The αvβ6 integrin is a highly specific immunohistochemical marker for cholangiocarcinoma

doi:10.1016/j.jhep.2009.12.006

Journal of Hepatology

Volume 52, Issue 3, March 2010, Pages 362-369

https://doi.org/10.1016/j.jhep.2009.12.006 Get rights and content

Background & Aims

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are common primary hepatic malignancies. Their immunohistological differentiation using specific markers is pivotal for treatment and prognosis. We found αvβ6 integrin strongly upregulated in biliary fibrosis, but its expression in primary and secondary liver tumours is unknown. Here, we aimed to evaluate the diagnostic applicability of αvβ6 integrin in differentiating primary liver cancers.

Methods

Expression of αvβ6 integrin was evaluated in liver tissues from patients with CC, HCC, fibrolamellar HCC, combined CC/HCC, hepatic metastases of colorectal and pancreatic carcinomas, primary sclerosing cholangitis (PSC), and in human primary and tumour-derived liver cell lines by immunohisto- and cytochemistry, and by TaqMan PCR. Diagnostic performance of the β6 subunit was compared with CK7, CK20, and HepPar 1.

Results

In CC cells β6 mRNA levels were induced 125-fold compared to primary cholangiocytes, while it was completely absent in hepatoma cells. In human tissues, β6 transcripts were more than 100-fold upregulated in CC compared to normal liver. By immunohistochemistry, 88% of CC, 50% of PSC, 13% of colorectal carcinoma metastases, and 80% of pancreatic carcinoma metastases presented αvβ6, whereas all HCC, combined CC/HCC and fibrolamellar HCC stained negative. Specificity of β6 immunohistochemistry for CC (100%) surpassed all other tested markers and sensitivity was equal to CK7 (86% vs. 90%).

Conclusion

The αvβ6 integrin is strongly expressed in human CC but not in HCC and therefore can be considered as a specific immunohistochemical marker in the differential diagnosis of primary liver tumours.

Introduction

Cholangiocarcinoma (CC) is among the most deadly malignancies in the gastrointestinal tract with a rising incidence worldwide for as of yet unknown reasons. Treatment options are discouragingly limited and a cure can only be achieved with timely and radical surgical resection or liver transplantation in highly selected cases [1], [2]. Although radio- and chemotherapy provide palliation for patients with unresectable disease, they fail to prolong patient survival [3], [4]. Therefore, early and precise diagnosis remains crucial. Diagnostic markers that allow timely detection of CC as well as its differentiation from other primary and secondary malignant liver lesions are too unspecific. Particularly the distinction between CC and hepatocellular carcinoma (HCC) can be difficult due to morphological similarities [5], [6], [7], [8]. In addition, commonly used immunohistochemical markers for CC and HCC such as cytokeratins (CK)-7, -20, and Hepatocyte Paraffin 1 (HepPar 1) have limited specificity as they can be expressed by both cancers [9], [10]. Therefore, novel diagnostic markers with better diagnostic performance are required.

Integrins comprise a large family of cell-surface glycoproteins which consist of α and β subunits and that regulate cell adhesion, migration, proliferation and apoptosis [11]. Changes in integrin expression and/or function are directly involved in tumour growth, metastatic spread, and angiogenesis, rendering these receptors promising diagnostic markers and potential therapeutic targets [12], [13], [14], [15], [16].

Alpha v beta 6 (αvβ6) integrin is exclusively expressed on epithelial cells and upregulated during morphogenesis, tumourigenesis, epithelial repair and experimental and human liver fibrosis [17], [18], whereas it is not constitutively expressed in normal tissues [19], [20]. Since the β6 subunit of the entire heterodimer only combines with the ubiquitously expressed αv chain, its synthesis is rate limiting for αvβ6 expression [17]. Therefore, measurement of the β6 subunit reflects the entire molecule.

Recently, αvβ6 integrin has been described as a marker for ongoing epithelial-to-mesenchymal transition (EMT), which is considered to be a crucial step in the development of several tumours [21]. The αvβ6 integrin was found expressed in pancreatic, gastric, lung, duodenal and colorectal adenocarcinomas [22], and indicated aggressive tumour growth [23], [24], [25]. Since the role of αvβ6 in primary liver tumours has not been studied yet, we investigated its expression in primary benign and malignant liver cells and in a panel of primary and secondary liver cancers to evaluate the potential of αvβ6 integrin as a diagnostic marker in the differential diagnosis of malignant liver lesions.

Section snippets

Cell culture

Human CC (TFK-1) [26] and HCC cells (HepG2; DSMZ, Braunschweig, Germany), and immortalized human cholangiocytes (MMNK-1; kindly donated by Dr. N. Kobayashi, Dept. of Surgery, Okayama, Japan) [27] were cultured in RPMI medium containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin. Cells were maintained at 37 °C in a 5% CO₂ humidified atmosphere. Primary bile duct epithelial cells (BDEC) were isolated from human liver resections and cultured as described [28].

Human samples

β6 Expression in normal and tumour-derived cell lines

Expression of the β6 integrin was measured at mRNA and protein levels in human primary BDEC, immortalized BDEC (MMNK-1), CC cells (TFK-1), and hepatoma cells (HepG2). A 125-fold increase of the β6 mRNA was detected in TFK-1 compared to immortalized and primary BDEC. In HepG2 cells β6 expression was barely detectable (Fig. 1A). Immunocytochemistry corresponded to mRNA levels as TFK-1 showed strong β6 immunoreactivity, whereas MMNK-1 and HepG2 stained negative (Fig. 1B–D).

β6 mRNA expression in primary liver tumours

Quantification of the β6

Discussion

A number of immunohistochemical markers are established for routine histopathology assessment of HCC and CC including several cytokeratins, HepPar 1 and others [5], [6], [7], [8], [9], [12]. However, none of these markers are exclusively specific for either CC or HCC. Overlapping or loss of several markers during neoplastic transformation frequently occurs, rendering a clean distinction between the two tumours and a precise diagnosis difficult [32], [33].

Integrin αvβ6 is expressed by activated

Acknowledgements

The authors who have taken part in this study declared that they do not have anything to declare regarding funding from industry or conflict of interest with respect to this manuscript. This work was supported by Grant 3100 A0 – 122114/1 from the Swiss National Fonds and by the Werner- and Hedy-Berger-Foundation for Cancer Research (Grant 1/2007).

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Research ArticleThe αvβ6 integrin is a highly specific immunohistochemical marker for cholangiocarcinoma

Background & Aims

Methods

Results

Conclusion

Introduction

Section snippets

Cell culture

Human samples

β6 Expression in normal and tumour-derived cell lines

β6 mRNA expression in primary liver tumours

Discussion

Acknowledgements

Lancet

J Hepatol

Cell

Hum Pathol

Gastroenterology

J Hepatol

Biochem Biophys Res Commun

Oral Oncol

Pathol Res Pract

Hum Pathol

J Hepatol

J Hepatol

Am J Pathol

Lab Invest

Cholangiocarcinoma

Nat Clin Pract Gastroenterol Hepatol

Guidelines for the diagnosis and treatment of cholangiocarcinoma: consensus document

Gut

The significance of alpha-fetoprotein and other tumour markers in differential immunocytochemistry of primary liver tumours

Histopathology

Histologic markers in primary and metastatic tumors of the liver

Cancer

Blood-group-related antigen Lewis(x) and Lewis(y) in the differential diagnosis of cholangiocarcinoma and hepatocellular carcinoma

Arch Pathol Lab Med

Well-differentiated cholangiocarcinoma: diagnostic significance of morphologic and immunohistochemical parameters

Am J Surg Pathol

Research Article
The αvβ6 integrin is a highly specific immunohistochemical marker for cholangiocarcinoma