Title: Human bile contains MicroRNA-laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis
Abstract: HepatologyVolume 60, Issue 3 p. 896-907 Hepatobiliary MalignanciesFree Access Human bile contains MicroRNA-laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis Correction(s) for this article Correction Volume 60Issue 6Hepatology pages: 2135-2135 First Published online: November 24, 2014 Ling Li, Ling Li Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, China Division of Gastroenterology, Third Hospital of Peking University Health Science Center, Beijing, ChinaSearch for more papers by this authorDavid Masica, David Masica Department of Biomedical Engineering and Institute for Computational Medicine Johns Hopkins University, Baltimore, MDSearch for more papers by this authorMasaharu Ishida, Masaharu Ishida Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorCiprian Tomuleasa, Ciprian Tomuleasa Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, China Center for Genomics and Translational Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, and Department of Hematology, Ion Chiricuta Comprehensive Cancer Center, Cluj Napoca, RomaniaSearch for more papers by this authorSho Umegaki, Sho Umegaki Tohoku University School of Medicine, Sendai, JapanSearch for more papers by this authorAnthony N. Kalloo, Anthony N. Kalloo Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorChristos Georgiades, Christos Georgiades Radiology Vascular & Interventional Radiology, American Medical Center, Nicosia, CyprusSearch for more papers by this authorVikesh K. Singh, Vikesh K. Singh Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorMouen Khashab, Mouen Khashab Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorStuart Amateau, Stuart Amateau Division of Gastroenterology and Hepatology, University of Colorado, Denver, COSearch for more papers by this authorZhiping Li, Zhiping Li Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorPatrick Okolo, Patrick Okolo Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorAnne-Marie Lennon, Anne-Marie Lennon Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorPayal Saxena, Payal Saxena Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorJean-Francois Geschwind, Jean-Francois Geschwind RadiologySearch for more papers by this authorTodd Schlachter, Todd Schlachter RadiologySearch for more papers by this authorKelvin Hong, Kelvin Hong RadiologySearch for more papers by this authorTimothy M. Pawlik, Timothy M. Pawlik SurgerySearch for more papers by this authorMarcia Canto, Marcia Canto Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorJoanna Law, Joanna Law Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorReem Sharaiha, Reem Sharaiha Division of Gastroenterology and Hepatology, Weill Cornell Medical College, New York, NYSearch for more papers by this authorClifford R. Weiss, Clifford R. Weiss RadiologySearch for more papers by this authorPaul Thuluvath, Paul Thuluvath The Institute for Digestive Health & Liver Disease at Mercy, Baltimore, MDSearch for more papers by this authorMichael Goggins, Michael Goggins Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorEun Ji Shin, Eun Ji Shin Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorHaoran Peng, Haoran Peng Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorVivek Kumbhari, Vivek Kumbhari Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorSusan Hutfless, Susan Hutfless Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorLiya Zhou, Liya Zhou Division of Gastroenterology, Third Hospital of Peking University Health Science Center, Beijing, ChinaSearch for more papers by this authorEsteban Mezey, Esteban Mezey Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorStephen J. Meltzer, Stephen J. Meltzer Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorRachel Karchin, Rachel Karchin Department of Biomedical Engineering and Institute for Computational Medicine Johns Hopkins University, Baltimore, MDSearch for more papers by this authorFlorin M. Selaru, Corresponding Author Florin M. Selaru Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, China Sidney Kimmel Cancer Center, Johns Hopkins Hospital, Baltimore, MDAddress reprint requests to: Florin M. Selaru, M.D., Johns Hopkins University, 720 Rutland Avenue, Suite 950, Baltimore, MD 21205. E-mail: [email protected]; fax: 410-614-9612.Search for more papers by this author Ling Li, Ling Li Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, China Division of Gastroenterology, Third Hospital of Peking University Health Science Center, Beijing, ChinaSearch for more papers by this authorDavid Masica, David Masica Department of Biomedical Engineering and Institute for Computational Medicine Johns Hopkins University, Baltimore, MDSearch for more papers by this authorMasaharu Ishida, Masaharu Ishida Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorCiprian Tomuleasa, Ciprian Tomuleasa Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, China Center for Genomics and Translational Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, and Department of Hematology, Ion Chiricuta Comprehensive Cancer Center, Cluj Napoca, RomaniaSearch for more papers by this authorSho Umegaki, Sho Umegaki Tohoku University School of Medicine, Sendai, JapanSearch for more papers by this authorAnthony N. Kalloo, Anthony N. Kalloo Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorChristos Georgiades, Christos Georgiades Radiology Vascular & Interventional Radiology, American Medical Center, Nicosia, CyprusSearch for more papers by this authorVikesh K. Singh, Vikesh K. Singh Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorMouen Khashab, Mouen Khashab Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorStuart Amateau, Stuart Amateau Division of Gastroenterology and Hepatology, University of Colorado, Denver, COSearch for more papers by this authorZhiping Li, Zhiping Li Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorPatrick Okolo, Patrick Okolo Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorAnne-Marie Lennon, Anne-Marie Lennon Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorPayal Saxena, Payal Saxena Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorJean-Francois Geschwind, Jean-Francois Geschwind RadiologySearch for more papers by this authorTodd Schlachter, Todd Schlachter RadiologySearch for more papers by this authorKelvin Hong, Kelvin Hong RadiologySearch for more papers by this authorTimothy M. Pawlik, Timothy M. Pawlik SurgerySearch for more papers by this authorMarcia Canto, Marcia Canto Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorJoanna Law, Joanna Law Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorReem Sharaiha, Reem Sharaiha Division of Gastroenterology and Hepatology, Weill Cornell Medical College, New York, NYSearch for more papers by this authorClifford R. Weiss, Clifford R. Weiss RadiologySearch for more papers by this authorPaul Thuluvath, Paul Thuluvath The Institute for Digestive Health & Liver Disease at Mercy, Baltimore, MDSearch for more papers by this authorMichael Goggins, Michael Goggins Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorEun Ji Shin, Eun Ji Shin Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorHaoran Peng, Haoran Peng Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorVivek Kumbhari, Vivek Kumbhari Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorSusan Hutfless, Susan Hutfless Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorLiya Zhou, Liya Zhou Division of Gastroenterology, Third Hospital of Peking University Health Science Center, Beijing, ChinaSearch for more papers by this authorEsteban Mezey, Esteban Mezey Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorStephen J. Meltzer, Stephen J. Meltzer Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, ChinaSearch for more papers by this authorRachel Karchin, Rachel Karchin Department of Biomedical Engineering and Institute for Computational Medicine Johns Hopkins University, Baltimore, MDSearch for more papers by this authorFlorin M. Selaru, Corresponding Author Florin M. Selaru Division of Gastroenterology and Hepatology, Department of Medicine, Beijing, China Sidney Kimmel Cancer Center, Johns Hopkins Hospital, Baltimore, MDAddress reprint requests to: Florin M. Selaru, M.D., Johns Hopkins University, 720 Rutland Avenue, Suite 950, Baltimore, MD 21205. E-mail: [email protected]; fax: 410-614-9612.Search for more papers by this author First published: 04 February 2014 https://doi.org/10.1002/hep.27050Citations: 148 Potential conflict of interest: Dr. Kaloo owns stock and has intellectual property rights in Apollo. He consults for Checkmed and Pentax. Dr. Saxena consults for and received grants from Boston Scientific and also received grants from Cook Medical. Dr. Geschwind consults for and received grants from Biocompatibles/BTG, Bayer, Guerbet, Nordion/BTG, and Phillips. He consults for Jennerex and received grants from Theshold. He is the founder and CEO of PreScience Labs, LLC. Dr. Thuluvath advises, is on the speakers' bureau for, and received grants from Vertex and Gilead. He advises Janssen and is on the speakers' bureau for Onyx. He received grants from Boehringer Ingelheim, Novartis, Bristol-Myers Squibb, Eisai, and Salix. This study was supported by a K08 Award (DK090154-01) from the National Institutes of Health (NIH; to F.M.S.) and by an Early Research and Detection Network (EDRN) Associate Membership supported by an U01 Award (CA086402) from the NIH. Dr. Meltzer is an American Cancer Society Clinical Research Professor. See Editorial on Page 782 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract 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) Abbreviations CA19-9 carbohydrate antigen 19-9 CCA cholangiocarcinoma CT computed tomography Ct cycle passing threshold CTRL control dCCA distal cholangiocarcinoma ERCP endoscopic retrograde cholangiopancreatography EV extracellular vesicle EUS endoscopic ultrasound HCC hepatocellular carcinoma iCCA intrahepatic cholangiocarcinoma IR interventional radiology MRI magnetic resonance imaging miR microRNA MOCA multivariate organization of combinatorial alterations NTA nanoparticle tracking analysis pCCA perihilar cholangiocarcinoma PSC primary sclerosing cholangitis qRT-PCR quantitative reverse-transcription polymerase chain reaction RF random forests SVMs support vector machines TNM tumor node metastasis TSG101 tumor susceptibility gene 101 Cholangiocarcinoma (CCA) is a cancer that arises in the biliary tree.1 Anatomically, CCA is divided into intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA) tumors.2 Surgery is the only curative option.3 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.3 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.2 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.4 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.3 Therefore, the sensitivity of cytology performed on brush biopsy specimens is, at best, only 20%.2 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.5 Additional studies have focused on RNA expression profiles in biliary brushings6 or on microRNA (miR) profiles in whole human bile.7 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.8 Therefore, it is not surprising that studies published to date present contradictory information regarding specific miR-based markers of cancer.9 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.7 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.12 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. Materials and Methods Bile Samples Bile samples from CCA and control (CTRL) patients were obtained from the Johns Hopkins Hospital (Baltimore, MD) under an institutional review board IRB-approved protocol. Bile samples were obtained at ERCP or at the time of percutaneous manipulation of biliary tubes by interventional radiology (IR). Aspiration of bile was performed after cannulation of the biliary tree before injection of contrast. CCA diagnosis was established based on pathologic and radiologic evidence. Table 1A contains demographic and clinical patient information. Table 1B contains the tumor node metastasis (TNM) stage for CCA patients who had carbohydrate antigen 19-9 (CA19-9) measured. Numerous patients with benign biliary obstruction were included in the control group. These patients were added because, from a clinical perspective, they present with a clinical picture indistinguishable from CCA. Thus, our marker panel was envisioned as particularly useful in this setting. Supporting Table 1 provides detailed information regarding control patients. For example, there were 13 patients with primary sclerosing cholangitis (PSC). These patients were followed for 5 years after bile specimens were collected to ensure that they were not already harboring early undiagnosed CCA. In addition, we included 15 patients with benign biliary tree obstruction and 3 with benign bile leaks. Table 1A. Clinical and Epidemiologic Information Identifier Gender Race Age CA19-9 Source Disease TNM CCA1 M C 57 147.9 Endoscopy pCCA T4N1M0 CCA2 M C 50 70.2 IR iCCA T3N0M0 CCA3 F C 55 38.1 IR pCCA T4N0M0 CCA4 F AA 32 NA Endoscopy PSC-iCCA T4N1M1 CCA5 F A 60 3,330.2 Endoscopy pCCA T3N0M0 CCA6 M C 85 360.2 Endoscopy dCCA T3N1M0 CCA7 M C 45 165.6 Endoscopy iCCA T4N1M1 CCA8 F AA 68 NA IR icCA T4N0M1 CCA9 M C 73 1,969.1 Endoscopy pCCA T3N1M0 CCA10 F C 69 481.6 IR pCCA T3N0M0 CCA11 M C 65 38.1 IR dCCA T3N0M0 CCA12 M H 64 4,872.7 IR pCCA T2N0M0 CCA13 F C 50 108.7 IR iCCA T4N0M0 CCA14 F C 75 12,352.2 IR pCCA T4N0M1 CCA15 F C 68 571.8 IR iCCA T4N0M1 CCA16 M C 51 10,692.7 IR pCCA T4N0M0 CCA17 M C 69 36.5 Endoscopy dCCA T4N1M0 CCA18 F C 72 3,610.5 IR iCCA T4N1M0 CCA19 F C 67 81 IR iCCA T1N0M0 CCA20 F C 69 1 Endoscopy pCCA T3N1M0 CCA21 F C 69 371.7 Endoscopy pCCA T4N2M1 CCA22 F C 57 167 Endoscopy pCCA T2N0M0 CCA23 F C 67 81 IR iCCA T1N0M0 CCA24 F C 47 496.2 Endoscopy iCCA T4N2M0 CCA25 M AA 63 0.1 IR pCCA T2N0M0 CCA26 F C 61 30.9 IR pCCA T3N0M0 CCA27 M C 70 NA IR iCCA T4N0M1 CCA28 F C 39 NA Endoscopy PSC-pCCA T3N0M0 CCA29 F A 68 73.7 Endoscopy pCCA T4N0M0 CCA30 F C 55 74.3 IR pCCA T4N0M0 CCA31 F H 56 15.5 Endoscopy pCCA T2N0M0 CCA32 F C 67 58.6 Endoscopy iCCA T3N1M0 CCA33 M AA 63 <1.0 IR pCCA T2N0M0 CCA34 M C 76 NA IR pCCA T1N0M0 CCA35 M C 63 NA IR pCCA T4N2M0 CCA36 F C 71 325.4 Endoscopy pCCA T4N0M1 CCA37 M C 68 2,119.9 Endoscopy iCCA T2N2M0 CCA38 F AA 75 77.8 Endoscopy PSC-dCCA T4N0M0 CCA39 F C 71 1,315.4 Endoscopy iCCA T4N1M1 CCA40 F C 69 574.6 Endoscopy PSC-pCCA T4N2M0 CCA41 M C 50 150 Endoscopy pCCA T2N0M0 CCA42 F AA 44 <1 Endoscopy pCCA T3N0M1 CCA43 F C 77 NA Endoscopy pCCA T2N0M0 CCA44 M C 56 114.4 Endoscopy pCCA T4N2M0 CCA45 M C 46 97.9 IR dCCA T2N0 M1 CCA46 F H 43 109.3 Endoscopy pCCA T4N0M0 CTRL1 M H 79 NA Endoscopy Biliary obstruction CTRL2 M C 54 NA Endoscopy Stent removal CTRL3 F AA 68 NA Endoscopy Biliary obstruction CTRL4 F C 55 NA Endoscopy SOD CTRL5 F C 65 309.5 Endoscopy Biliary obstruction CTRL6 M C 61 NA Endoscopy Cirrhosis CTRL7 M C 59 NA Endoscopy Bile leak CTRL8 M C 66 37.8 Endoscopy Biliary obstruction CTRL9 F A 82 NA Endoscopy Biliary obstruction CTRL10 F C 63 20.7 Endoscopy CP CTRL11 F C 50 NA Endoscopy SOD CTRL12 F C 53 NA Endoscopy Biliary obstruction CTRL13 M AA 69 NA IR Biliary obstruction CTRL14 M C 61 NA IR Bile leak CTRL15 M C 56 NA IR Biliary obstruction CTRL16 F AA 41 NA Endoscopy Bile leak CTRL17 M C 60 NA Endoscopy Biliary obstruction CTRL18 M C 52 NA Endoscopy Stent removal CTRL19 F C 39 8.6 Endoscopy CP CTRL20 F C 33 7 Endoscopy CP CTRL21 F AA 49 NA Endoscopy SOD CTRL22 F C 47 NA Endoscopy SOD CTRL23 F C 55 NA Endoscopy SOD CTRL24 M C 27 NA IR Cholangitis CTRL25 F C 45 NA Endoscopy SOD CTRL26 F C 63 NA Endoscopy SOD CTRL27 F C 66 NA Endoscopy SOD CTRL28 M C 65 <1.0 Endoscopy CBD stricture CTRL29 F AA 42 NA Endoscopy SOD CTRL30 F C 49 NA Endoscopy SOD CTRL31 M A 30 4.8 Endoscopy PSC CTRL32 M C 35 20.8 Endoscopy PSC CTRL33 M C 53 257.1 Endoscopy PSC CTRL34 F C 57 1 Endoscopy PSC CTRL35 M C 39 7.9 Endoscopy PSC CTRL36 F C 22 8.4 Endoscopy PSC CTRL37 M C 66 25.2 Endoscopy PSC CTRL38 F AA 41 188.4 Endoscopy PSC CTRL39 M A 42 NA Endoscopy PSC CTRL40 M C 68 230.6 Endoscopy CBD stricture CTRL41 F C 58 <1 Endoscopy CP CTRL42 M A 57 NA Endoscopy Biliary obstruction CTRL43 F C 39 NA IR CBD stricture CTRL44 M C 74 <1 Endoscopy Stent removal CTRL45 F C 49 NA Endoscopy PSC CTRL46 M A 31 16.4 Endoscopy PSC CTRL47 M C 31 35 Endoscopy PSC CTRL48 M A 31 64.2 Endoscopy PSC CTRL49 M C 73 NA endoscopy Cholangitis CTRL50 F AA 56 <1.0 Endoscopy Biliary obstruction Gender, race, age, level of CA19-9 (where available) diagnosis, and TNM classification are presented. Abbreviations: M, male; F, female; C, Caucasian; AA, African American; A, Asian; H, Hispanic; NA, not available; PSC-CCA, PSC arising CCA; SOD, sphincter of Oddi dysfunction; CP, chronic pancreatitis; CBD, common bile duct. Bile Extracellular Vesicle Isolation Initially, we experimented with EVs isolation from 1 mL of fresh bile. Once the experimental procedures were carefully delineated, we started extracting EVs from 400 µL of bile. Bile samples were centrifuged at 300×g for 10 minutes at 4°C to pellet cells and debris. The supernatant was then centrifuged at 16,500×g for 20 minutes at 4°C to further remove cellular debris and then filtered through a 200-nm filter. Next, the supernatant was centrifuged at 120,000×g for 70 minutes at 4°C to pellet EVs.13 EVs were utilized for immediate RNA extraction or resuspended in 50-150 µL of phosphate-buffered saline and stored in −80°C for future use. Statistical Analyses We used three computational packages to assess the predictive value of selected miR species for CCA diagnosis: random forests (RFs), support vector machines (SVMs), and our recently developed MOCA algorithm (see details below).14, 15 For details, as well as for materials and methods, please see the Supporting Information (Methods; Supporting Figs. 1 and 2; Supporting Table 2). Results Delineation of the Source of miRs in Human Bile The sole published report utilizing human bile for miR-based CCA diagnostic panels employed whole bile.7 Based on RNA gel electrophoresis, as well as quantitative reverse-transcription polymerase chain reaction (qRT-PCR) values for a well-expressed miR species (miR-21), we determined that free-floating cells in bile contribute to the RNA extracted from bile (Supporting Fig. 3A). Unfortunately, the quantity and quality of the RNA contributed by free-floating cells likely depends on the number of free-floating cells in a specific bile specimen and also on the degree of cell viability and therefore is unpredictable. In addition, we demonstrate that the RNA contributed by free-floating cells is rapidly degraded at RT, as well as from a single freeze-thaw cycle (Supporting Fig. 3A,B). These data strongly argue against using whole bile for developing a miR-based disease marker panel. Isolation and Characterization of Human Bile Extracellular Vesicles Extracellular vesicle preparations from human bile were imaged by using TEM. We noted the presence of 30-110 nM of vesicles, consistent with previously reported features of EVs (Fig. 1A).16 To further confirm that these spherical structures are EVs, we assayed for presence of tumor susceptibility gene 101 (TSG101) and CD63, molecules frequently used as extracellular vesicle markers.17 Western blotting confirmed that the biliary-derived extracellular vesicle preparations are rich in TSG101 and CD63 (as shown in Fig. 1B). To further define human biliary EVs, we employed multiparameter nanoparticle tracking analysis (NTA). First, we noted the presence of round vesicles, displaying typical Brownian motion (Fig. 1C; Supporting Movie 1). Next, we found that the majority of EVs in human bile were between 30 and 110 nM and that the mode of EV sizes was 84 nM (Fig. 1D), suggesting that EVs isolated are most likely exosomes. Based on NTA analysis, we also determined that bile from CCA patients contained approximately 3 × 10−11 EVs/mL of bile, and bile from control patients contained approximately 2.5 × 10−10 EVs/mL of bile. To further substantiate the presence of EVs in preparations from human bile, we stained EVs with PKH67, as previously described.18 Next, we added stained EVs or control extracts to cells in culture. Cells took up stained EVs from human bile, but not from the controls as shown in (Supporting Fig. 4; Supporting Movie 2). Figure 1Open in figure viewerPowerPoint Human biliary EV characterization. (A) Typical transmission electron microscopy picture demonstrating the presence of 30-110 nM of spherical structures in human bile. Further characterization demonstrates that these vesicles display EV characteristics. (B) Presence of typical EV proteins (TSG101 and CD63) in EV preparations from human bile. (C) The same 30-110 nM of vesicles as visualized with NTA. (D) Mode of EVs isolated from human bile was determined to be approximately 84 nm (x-axis depicts EV size and y-axis depicts EV concentration for each size). Presence and Isolation of miR Species From Biliary Extracellular Vesicles EVs isolated from serum were demonstrated to contain miR species, but no studies to date have investigated human bile EVs.19 We performed qRT-PCR miR arrays on EV RNA isolated from 1 CCA bile specimen (Fig. 2A). According to the manufacturer's recommendation, we utilized a cycle passing threshold (Ct) value threshold of 40 cycles to establish which miR species existed in quantities high enough to be detected. Utilizing a Ct value threshold of 40 cycles, we were able to detect 137 miR species. From these 137 miR species with amplification, a number of 74 miR species were amplified at a Ct value of 32 or less, which is considered by the manufactur