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

Abstract

The combination of liquid biomarkers from a single blood tube can provide more comprehensive information on tumor development and progression in cancer patients compared to single analysis. Here, we evaluated whether a combined analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating cell-free microRNA (miRNA) in total plasma and extracellular vesicles (EV) from the same blood sample is feasible and how the results are influenced by the choice of different blood tubes. Peripheral blood from 20 stage IV melanoma patients and five healthy donors (HD) was collected in EDTA, Streck, and Transfix tubes. Peripheral blood mononuclear cell fraction was used for CTC analysis, whereas plasma and EV fractions were used for ctDNA mutation and miRNA analysis. Mutations in cell-free circulating DNA were detected in 67% of patients, with no significant difference between the tubes. CTC was detected in only EDTA blood and only in 15% of patients. miRNA NGS (next-generation sequencing) results were highly influenced by the collection tubes and could only be performed from EDTA and Streck tubes due to hemolysis in Transfix tubes. No overlap of significantly differentially expressed miRNA (patients versus HD) could be found between the tubes in total plasma, whereas eight miRNA were commonly differentially regulated in the EV fraction. In summary, high-quality CTCs, ctDNA, and miRNA data from a single blood tube can be obtained. However, the choice of blood collection tubes is a critical pre-analytical variable.

Keywords: CTC; EV; ctDNA; liquid biopsy; melanoma; miRNA.

Fig. 1

ctDNA concentrations and mutation detection in plasma samples from MM patients. (A) ctDNA concentration (ng·µL⁻¹) from 2 mL plasma of individual MM patients in EDTA and Streck tubes. (B) Boxplot depicting the dispersion of ctDNA concentration (ng·µL⁻¹) of MM patients (n = 20) in EDTA and Streck tubes. No significant difference in ctDNA concentration was found (P = 0.96, Wilcoxon signed‐rank test). (C) VAF analysis of patients MM10 and MM18 who showed similar frequencies of somatic mutations for DPH3 ‐8 C>T and MAP2K1 N382H in both EDTA and Streck tubes.

Fig. 2

Images of spiked SK‐MEL28 cells in HD blood and CTCs identified in the MM patients using a multicolor immunofluorescence approach. After the isolation with the ClearCell system, the immunofluorescence staining was performed using a combination of four different MM markers (MCAM and NG2 in the PE and MART1 and HMB45 with Alexa 488). As an exclusion marker, CD45 (APC) was used. For nuclear staining, DAPI was used (blue channel). CTCs were positive for the melanoma markers and negative for CD45, while leukocytes were only positive for CD45 and negative for the melanoma markers.

Fig. 3

NTA and transmission EM imaging of EVs (A) EV concentration (particles·mL⁻¹) of individual MM patients and HDs in EDTA and Streck tubes. (B) Boxplots showing the particle concentration per mL of MM patients (n = 10) and HDs (n = 5). No significant differences were found between blood collected in EDTA and Streck tubes of HD and MM [P = 0.31 (HD), P = 0.92 (MM); Wilcoxon signed‐rank test]. (C) EM imaging of EVs isolated from EDTA and Streck tubes from HD3 and MM03 show similarities regarding morphology, size, and sample purity. (D) Venn diagram depicting the number of differentially regulated miRNA from NormFinder and geNorm analyses analyzed in blood collected in either EDTA or Streck tubes.

Fig. 4

Results of significantly differentially regulated miRNA in MM patients compared to HD (A) After normalization with NormFinder and application of FC (> 2) and P‐value (< 0.05) cutoff values, volcano plots illustrate significant differentially expressed genes. Green dots represent statistically significant and differentially regulated miRNA, while miRNA shown as black dots are below these defined thresholds of P‐value and FC. In EDTA plasma and EV samples, eight and 46 miRNA were identified with significantly different expression levels, while in Streck plasma and EV samples five and 62 significant miRNA are shown, respectively. (B) Dynamic PCA results after applying thresholds of FC (> 2) and P‐value (< 0.05). Blue dots represent HDs; MM samples are shown with a green dot. Since both groups are spatially isolated, a good separation of HDs and MM patients is achieved in both EDTA and Streck samples.