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Pre-analytical factors affecting the establishment of a single tube assay for multiparameter liquid biopsy detection in melanoma patients.

Author(s): Schneegans S, Lück L, Besler K, Bluhm L, Stadler JC, Staub J, Greinert R, Volkmer B, Kubista M, Gebhardt C, Sartori A, Irwin D, Serkkola E, Af Hällström T, Lianidou E, Sprenger-Haussels M, Hussong M, Mohr P, Schneider SW, Shaffer J, Pantel K, Wikman H

Publication: Mol Oncol, 2020, Vol. , Page

PubMed ID: 32246814 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared EV and ctDNA concentration, EV morphology, plasma and EV miRNA content, and ctDNA VAF between specimens collected in EDTA and Streck tubes from patients with melanoma and healthy donors. The effect of collection tube (Streck, EDTA, or TransFix) and plasma removal on CTC detection was also investigated. Blood was collected from 20 patients with stage IV melanoma (11 women and 9 men, mean age 74) into EDTA, Streck, and Transfix tubes and from five healthy donors into Streck and EDTA tubes. Blood in Streck and EDTA tubes was separated into PBMC and plasma by centrifugation at 500 x g for 10 min, 2-5 h after collection. Plasma was not obtained from specimens in Transfix tubes but CTCs were isolated from the PBMC the day after collection. CTCs were obtained from PBMC from Streck and EDTA tubes and from whole blood in Transfix tubes using the Clear-Bridge ClearCell FX1 System. To determine the effect of plasma removal, specimens were spiked with 50 H1975 GFP-positive cells or 100 SK-MEL-28 cells and stained for Melan-A (MCAM) and neural-glial antigen-2 (NG2), melanin-A (MART1) and the antibody HMB45, or CD45(10 patients each). The plasma from Streck and EDTA tubes was divided into two aliquots for cfDNA and miRNA. Plasma for cfDNA analysis (20 melanoma patients) was centrifuged at 6000 x g (Streck tube) or 2500 x g (EDTA tube) for 10 min and stored frozen until extraction using the QIAamp MinElute ccfDNA Mini Kit. cfDNA was quantified using the Qubit Fluorometric Quantitation System and evaluated using High Sensitivity D5000 ScreenTape. Specimens were enriched using Ultra-SEEK Melanoma Panel, loaded onto SpectroCHIP-96 Arrays, and analyzed using matrix assisted laser desorption/ionization time-of-flight mass spectrometry-based MassARRAY System. Plasma for miRNA (10 melanoma patients and 5 healthy donors) and EV analysis (from melanoma patients) was centrifuged twice at 2500 x g for 10 min and stored at -80°C. EVs were obtained by ultracentrifugation (100,000 x g for 2 h) and pellet was suspended in PBS, stored at -80°C, and then analyzed by nanoparticle tracking analysis (NTA) and electron microscopy. miRNA was extracted from plasma and EVs using the miRNeasy Serum/Plasma Advanced Kit. Libraries were constructed using a QIAseq miRNA Library Kit and sequenced on an Illumina NextSeq 550. Differential expression of miR-375, miR-215-5p, and miR-200c-3p were validated using miRCURY LNA miRNA PCR assays using the average of miR-103a-3p and miR-93-5p as reference.

   Summary of Findings:

   ctDNA concentrations were comparable in plasma from Streck and EDTA tubes (3.0 ng/µL ±4.7 and 3.3 ng/µL ± 4.6, respectively). ctDNA mutations were detected in 8 of 12 patients, with seven patients having identical mutations identified and comparable VAF between tube types. The discordant case was found to harbor different mutations in the different tubes, with BRAF K601E at VAF of 0.5 found in the EDTA specimen and MAP2K1 I111S at a VAF of 0.3 found in the Streck specimen. Recovery of spiked-in SK-MEL-28 or H1975 cells were comparable before and after plasma removal in both Streck and EDTA specimens. CTCs were identified in 3 of 20 patient specimens and CTCs were only identified in the EDTA tube for each patient. The mean EV particle concentrations were comparable in EDTA and Streck specimens (3.33 x 10¹¹ ± 2.77 x 10¹¹ and 2.84 x 10¹¹ ± 2.31 x 10¹¹, respectively) with more inter-individual variability than between-tube variability found for all but one patient. Similarly, the particle size distributions were generally comparable between the tubes types with more variability noted between individuals. There was no difference in mean particle concentration, size distribution, or EV morphology between healthy donors and those with melanoma. There was a trend toward more unique molecular identifier reads in EDTA than Streck plasma (average 1.8-fold for healthy patients and 2.6-fold for melanoma patients) and EVs (average 4.0-fold for healthy patients and 6.5-fold for melanoma patients), but the differences were not significant. Significantly higher miR-4799-3p and lower miR-205-5p, miR-4529-3p, and miR-141-3p levels were found in EDTA plasma from patients with melanoma than healthy donors after geNorm normalization. In contrast, significantly higher miR-506-3p and lower piR-003731 levels were found in Streck plasma from patients with melanoma than healthy donors after geNorm normalization. After normalization using NormFinder, six miRNAs were found upregulated (piR_000753, miR-486-3p, piR_018849, miR-4799-3p, piR_018573, and miR-4257) and two were downregulated (miR-499a-3p and miR-141-3p) in EDTA plasma from melanoma patients while three miRNAs were upregulated (miR-320c, miR-4511, and miR-506-3p) and two downregulated (miR-4757-5p and piR_003731) in Streck plasma from melanoma patients with no overlap in miRNAs identified between tube types. Similar to results with plasma from melanoma patients, EVs had higher levels of 8 miRNAs in EDTA specimens and 11 miRNAs in Streck specimens and lower levels of 12 miRNAs in EDTA specimens and 12 miRNAs in Streck specimens. After geNorm normalization, seven of the miRNAs (miR-375, miR-215-5p, miR-141-3p, miR-200a-3p, miR-200b-3p, miR-200c-3p, and miR- 584-5p) were common to both tubes. Using NormFinder, melanoma patients had higher levels of 18 miRNAs in EDTA EVs and 24 miRNAs in Streck EVs and lower levels of 28 miRNAs in EDTA EVs and 38 miRNAs in Streck EVs. Nine of the identified miRNAs were common to both tubes and these included six found using geNorm (miR-375, miR-215-5p, miR-141-3p, miR-200a-3p, miR-200b-3p, and miR-200c-3p). Real-time PCR confirmed downregulation of miR-200c-3p in melanoma patients in both Streck and EDTA EVs (1.97 and 2.48-fold, respectively) and miR-375 in EDTA EVs (2.23-fold) but found no difference in miR-215-5p in melanoma patients, regardless of EV type.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen
   • Other Preservative
   • Streck/BCT

   Diagnoses:

   • Neoplastic - Melanoma
   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	MALDI-TOF MS
   RNA	Next generation sequencing
   RNA	Real-time qRT-PCR
   Cell count/volume	Light scattering
   Cell count/volume	Immunoassay
   Morphology	Electron microscopy
   Morphology	Light scattering
   Cell count/volume	Electron microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Melanoma Healthy donor
   Biospecimen Acquisition	Type of collection container/solution	Streck BCT EDTA tube Transfix tube
   Next generation sequencing Specific	Data handling	Normalized with geNorm Normalized with NormFinder
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-375 miR-215-5p miR-200c-3p
   Biospecimen Preservation	Type of fixation/preservation	Blood collection tube additive EDTA

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