The challenge of cholangiocarcinoma diagnosis: The turning point is in extracellular vesicles?
Authors: Domenico Alvaro
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. See Article on Page 1125 Extracellular vesicles (EVs) are membrane‐derived vesicles of nanometer size, released by normal and diseased cells into the extracellular milieu.1 It is now evident that EVs are involved, together with cell‐to‐cell contact and soluble factors, in modulating intercellular communications.1 EVs have been the object of extensive investigations during recent years and have been identified in virtually all body fluids. They are classified into three groups based on their cellular biogenesis: exosomes, microvesicles, and apoptotic bodies. Exosomes (40‐150 nm in diameter) derive from intracellular trafficking through the endolysosomal pathway.1 EVs are released in a regulated and/or domain‐specific manner and participate in many important biological processes other than being involved in disease pathogenesis. Once released, EVs modulate the behavior of neighboring cells through either paracrine signals or horizontal transfer of bioactive agents. The bulk of the recent literature deals with the role of EVs in oncology because they are aberrantly produced by cancer cells and may convey oncogenic factors that are able to induce tumorigenic conversion and promote tumor growth, survival, and spreading.1 In addition, it was shown how tumors may modulate their own niche through EVs, by which the immune system can be suppressed while tumor neo‐angiogenesis is activated.3 In this issue of hepatology, Arbelaiz et al.5 evaluated the serum concentration of EVs and performed a careful analysis of the protein content in patients with cholangiocarcinoma (CCA), primary sclerosing cholangitis (PSC), and hepatocellular carcinoma. Without large differences among the investigated groups in their serum concentration, EVs, unequivocally characterized as exosomes, contain several proteins with high diagnostic values for discriminating between CCA, PSC, hepatocellular carcinoma, and normal subjects specifically. Therefore, the proteomic signatures of serum EVs detected in CCA, PSC, and hepatocellular carcinoma patients show potential usefulness as diagnostic tools. As far as CCA is concerned, it is of particular note that the authors5 demonstrated how several of the best biomarker candidates for the differential diagnosis of CCA versus PSC showed higher diagnostic values in the comparison between early‐stage CCA (stages I and II) and PSC. In particular, ficolin‐2, inter‐alpha‐trypsin inhibitor heavy chain H4, and fibrinogen gamma chain showed a better diagnostic value with respect to carbohydrate antigen 19‐9, especially when CCA stage I‐II was compared with PSC. Interestingly, and as a confirmation that the EV cargo reflects the pathobiology of CCA, the authors demonstrated that CCA cell‐derived EVs contain a number of proteins previously identified as CCA prognostic factors that are up‐regulated in CCA cells or that drive important carcinogenic processes in CCA cells, such as epidermal growth factor receptor, integrin beta‐4, agrin, epithelial cell adhesion molecule, epidermal growth factor receptor kinase substrate 8, and tumor‐associated calcium signal transducer 2. This is an additional clue to the accumulating evidence that EVs reflect the physiopathological state of the cells by which they are released and can be important mediators for cancer progression.1 From a pathogenic point of view, direct cell‐to‐cell transfer of oncogenic proteins through exosomal pathway has been proposed as a novel mechanism for CCA progression and metastasis.6 In addition, Haga et al.7 have demonstrated how CCA‐derived EVs may modulate the cellular behavior of tumor stroma to promote tumor cell growth. Specifically, CCA‐derived EVs suspended in culture medium induced fibroblastic differentiation of mesenchymal stem cells, which in turn displayed enhanced secretion of soluble factors that promoted CCA cell proliferation. As polarized cells, cholangiocytes release EVs into bile from their apical membranes (in close contact with primary cilia), where the intercellular communication involves cholangiocytes located downstream in the biliary tree (Fig. 1). In contrast, EVs released from the cholangiocyte basolateral pole into the interstitial space and/or the peribiliary plexus target for intercellular communication cholangiocytes located upstream in the biliary tree or hepatocytes. The EV cargo in the two distinct EV populations (i.e., basolateral and apical) is evidently different as a large difference exists between the protein content of EVs released by normal cholangiocytes and cholangiocytes involved in chronic inflammation (i.e., PSC) or neoplastic transformation (i.e., CCA). Of note, this study5 identified a differential EV protein cargo between human CCA cells and normal cholangiocytes in vitro, as well as their particular apical versus basolateral secretion pattern.Figure 1: Release of EVs (exosomes and microvesicles) from the basolateral and apical poles of cholangiocytes. Cholangiocytes release EVs into bile from their apical membrane, where prima cilia are seen and where the intercellular communications involve cholangiocytes located downstream in the biliary tree. On the opposite, exosomes and microvesicles released from the cholangiocyte basolateral pole into the interstitial space and/or the peribiliary plexus target for intercellular communication cholangiocytes located upstream in the biliary tree or hepatocytes. The cargo of two distinct EV populations (i.e., basolateral or apical) is different as a large difference exists between the protein content of EVs released by normal cholangiocytes and cholangiocytes involved in chronic inflammation or neoplastic transformation. Abbreviations: MVB, multivesicular bodies; N, nucleus; PBP, peribiliary plexus.In the setting of a CCA diagnosis, the most challenging clinical scenario concerns the identification of malignant strictures in PSC patients or the differential diagnosis between benign and malignant strictures in non‐PSC patients. As far as PSC is concerned, there is no evidence‐based method for patient surveillance in relation to CCA emergence.8 The most widely used biomarker, carbohydrate antigen 19‐9, shows a frustratingly low sensitivity, particularly for small cancers. Additional noninvasive biomarkers taken alone or in conjunction with carbohydrate antigen 19‐9 scarcely improve the accuracy for CCA early diagnosis.8 Imaging techniques including magnetic resonance and positron emission tomography tend to have poor sensitivity and specificity. Usually these patients are submitted to endoscopic retrograde cholangiopancreatography where brush cytology, pinch biopsies, cholangioscopy, and/or confocal microscopy are performed; but the added benefits are questionable even if combined with specialized tests including fluorescent in situ hybridization for chromosomal abnormalities.8 In substance, there is no evidence that current approaches to surveillance for CCA are effective at prolonging PSC patient survival.8 A similar approach (endoscopic retrograde cholangiopancreatography plus other diagnostic tools) is followed in non‐PSC patients presenting with isolated bile duct stenosis where a differential diagnosis of benign versus malignant nature of the stricture is requested. The hope emerging from current investigations concerning serum EVs is that, very soon, a noninvasive method could allow detection of CCA at an early stage or, even better, could represent an effective tool for the surveillance of these patients. In this setting, additional inputs come from studies of EVs in bile. Indeed, a few years ago, Li et al.9 detected EVs in human bile and demonstrated that they contain abundant microRNA species. Using multivariate organization of combinatorial alterations, these authors9 defined a biliary vesicle microRNA‐based panel for CCA diagnosis, which demonstrated high sensitivity and specificity. Most importantly these findings were controlled versus patients with PSC or with biliary obstruction of varying etiologies (benign biliary stricture, papillary stenosis, choledocholithiasis, extrinsic compression from pancreatic cysts, and cholangitis). Therefore, EVs in human bile may serve as an effective tool for the diagnosis of CCA because they contain a CCA‐specific panel of microRNAs. More recently, Severino et al.10 evaluated the concentration of EVs in bile of patients with malignant and nonmalignant common bile duct stenosis. Specifically, in bile samples collected from patients undergoing therapeutic endoscopic retrograde cholangiopancreatography and with a diagnosis of malignant (pancreatic cancer or CCA) or nonmalignant stenosis, the authors10 demonstrated that the concentration of EVs in bile samples absolutely discriminates between patients with malignant and nonmalignant common bile duct stenosis. Indeed, the authors10 were able to correctly diagnose CCA in all of the patients included in the verification cohort with a diagnostic accuracy of 100%. Summing up, the quantitative and qualitative analyses of EVs in serum or bile could represent an important diagnostic advance in the field of neoplastic cholangiopathies. Of course, a methodology for EV detection and for the analysis of their cargo that is more accessible to common laboratories could represent a further desirable advance for these discoveries. In this sense, there is an important need both to validate promising EV‐related biomarker candidates for CCA found in serum and bile in larger and independent cohorts and to implement new methods for identifying these biomarkers in a hospital setting with assumable cost‐effectiveness in routine patient care. Author names in bold designate shared co‐first authorship.