Extracellular vesicle noncoding RNA: New players in the diagnosis and pathogenesis of cholangiocarcinoma
Authors: Tusher Patel
Journal: Hepatology (2014)

Several decades have passed since the recognition and initial description of the release of extracellular vesicles (EVs) from cells, but their characterization as potential biomarkers of disease is only now gaining momentum. Recent studies have reported that a variety of EVs, such as exosomes and microvesicles, can be isolated from several types of body fluids. The ability to characterize the content and identify disease-specific molecules, such as proteins, messenger RNA, or noncoding RNA that are expressed on the surface or present within these membrane bound vesicles, provides the rationale for their potential value as biomarkers of disease.1, 2 Reports of the identification of EVs within bile and their isolation set the stage for analysis of their biomarker potential for biliary tract diseases.3 By examining the microRNA (miRNA) content within EVs isolated from bile from 3 patients with cholangiocarcinoma (CCA) and 3 controls, Li et al. identified a panel of five miRNAs (191, 486-3p, 1274b, 16, and 484) with potential clinical utility as an accurate biomarker for CCA.4 A sensitivity of 67% and specificity of 96% was reported for diagnosis of a mixed group of 14 patients with intrahepatic, 5 patients with distal, and 27 patients with perihilar CCA, compared to a heterogeneous group of 50 controls with nonmalignant diseases. The early diagnosis of malignancies that arise from the large biliary ductal system, such as perihilar or distal CCA, is difficult.5 When these occur in the setting of biliary tract strictures, it is among the most perplexing and challenging aspects of medical practice. Current approaches, such as bile duct biopsy or brushings of strictures during endoscopic or radiologic procedures, are limited by poor cell yields as a consequence of the desmoplastic nature of the tumor. Attempts to improve the diagnostic yield of cytology, such as by the use of fluorescent in situ hybridization, have had modest effect, but continue to be refined. Indeed, existing approaches may be negative in up to 50% of patients with malignancy. By the time more definite signs of cancer arise, tumors may have progressed with spread that precludes curative intervention. Circulating tumor markers, such as carbohydrate antigen 19-9 (CA 19-9), are not specific and have not been helpful. An alternative approach has been to examine potential biomarkers within bile. Sampling of bile for detection of tumor-related biomarkers is appealing for several reasons. First, bile can be obtained at the time of endoscopic retrograde cholangiopancreatography that may be performed for diagnosis or therapy of biliary tract strictures or suspected biliary tract disease. Next, stricturing disease may enhance local concentrations of potential biomarkers that are secreted or released from tumor cells. Concentrations of potential markers may be increased in bile because of either impaired bile flow as a result of strictures or smaller sampling volume of distribution when compared with the systemic circulation. Furthermore, local sampling may have a greater likelihood of detecting candidate biomarkers that are directly related to the tumor. Finally, local sampling may enhance specificity, when compared with circulating biomarkers, by minimizing the potential for nonspecific release from other cells and tissues. Proteomic analysis has identified several biomarkers within bile, including proteins such as CA19-9, neutrophil gelatinase-associated lipocalin, carcinoembryonic cell adhesion molecule 6, S100, and RNU2-1f.6-8 The diagnostic utility of several of these markers in bile is superior to that of the same markers in serum. Markers such as biliary vascular endothelial growth factor 1 may also be useful to exclude CCA in the distal common bile duct. Although the study by Li et al. is not the first report of the identification of miRNA within bile,9 or even of their potential as biomarkers, their analysis involved a systematic examination of analysis of miRNA within EVs obtained from bile and resulted in the development of a predictive marker comprised of 5-miRNA that has an improved accuracy for the diagnosis of malignancy within the biliary tract, compared with conventional approaches. Their study represents a joint effort involving individuals from several institutions that highlight the advantage and value of such collaborative efforts for ensuring progress in uncommon conditions such as CCA, where the experience in a single center may be limited. These studies emphasize the utility of bile as a biofluid as well as the analysis of EVs for biomarker studies. There are several challenges and hurdles to be overcome before integration of these findings into clinical practice. The effect of bile on either the stability of EVs or on the integrity of miRNAs are not known. Bile collection and sampling time with respect to timing of biliary drainage procedures, stability, storage, and the presence of biliary infection are potential confounders that can influence biliary protein concentrations. Whether or not these factors may modulate the release of EVs, their specific RNA content or relative RNA concentrations remain to be examined. This is a critical issue for the use of a biomarker assay based on the normalization to input volume, as reported by Li et al. Normalization to endogenous invariant controls would be an optimal choice, but data to guide these are lacking. Although small RNA U6 may not be an appropriate choice for analysis of circulating miRNA, we have identified low variance of RNU6B expression within EVs that are released from normal, as well as malignant, hepatocytes and cholangiocytes. In addition to these issues, it is necessary to precisely define the nomenclature and ontology of EVs. There exists an extensive literature on the role of microvesicles and microparticles in liver and other diseases. However, these studies encompass a very limited population of EVs. The methodologies used for the majority of these studies do not detect all EVs, or may include nonvesicular proteins, and may thereby exclude other EVs that may have biomarker potential or contribute to functional biological effects. Furthermore, standardization of ontology, as well as of techniques for the identification and characterization of EVs, are needed. Ongoing efforts to do so are being conducted under the auspices of the National Institutes of Health Common Fund–supported Extracellular RNA consortium and other international groups. Cancer-related miRNA signatures may have clinical utility as markers of disease prognosis. Indeed, circulating miRNAs have been implicated in several biological processes that may be involved and contribute to disease prognosis. Similarly, future studies should be considered to examine the potential for biliary miRNAs as prognostic biomarkers for CCA (Fig. 1). In addition to their biomarker potential, the study of microRNA as well as other noncoding RNA within EVs has implications for understanding disease pathobiology. Uptake of EVs and release of their biologically active contents by recipient cells provides a mechanism by which cells can interact with each other. Study of intercellular signaling within the local cellular microenvironment has previously been focused on protein mediators, such as cytokines and other biologically active factors. The ability of EVs to transfer biologically active RNA molecules, such as microRNA, across cells provides a potent mechanism for intercellular interactions that could be equally as important. Deregulated expression of microRNA in CCA has now been shown in several studies and justifies further study of the contribution of their transfer by EVs on disease pathogenesis and spread. Indeed, EV noncoding RNA content has been reported to vary between malignant and nonmalignant hepatocytes. Noncoding RNA that are selectively enriched within EVs in malignant cells, such as long non-coding RNA regulator of reprogramming, have been shown to modulate cellular responses to hypoxia.10 The concept that EV RNA can act as signals to modulate the phenotype within target cells is a novel and emerging paradigm in intercellular signaling. This concept has profound implications for understanding the role of intercellular interactions involved in promoting CCA formation or growth, as well as for other biliary tract diseases. Studies to elucidate the mechanisms by which noncoding RNA within EVs are released from tumor cells into bile and their biological relevance are warranted. Despite several advances in understanding the molecular pathogenesis of CCA in recent years, their early diagnosis remains an extremely vexing clinical challenge. The release of noncoding RNA within EVs into bile may be useful for early diagnosis of malignancy in addition to subserving an important functional biological effect as a mechanism of signaling that mediates cholangiocyte responses to growth stimuli. Further studies to examine other tumor-associated markers, such as gene mutations in isocitrate dehydrogenase 1 and 2, BRCA1 associated protein-1 (ubiquitin carboxy-terminal hydrolase), or fibroblast growth factor receptor 2 fusions within EVs, may allow us to further improve sensitivity for early diagnosis of CCA. Tushar Patel, M.B.Ch.B. Departments of Transplantation and Cancer Biology Mayo Clinic Jacksonville, FL