Impact of blood collection tubes on CTC-,ctDNA- and miRNA recoveries in malignant melanoma patients

Liquid biopsy represents a powerful clinical tool that makes use of the detection of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and exosomes in peripheral blood. Each different analysis is complementary to each other and can give additional valuable clinical information. These biomarkers are susceptible to quick degradation, presenting a challenge in a clinical environment and may require different blood collection tubes. The aim of this CANCER-ID study was to analyze the influence of different blood tubes on quantity and quality of CTCs, ctDNA and total and exosomal miRNA isolated from the same blood tube.

After confirming that plasma removal does not influence CTC recovery rates, peripheral blood from 20 cases of metastatic melanoma patients was collected in EDTA, Streck and Transfix tubes. CTC isolation from the PBMC fraction was performed by the ClearCell device (Clearbridge BioMedics). CTCs were identified by immunofluorescence staining. CtDNA was extracted from Streck and EDTA plasma samples, quantity (Qubit) and quality (Tapestation) measurements were performed before analyzing 86 hot-spot mutations in 13 genes by the UltraSEEK chemistry (Agena Bioscience). Ten of the 20 metastatic melanoma patients and 5 healthy donors were chosen for miRNA analysis. MiRNAs were extracted with miRNeasy Serum/Plasma Advanced Kit and miRNeasy Serum/Plasma Kit (QIAGEN) from total plasma and from extracellular vesicles (EVs), respectively. EVs were isolated by ultracentrifugation. QIAseq miRNA libraries were produced and sequenced (Illumina NextSeq 550). The reads were mapped to miRBase and normalized (geNorm). Volcano plots of fold change versus p-value were used to display the miRNAs that are significantly regulated.

The CTC enrichment results showed that 3/20 of the EDTA blood samples were positive, whereas samples from Streck and Transfix tubes were negative. The quantity or quality of the ctDNA did not significantly differ between the EDTA and Streck tubes. Mutation analysis of ctDNA performed so far showed similar detection sensitivities between the two tubes. The morphology, particle concentration and size distribution of EVs did not differ between the two tubes. MiRNA-NGS analyses from plasma revealed that seven (EDTA) and four (Streck) miRNAs are significantly differentially expressed in patients compared with healthy donors. No overlap of these miRNAs was found between the two tubes. In the EV fraction 24 and 31 miRNAs were found significantly differentially expressed in the EDTA and Streck tubes, respectively. Here, one EV miRNA is up-regulated and six are down-regulated in both tubes.

In conclusion, the CTC recovery rates and minimal overlap of circulating miRNAs indicate that the different tubes affect CTC, ctDNA and miRNA results. Thus, the outcome of liquid biopsy analyses strongly depends on the choice of blood collection tubes an important pre-analytical variable.

Svenja Schneegans1, Lelia Lück1, Leonie Bluhm2, Janina Staub3, Rüdiger Greinert2, Beate Volkmer2, Alexander Sartori4, Darryl Irwin4, Taija af Hallstrom5, Melanie Hussong6, Jonathan Shaffer6, Markus Sprenger-Haussels7, Stefan W. Schneider3, Peter Mohr2, Klaus Pantel1, Harriet Wikman1. 1 University Medical Center Hamburg-Eppendorf, Department of Tumor Biology, Hamburg, Germany; 2 Elbe Clinics, Centre of Dermatology, Buxtehude, Germany; 3 University Medical Center Hamburg-Eppendorf, Department of Dermatology and Venereology, Hamburg, Germany; 4 Agena Bioscience GmbH, Hamburg, Germany; 5 Orion Pharma, Orion Corporation, Espoo, Finland; 6 QIAGEN Inc, Frederick, MD; 7 QIAGEN GmbH, Hilden, Germany

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