The ELIMA study: evaluation of multiple liquid biopsy analytes including CTCs, EVs and cfDNA in metastatic breast cancer patients all from one blood sample

Background: To gain comprehensive insights into the genomic and transcriptomic complexity useful for therapy management in metastatic breast cancer (MBC), we aimed to isolate and analyze mRNA and gDNA from circulating tumor cells (CTCs), mRNA from extracellular vesicles (EVs) and cell-free DNA (cfDNA) from a minimized blood volume.

Patients and methods: EDTA blood (2x 9 ml) was drawn from 35 MBC patients with hormone receptor-positive and HER2 negative primary tumors at the time of disease progression and at two consecutive staging time points. CTCs and their mRNA were isolated using the AdnaTest EMT2/StemCell Select/Detect. Plasma of CTC-depleted blood was used for cfDNA isolation, while mRNA from EVs was isolated by exoRNeasy using the remaining blood. The mRNA purified from CTCs and EVs was analyzed by a multimarker qPCR panel. gDNA from CTCs was isolated from the mRNA-depleted CTC lysates using the AllPrep DNA/RNA Nano Kit prototype. CTC gDNA and cfDNA were analyzed with a customized QIAseq Targeted DNA Panel for Illumina with unique molecular indices.

Results: Isolation of mRNA and gDNA from CTCs, mRNA from EVs and cfDNA was successfully established in a condensed workflow. CTC and EV mRNA profiles showed substantial differences synergizing with regard to their clinical relevance. Appearance of pathogenic ESR1 and PIK3CA mutations in cfDNA correlated with therapy failure. 57% of all cfDNA variants were recovered in the matched CTC gDNA, but 84% of all variants were unique in either cfDNA or CTC gDNA. The analysis of each single analyte identified a similar number of patients (50%-64%) with actionable markers, but the multi-parametric evaluation of all four analytes identified actionable markers in 93% of the patients.

Conclusions: We established a workflow for parallel isolation of multiple liquid biopsy analytes from a minimized blood volume. Each analyte showed additive value for therapy management, thus, the comprehensive picture of the genomic and transcriptomic complexity might enable to identify the most suitable therapy regimen in each individual patient in the future.