Abstract PS2-24: Dynamics of a multi-parametric liquid biopsy, including CTCs, EVs, and cfDNA, during therapy in metastatic breast cancer patients provide useful insights for therapy management
Authors: Kasimir-Bauer Sabine , Storbeck Markus , Hahn Peter , Hauch Siegfried , Sprenger-Haussels Markus , Hoffmann Oliver , Kimmig Rainer , Keup Corinna 
Published: February 2021

Abstract
Background: Extensive knowledge about the molecular complexity could benefit therapy management in metastatic breast cancer (MBC). Consequently, we isolated and analyzed mRNA from circulating tumor cells (CTCs), mRNA from extracellular vesicles (EVs), and cell-free DNA (cfDNA) from a minimized blood volume and aimed to assess the dynamics of these analytes to elucidate the relevance of multi-parametric liquid biopsies for therapy monitoring and therapeutic decision-making by studying them at various points in time during the course of the therapy. Methods: 2x 9 ml of EDTA blood 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 from CTC-depleted blood was used for cfDNA isolation, while mRNA from EVs was isolated using exoRNeasy and the remaining blood. mRNA purified from CTCs and EVs was analyzed by a multimarker qPCR panel targeting 17 transcripts. cfDNA was analyzed with a customized QIAseq Targeted DNA Panel (targeting 17 genes) for Illumina with unique molecular indices. Consumables: QIAGEN, Germany. Results: Data from 35 patients at three time points (105 samples) and 51 parameters [results of 17 genes in three analytes (CTCs, EVs, and cfDNA)] were correlated with therapy outcomes defined by visual staging to examine their value for monitoring. Among the top 15 parameters with the best sensitivity and specificity - regardless of whether or not sensitivity and specificity were jointly examined - most parameters originated from the analysis of CTCs. Among these parameters, a two-tailed Fisher’s exact test revealed ERBB3 CTC signals (96% spec; 22% sens; p=0.022) or a combination of ERBB3 CTC signals or ERBB2 CTC signals (87% spec; 37% sens; p=0.008) to be highly correlated with the time of disease progression. Interestingly, an evaluation of the development of signals during the therapy resulted in EV parameters having the best sensitivity and specificity. The appearance of signals associated with resistance (ERCC1) in EVs was significantly correlated with worse therapy outcomes reaching a specificity of 98% (24% sens; p=0.007). A visualization of all results from seven index patients demonstrated the diversity within CTC signals and the dynamics of EV signals during treatment. Some of the CTC signals and their matched EVs (e.g. BRCA1 signals) showed opposed behavior during the treatment for a given patient. PIK3CA and ESR1 variants, known to be associated with resistance, were frequently found in those index patients receiving anti-hormonal treatment. Allele frequencies of PIK3CA variants in cfDNA and PIK3CA CTC signals showed a similar reduction after successful chemotherapy. In contrast, a switch to chemotherapy induced ERCC1 CTC signals in two cases. In three other cases, ERCC1 CTC signals persisted despite the patients having successfully responded to the chemotherapy administered. In individual patients, only signals from a single analyte were particularly prominent at all time points. Conclusion: Among the parameters tested for the purposes of monitoring, most of the top 15 originated from the analysis of CTCs and therefore, underscore their salience in the field of liquid biopsy. Additionally, the dynamic development of overexpression signals in EVs during the course of therapy proved to be an important indicator of disease progression and might therefore also be useful for therapy monitoring. Combination of these results with the analysis of cfDNA variants over time, as done with our index patients, underlines the usefulness of this multimodal liquid biopsy approach for therapeutic decision-making in the future.
Citation Format: Sabine Kasimir-Bauer, Markus Storbeck, Peter Hahn, Siegfried Hauch, Markus Sprenger-Haussels, Oliver Hoffmann, Rainer Kimmig, Corinna Keup. Dynamics of a multi-parametric liquid biopsy, including CTCs, EVs, and cfDNA, during therapy in metastatic breast cancer patients provide useful insights for therapy management [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-24.