Cholangiocarcinoma (CCA) presents significant diagnostic challenges, resulting in late patient diagnosis and poor survival rates. Primary sclerosing cholangitis (PSC) patients pose a particularly difficult clinical dilemma because they harbor chronic biliary strictures that are difficult to distinguish from CCA. MicroRNAs (miRs) have recently emerged as a valuable class of diagnostic markers; however, thus far, neither extracellular vesicles (EVs) nor miRs within EVs have been investigated in human bile. We aimed to comprehensively characterize human biliary EVs, including their miR content. We have established the presence of extracellular vesicles in human bile. In addition, we have demonstrated that human biliary EVs contain abundant miR species, which are stable and therefore amenable to the development of disease marker panels. Furthermore, we have characterized the protein content, size, numbers, and size distribution of human biliary EVs. Utilizing multivariate organization of combinatorial alterations (MOCA), we defined a novel biliary vesicle miR-based panel for CCA diagnosis that demonstrated a sensitivity of 67% and specificity of 96%. Importantly, our control group contained 13 PSC patients, 16 with biliary obstruction of varying etiologies (including benign biliary stricture, papillary stenosis, choledocholithiasis, extrinsic compression from pancreatic cysts, and cholangitis), and 3 with bile leak syndromes. Clinically, these types of patients present with a biliary obstructive clinical picture that could be confused with CCA. Conclusion: These findings establish the importance of using extracellular vesicles, rather than whole bile, for developing miR-based disease markers in bile. Finally, we report on the development of a novel bile-based CCA diagnostic panel that is stable, reproducible, and has potential clinical utility. (Hepatology 2014;60:896–907)
Cholangiocarcinoma (CCA) is a cancer that arises in the biliary tree. Anatomically, CCA is divided into intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA) tumors. Surgery is the only curative option. Unfortunately, because of the nonspecific nature of symptoms, as well as to failure of currently available tests, patients are usually diagnosed late in disease progression, when they are no longer surgical candidates. All three types of CCA present diagnostic dilemmas. For example, the diagnosis of iCCA is, in part, based on lack of liver cirrhosis and absence of any other known primary solid tumors. However, iCCA can also develop in cirrhotic livers, and a small size iCCA arising in a cirrhotic liver may mimic hepatocellular carcinoma (HCC) in terms of its rapid uptake of contrast material. Diagnosing pCCA is equally difficult, despite a variety of available diagnostic tools, including magnetic resonance imaging (MRI), computed tomography (CT), endoscopic retrograde cholangiopancreatography (ERCP), cholangioscopy, and endoscopic ultrasound (EUS). pCCA tends to display a strong desmoplastic reaction, which poses a significant diagnostic challenge, because obtaining cells from these lesions for cytologic examination is exceedingly difficult. Therefore, the sensitivity of cytology performed on brush biopsy specimens is, at best, only 20%. Similar to pCCA, cancers located in the distal bile duct (dCCA) display low celullarity and a strong desmoplastic reaction, rendering cytologic diagnosis very difficult.
Multiple recent studies have sought to develop more-precise markers of CCA. One such approach aimed at diagnosing CCA is based on serum proteomics. Additional studies have focused on RNA expression profiles in biliary brushings or on microRNA (miR) profiles in whole human bile. However, there are several limitations in interpreting the results from these studies. First, these studies tended to include small numbers of patients. In addition, standardization of specimen collection, specimen manipulation, and marker derivation have received inadequate attention. For example, in previous bile-based studies, there has been scant information regarding standardization of bile processing to ensure reproducible and reliable results. The issue of unreliable and/or conflicting results is paramount in cancer marker development. Therefore, it is not surprising that studies published to date present contradictory information regarding specific miR-based markers of cancer. In addition to which methodologies are best for body fluid collection, storage, and processing, there are several unanswered questions, including the appropriate reference gene(s) for normalization. Serum/plasma studies thus far have employed a variety of reference genes, including miR-16, miR-142-3p, let-7a, and small RNA U6. Whereas it is difficult to predict which of these RNAs serves as the best normalizer, it is apparent that some are worse than others. U6, in particular, which is approximately 4 times longer than any miR, should be avoided in miR-based marker panels in biologic fluids because it is less stable than miR species in body fluids and displays a different dynamic of degradation.[10, 11] Unfortunately, the only previous bile-based miR panel for CCA diagnosis employed U6 as a normalizer. Last, given the complex makeup of biologic fluids, it is naïve to hypothesize that a single RNA exhibits constant expression across various physiologic and pathologic states. Recent evidence suggests that in order to circumvent the need for an internal control, synthetic miR sequences can be spiked into biologic fluids before RNA extraction.
We hypothesized that because CCAs are in direct contact with bile, an accurate tumor-derived miR profile is more likely to exist in bile than in serum. In the current study, we present analyses of human bile geared toward developing a reliable, reproducible miR-based CCA diagnostic panel. We investigated the source of miRs in human bile, the stability of miR profiles in human bile, the best bile-processing procedures, and the most stable miR panel for diagnosing CCA from human bile.
The field of CCA is in urgent need of better diagnostic methods. Whereas the overall survival of CCA patients is dismal, there is a large discrepancy between survival of patients diagnosed early and the vast majority of patients, who are diagnosed late in their disease. The data presented herein delineate a 5-miR panel with superior diagnostic accuracy for CCA, when compared to the currently available diagnostic methods. In addition, these studies are the first to identify and characterize EVs in human bile. The presence of miR-laden EVs in human bile has physiologic, as well as potential pathologic, implications. It was recently demonstrated that HCC cells release EVs rich in miR species, which are believed to function in intrahepatic cell-cell signaling. In addition, it was shown that exosomes, a type of EVs, exist in rat bile, interact with cholangiocytes, and are able to modulate intracellular growth mechanisms. These findings suggest a new paradigm, wherein liver and biliary tree cells communicate through EVs and extracellular vesicle-transported miR species.[21, 22] Our study adds to this paradigm and is the first to put forward the hypothesis that human bile acts as a physiologic and pathologic conduit allowing the communication of information, in the form of EV-transported miR species, between various cells within the liver and biliary tree. These findings open a broad new avenue of investigation for understanding normal physiologic signaling, as well as potential implications in disease, such as CCA.
The current study furnishes strong evidence that RNA isolated from human bile derives from free-floating cells, as well as biliary EVs. Although our vesicle isolation protocol is geared toward exosome isolation through employment of differential ultracentrifugation, further studies are needed to definitely conclude that our findings are specific for exosomes, and not extracellular vesicles in general. In addition, this study establishes that RNA originating from these free-floating cells is rapidly degraded, both by storing bile at room temperature (even for 1 hour) and by a single freeze-thaw cycle. We conclude that any bile-based miR panel developed from whole, cell-containing bile will be unpredictably biased by bile processing and therefore destined to have limited clinical applicability. In contrast, we demonstrate that bile EVs contain abundant miR species that are stable and therefore usable for the development of bile-derived miR-based diagnostic panels.
Utilization of bodily fluids for the development of disease markers is appealing because these fluids can be obtained by noninvasive (urine), minimally invasive (blood), or moderately invasive (bile) procedures. Nevertheless, the mere presence of EVs in these bodily fluids, along with the fact that they contain miR species, is not sufficient to develop stable, reproducible diagnostic panels. The field of miR-based disease markers is plagued by contradictory, and often irreproducible, results. An intuitive explanation for this problem is a lack of standardization in specimen collection, storage, and processing. An additional difficulty is normalization of miR values in biologic fluids. In contrast to tissues, biologic fluids have little long RNA species (such as messenger RNA), and these species are quickly degraded. Therefore, attempts to normalize to a standard housekeeping gene are destined to failure. A multitude of biologic fluid-derived miR panels, including the sole bile-based miR panel published to date, utilize as normalizer the small RNA, U6. However, there is a growing body of evidence suggesting that utilization of U6 to normalize miR expression in body fluids introduces biases that have the potential of rendering the results unreproducible and/or unusable in a clinical laboratory setting. Although there is no indication that any other intrinsic RNA species performs better than U6, there is accumulating evidence that U6 is not appropriate as a normalizer.[10, 11] In addition, the use of any other intrinsic miR species as a normalizer is fundamentally based on the assumption that such a miR with constant expression across physiologic and pathologic states exists. There is no evidence to date to suggest that such an miR species exists in biliary EVs. Presently, there are no data to suggest that any intrinsic RNA normalizer, whose expression is stable in normal and, more importantly, in diseased states, exists in biologic fluids. Therefore, we propose that biologic fluid-derived miR levels should be normalized to initial volume of fluid analyzed. In addition, our study establishes that RNA extraction efficiency varies significantly among specimens, arguing that studies lacking a spike-in control may produce unreliable results.
Cancer diagnosis and clinical outcome prediction studies have created a relatively new field for applied mathematics. However, many previous studies have been limited to a single analytic method. Our current study compares SVMs and RF, two of the most commonly used mathematical models for cancer diagnosis, to MOCA, a recently developed mathematical model. One limitation of SVMs and RF is a lack of transparency in the classifiers that result from training. Because these models are “black boxes,” it is difficult to apply real biological principles to subsequent clinical testing, and continued use of the initial training model is required. Conversely, MOCA returned the combination of miR species that optimized CCA diagnosis and the predictive value that each miR contributed to that combined biomarker. Furthermore, MOCA returned the same biomarkers regardless of how the 10-fold cross-validation data were split; SVM and RFs varied as a function of data split, and for RFs, the model was dependent on the “seed” employed (see Materials and Methods).
We deliberately tuned our analytic technique for high specificity because the clinical consequences of false-positive CCA diagnosis would be calamitous. With specificity at 96%, our 5-miR panel displayed a sensitivity of 67%. Thus, the overall performance of this panel is superior to CA 19-9, as well as to any CCA diagnostic method currently employed in clinical practice. Further studies are needed to verify these findings in larger cohorts and explore the potential utility of combining our new 5-miR panel with other marker panels or with established clinical modalities, such as cytology.
We foresee potential clinical utility of our marker panel in patients with obstruction in the biliary tree (Supporting Table 1). The typical patient who would benefit from this marker panel is a PSC patient. In our analyses, 12 of 13 patients with PSC and no cancer were correctly diagnosed as not having CCA. Please note that all patients were followed for 5 years since the date the bile was collected to ensure that, at least for 5 years, they would not develop CCA. The PSC patient who was diagnosed with CCA continues to be followed at our hospital. There is still no evidence of CCA. The two potential explanations for this seemingly false-positive result are (1) the method is not perfect and the results need to be integrated clinically and with other laboratory results and (2) the biologic state that induced a positive test is reversed, or reversible. Further studies are needed in order to answer this question.
Among patients with a known diagnosis of CCA, 4 had a preexisting diagnosis of PSC. Three of these four were diagnosed correctly by our marker panel (Supporting Table 3). The single patient misdiagnosed as CCA negative did not have CA19-9 levels drawn. Of the remaining 3 patients correctly diagnosed by our marker panel, 2 had CA19-9 levels recorded. One of these had a CA19-9 level of 574.6 units/mL, suggestive of CCA. However, the other had a CA19-9 level of only 77.8 units/mL, which, although above the upper normal limit, is below the accepted cut-off value of 129 units/mL currently utilized to diagnose CCA.
Our marker panel outperformed CA19-9 levels in non-PSC patients. Among 39 CCA patients with recorded CA19-9 values (Table 1B), our marker panel correctly diagnosed 28, translating to a sensitivity of 71%. The accepted cut-off CA19-9 value of 100 units/ML (in patients without PSC) correctly diagnosed only 23 patients, for a sensitivity of only 58%. This calculated sensitivity of CA19-9 is slightly higher than, but similar to, that found in a previous study (53%). Thus, among every 100 CCA patients, our method is expected to diagnose 13 patients more than would CA19-9 levels. Notably, among 11 CCA patients correctly diagnosed by our 5-miR panel, but misdiagnosed by CA19-9, there were 8 CCA patients without lymph node or distant metastatic implants (N0M0). These patients are potentially cured by resection. In contrast, of the 6 patients correctly diagnosed with CCA by CA19-9, but not by our 5-miR panel, only 2 were N0M0 (Supporting Table 4). In further support of our hypothesis that our panel can better identify early tumors, the two T1N0M0 cancers in our cohort were correctly diagnosed by our panel, but not by CA19-9 levels. Diagnosing these early cancers is crucial, because the only current curative option for CCA patients is surgery, which can only be performed early in the course of this disease. We conclude that CA 19-9 tends to diagnose advanced CCA, for which surgery may no longer be an option, whereas our marker panel tends to diagnose early CCA, where surgery is still an option. Therefore, the difference in sensitivity between our marker panel and CA19-9 levels (71% vs. 58%) may actually underestimate our marker panel's potential effect on patient survival. Clearly, prospective studies are needed to answer this important question. Finally, in our 39-patient CCA cohort, 36 were correctly diagnosed by either our marker panel or CA19-9, translating into a combined sensitivity of 89.7%. Thus, even if treatments for advanced CCA improve, combining our marker panel with CA19-9 levels may become a valuable diagnostic strategy.