Abstract 1594: A fast and sensitive workflow to screen therapy-relevant mutations in circulating tumor cells and quantification of cancer-associated extracellular vesicle-derived mRNA in plasma of cancer patients
Authors: Neumann Martin H. , Bender Sebastian , Krahn Thomas , Schlange Thomas 
Published: August 2018

Abstract
Liquid biopsy can be considered a test that detects rare cells or nucleic acids circulating in blood or in other biofluids. The concept of liquid biopsy aims to monitor the status of a disease or treatment efficacy in a simple, fast, cost efficient way and at any point in time with acceptable risk and burden for the patient. However, the volumes of available blood samples can be quite limited and rare cells as well as circulating nucleic acids are normally present in low abundance. Therefore, detection of rare events or the quantification of limited material requires robust, highly sensitive technologies like droplet digital PCR (ddPCR).
Here, a novel workflow is presented combining the effective enrichment of rare cells using the Parsortix microfluidic filtration system with the analysis by ddPCR. Ten or 100 cells of NSCLC cell line NCI-H441 were spiked into a suspension of 100k NCI-H1563 cells or healthy EDTA blood. The resulting cell suspensions were processed using the microfluidic device Parsortix to enrich cell factions by size and deformability. Enriched cell fractions were harvested or lysed directly in the Parsortix cassette. Genomic DNA was prepared from all cell fractions. NCI-H441 cells are heterozygous for KRAS G12V and TP53 R158L mutations, while NCI-H1563 cells do not contain the mutations. Ten cells or less were enough to detect KRAS G12V or TP53 R158L mutations in a standard blood sample (7-10 ml). This combined workflow enables quantification of circulating tumor cells and analysis for therapy relevant point mutations in a fast, EpCAM independent manner.
Additionally, extracellular vesicle derived mRNA (EVmRNA) isolated from either cell culture supernatant or plasma from cancer patients was analyzed to demonstrate the suitability of ddPCR for assessing specific EVmRNA as biomarkers in different cancer types. In 13 NSCLC and 17 metastatic breast cancer plasma samples the levels of epithelial and oncogene EVmRNA, including EpCAM, ERBB2 or FGFR3 were determined and compared to healthy control plasma. Levels of potentially cancer-associated EVmRNA were only detectable in plasma from cancer patients, but not in healthy controls. Samples from patients and healthy volunteers, respectively, were collected under signed informed consent.
We demonstrated, that ddPCR allows the verification of therapy relevant point mutations in CTCs has the potential to be used as a screening approach. Moreover, despite the small amounts of mRNA in extracellular vesicles from patient blood, using ddPCR differential mRNA levels can be determined in patient plasma and enable and expand the scope of biomarker analysis from EVs.
This work is supported by IMI JU & EFPIA (grand no. 115749).
Citation Format: Martin H. Neumann, Sebastian Bender, Thomas Krahn, Thomas Schlange. A fast and sensitive workflow to screen therapy-relevant mutations in circulating tumor cells and quantification of cancer-associated extracellular vesicle-derived mRNA in plasma of cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1594.