.V. Thomas H.C. Thursz M.R. Wasan H. British SocietyGuidelines for the diagnosis and treatment of cholangiocarcinoma: consensus document.Gut. 2002; 51: VI1-VI9Crossref PubMed Scopus (565) Google Scholar). Compared with other types of cancer, CCC is a relatively rare disease, accounting for about 3% of all gastrointestinal malignancies (2.Patel T. Cholangiocarcinoma.Nat. Clin. Pract. Gastroenterol. Hepatol. 2006; 3: 33-42Crossref PubMed Scopus (238) Google Scholar). However, its incidence is increasing, and as a result of poor patient outcomes it has overtaken hepatocellular carcinoma as the main cause of death from a primary hepatobiliary tumor (3.Khan S.A. Taylor-Robinson S.D. Toledano M.B. Beck A. Elliott P. Thomas H.C. Changing international trends in mortality rates for liver, biliary and pancreatic tumours.J. Hepatol. 2002; 37: 806-813Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). Reasons for the high mortality rate (5-year survival rate of about 5%) (4.Lazaridis K.N. Gores G.J. Cholangiocarcinoma.Gastroenterology. 2005; 128: 1655-1667Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar) are the difficult diagnosis and limited treatment options. At present, extensive surgical resection of the extrahepatic bile ducts and parts of the liver or liver transplantation remain the only potentially curative treatment options, although most patients are considered inoperable at the time of diagnosis (5.Khan S.A. Thomas H.C. Davidson B.R. Taylor-Robinson S.D. Cholangiocarcinoma.Lancet. 2005; 366: 1303-1314Abstract Full Text Full Text PDF PubMed Scopus (1007) Google Scholar). cholangiocellular carcinoma two-dimensional differential in-gel electrophoresis area under the curve 4-aminobutyrate aminotransferase, mitochondrial apolipoprotein A-IV betaine-homocysteine S-methyltransferase 1 confidence interval fatty acid–binding protein 1 14-3-3 protein σ stress-induced phosphoprotein 1. cholangiocellular carcinoma two-dimensional differential in-gel electrophoresis area under the curve 4-aminobutyrate aminotransferase, mitochondrial apolipoprotein A-IV betaine-homocysteine S-methyltransferase 1 confidence interval fatty acid–binding protein 1 14-3-3 protein σ stress-induced phosphoprotein 1. In general, the diagnosis of CCC is made based on histomorphological evaluation of core biopsies or cytological specimens. However, distinction between CCC and benign diseases such as reactive bile ductules or bile duct adenomas can be challenging when based on conventional histology alone. Additionally, it may be difficult to distinguish CCC from metastatic adenocarcinoma in the liver, especially when it originates from the pancreas like pancreatic ductal adenocarcinoma. Therefore, specific immunohistochemical tissue markers for CCC would be highly beneficial for further validation of the diagnosis. In routine immunohistochemical diagnosis of CCC, so far, the detection of p53 (a product of a tumor suppressor gene) has proven useful, although its application is limited because of low sensitivity (6.Riener M.O. [Diagnosis of tumours of the liver and the biliary tract: new tissue and serum markers].Pathologe. 2011; 32: 304-309Crossref PubMed Scopus (9) Google Scholar). The cytokeratins Ck7, Ck8, Ck18, and Ck19 have been reported to have sensitivities of between 80% and 97% for CCC cells, but at low specificities and a similar expression as in non-tumorous cholangiocytes (7.Langer F. von Wasielewski R. Kreipe H.H. [The importance of immunohistochemistry for the diagnosis of cholangiocarcinomas].Pathologe. 2006; 27: 244-250PubMed Google Scholar). In addition, the tumor marker carcinoembryonic antigen, which is a commonly applied serum marker, has been used for immunohistochemical staining of CCC tissue. Although this was reported to be positive in 100% of the tested CCC sections, it also was immunoreactive in 60% of hepatocellular carcinomas (8.Balaton A.J. 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