Human bile contains microRNA-laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis - PubMed (original) (raw)

. 2014 Sep;60(3):896-907.

doi: 10.1002/hep.27050. Epub 2014 Jul 25.

David Masica, Masaharu Ishida, Ciprian Tomuleasa, Sho Umegaki, Anthony N Kalloo, Christos Georgiades, Vikesh K Singh, Mouen Khashab, Stuart Amateau, Zhiping Li, Patrick Okolo, Anne-Marie Lennon, Payal Saxena, Jean-Francois Geschwind, Todd Schlachter, Kelvin Hong, Timothy M Pawlik, Marcia Canto, Joanna Law, Reem Sharaiha, Clifford R Weiss, Paul Thuluvath, Michael Goggins, Eun Ji Shin, Haoran Peng, Vivek Kumbhari, Susan Hutfless, Liya Zhou, Esteban Mezey, Stephen J Meltzer, Rachel Karchin, Florin M Selaru

Affiliations

Human bile contains microRNA-laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis

Ling Li et al. Hepatology. 2014 Sep.

Erratum in

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.

© 2014 by the American Association for the Study of Liver Diseases.

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Figures

Figure 1

Figure 1. Human biliary EVs characterization

Panel A includes a typical TEM picture demonstrating the presence of 30-110 nM spherical structures in human bile. Further characterization demonstrates that these vesicles display extracellular vesicle characteristics. Panel B demonstrates presence of typical extracellular vesicle proteins (TSG101 and CD63) in EV preparations from human bile. Panel C demonstrates the same 30-110 nM vesicles as visualized with NTA. Panel D demonstrates that the mode of EVs isolated from human bile was determined to be approximately 84 nm (X-axis depicts EVs size and Y-axis depicts EVs concentration for each size).

Figure 2

Figure 2. miR species extraction from human bile extracellular vesicles

Panel A shows amplification curves for miR species from EVs isolated from a bile specimen (X-axis – Cycle number and Y-axis – measured miR expression). Panel B depicts the large variability in measured Cel-miR-39 quantity after Cel-miR-39 was spiked at equal concentration in a cohort of 60 bile specimens (X-axis – bile specimens, Y-axis – measured qRT-PCR value for Cel-miR-39).

Figure 3

Figure 3. High stability and reproducibility of measured miR expression in bile EVs extracts

Panel A demonstrates that the levels of miR-21 and miR-638 are stable in EVs isolated from human bile kept at RT for 2-24 hours. Panel B demonstrates the stability of miR-21 and miR-638 after multiple freeze-thaw cycles.

Figure 4

Figure 4. Expression of 11 selected miR species across 96 samples

The heatmap segregates the 50 control samples (left side) and 46 CCA samples (right side). Bright red was used for all coordinates with a Z-score ≥ 0.5; therefore, these coordinates correspond to a CCA classification by MOCA. See Table 3B for mean expression values and diagnostic thresholds for all 11 miR species.

Figure 5

Figure 5. Bile specimen classification by multi-miR markers with high predictive value

Panels A and B display classification across 46 CCA samples by each of the markers from Table 3C (color bars in A) and expression for each of the corresponding miRs (heatmap in B). In B, bright red was used for all coordinates with a Z-score ≥ 0.5; therefore, these coordinates correspond to a CCA classification by MOCA. For each multi-miR marker, panel A displays the correct diagnosis with a solid rectangle. Panels C and D display classification across 50 control (CTRL) samples by each of the markers from Table 3C. For each multi-miR marker, panel D displays the correct diagnosis with a solid rectangle

Comment in

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