mRNA and gDNA from circulating tumor cells, extracellular vesicles and cell-free DNA

Establishment of a workflow for analysis of mRNA and gDNA from circulating tumor cells, extracellular vesicles and cell-free DNA from the same blood sample to mirror the genomic and transcriptomic complexity in metastatic breast cancer patients

Corinna Keup, Markus Storbeck, Peter Hahn, Siegfried Hauch, Markus Sprenger-Haussels, Ann-Kathrin Bittner, Oliver Hoffmann, Mitra Tewes, Rainer Kimmig and Sabine Kasimir- Bauer.

Background: Blood analytes derived from liquid biopsies are discussed as useful tools for therapy stratification and for monitoring of clonal evolution. To gain comprehensive insights into the genomic and transcriptomic complexity in metastatic breast cancer (MBC) useful for therapy management, 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 the same blood sample with minimized volume in a condensed workflow.

Patients and methods: EDTA blood (2x 9 ml) was drawn from 35 MBC patients with hormone receptor positive and HER2 negative primary tumor at time of disease progression and at two further consecutive staging time points. CTCs were isolated in duplicate from 5ml blood by immunomagnetic selection (AdnaTest EMT2/StemCell Select, QIAGEN). Plasma (4 ml) of the CTC-depleted blood was used for cfDNA isolation (QIAamp MinElute ccfDNA Kit, QIAGEN), plasma (4 ml) from the blood not used for CTC/cfDNA isolation was applied for EV isolation (exoRNeasy, QIAGEN). The mRNA purified from CTCs and EVs was analyzed by qPCR panel (AdnaPanel TNBC prototype, QIAGEN), while cfDNA was analyzed with a customized QIAseq Targeted DNA Panel for Illumina (QIAGEN) with unique molecular indices. We are working on a workflow to isolate gDNA from CTCs, starting from mRNA- depleted CTC lysates and analyzing the gDNA with a customized QIAseq Targeted DNA Panel. Isolation and mutation analysis of CTC gDNA was shown to be feasible in spiking experiments.

Results: Isolation of mRNA and gDNA from CTCs, mRNA from EVs and cfDNA was successfully established in a parallel workflow. CTC and EV mRNA profiles showed substantial differences synergizing with regard to their clinical relevance. Whereas overexpression of mTOR was related to therapy responsiveness in CTCs, mTOR signals in EVs related to therapy failure. ERBB2 overexpressing CTCs were found in one third of all MBC patients enabling new therapeutic options. Matched cfDNA revealed the appearance of pathogenic mutations (e.g. PIK3CA H1047R) across treatment indicating underlying resistance mechanisms. Moreover, we identified significant correlations of therapy outcome with the overexpression of transcripts/ presence of mutations in each of the isolated liquid biopsy analytes. Comparison of CTC gDNA and cfDNA is further conducted.

Conclusions: We were able to describe a complete workflow for parallel CTC mRNA, CTC gDNA, EV mRNA and cfDNA isolation from a minimized blood volume. In this research study each analyte showed synergistic potential for therapy management, thus the comprehensive picture of the genomic and transcriptomic complexity might in future enable to identify the most suitable therapy regiment in each individual patient.

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