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Serum outperforms plasma in small extracellular vesicle microRNA biomarker studies of adenocarcinoma of the esophagus.

Author(s): Chiam K, Mayne GC, Wang T, Watson DI, Irvine TS, Bright T, Smith LT, Ball IA, Bowen JM, Keefe DM, Thompson SK, Hussey DJ

Publication: World J Gastroenterol, 2020, Vol. 26, Page 2570-2583

PubMed ID: 32523312 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the size, miRNA profile, and performance on a miRNAbiomarker panel for esophageal adenocarcinoma of small extracellular vesicles (sEVs) isolated from plasma and serum. Blood was collected from 10 healthy patients and 10 patients with esophageal adenocarcinoma into Z Serum Separator Clot Activator and K₃EDTA Vacuette tubes and stored at room temperature for 16-24 h. Serum was obtained by a single centrifugation of blood at 650 x g for 15 min and plasma was obtained by dual centrifugation at 650 x g for 15 min. Plasma and serum were stored at -80°C. Small extracellular vesicles were isolated from plasma and serum by centrifugation at 16000 x g at 4 °C for 30 min followed by the ExoQuick Kit and resuspended in phosphate buffered saline. sEVs were analyzed using NanoSight LM10 Nanoparticle Analysis System. miRNA was extracted from sEVs using the miRNeasy Serum/Plasma Kit and expression of 758 miRNAs were profiled using the Taqman OpenArray Human microRNA panel.

   Summary of Findings:

   The size of particles isolated from serum and plasma were comparable. The average concentration of sEVs was 1.2-fold higher in serum than plasma of healthy individuals (P=0.047) but not cancer patients (P=0.56). More miRNAs were detected in plasma sEVs than serum sEVs (480 versus 412, P=0.005) and more of the miRNAs were unique to plasma than to serum (108 versus 40). Importantly, while 22 of the miRNAs unique to plasma were also detected in at least 50% of specimens, only one of the miRNAs unique to serum was found in at least 50% of the specimens. A total of 372 miRNAs were found in both plasma and serum 118 of which were found in all specimens. Correlations in expression between plasma and serum of healthy patients were stronger for the 118 miRNAs expressed in all specimens than the 372 miRNAs identified overall (R²=0.92 versus R²=0.87) and a similar trend was observed in specimens from cancer patients. Importantly, comparisons of the lists of the 20 most abundant miRNAs in serum and plasma revealed 16 in common, all of which were 2- to 11-fold higher in plasma sEVs (P<0.05). Among the 16 most detected miRNAs were miR-223-3p, miR-451a, miR-19b-3p, miR-17-5p, miR-30b-5p, miR-106a-5p, miR-150-5p, and miR-92a-3p; all of which are known to be present in blood cells. miR-223-3p and miR-451a were 9.6- and 2.5-fold higher, respectively; in plasma than serum (Wilcoxon signed-rank test P=0.0051 and P=0.01, respectively). miRNAs previously identified as uniquely vesicular were identified in plasma and serum sEVs (12 and 14, respectively), but only 1 miRNA unique to blood was found in plasma and only 4 were identified in serum. While Serum had a higher percentage of vesicle-associated miRNAs with CT values <25 than plasma (60% versus 44%), plasma had a higher percentage of protein associated miRNAs with CT<25 and <20 than serum (62% versus 31% and 22% versus 0%, respectively). Importantly, a five-miRNA biomarker panel for esophageal adenocarcinoma had higher accuracy in sEVs from serum than plasma (area under the ROC curve of 0.95 versus 0.80) and the difference was even more pronounced in eave-one-out cross validation (area under the ROC curve 0.90 versus 0.54).

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   Cell count/volume	Light scattering

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Esophageal adenocarcinoma Healthy
   Biospecimen Aliquots and Components	Blood and blood products	Serum vesicles Plasma vesicles

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