Abstract P4-01-10: The analysis of cell-free DNA and circulating tumor cells from one blood tube might empower treatment decisions in metastatic breast cancer patients
Authors: Kasimir-Bauer S , Bittner A-K , Hoffmann O , Hauch S , Sprenger-Haussels M , Storbeck M , Benyaa K , Hahn P , Mach P , Tewes M , Kimmig R , Keup C 
Published: May 2019

Abstract
Background: The detection and characterization of circulating tumor cells (CTCs) as one of the analytes in liquid biopsy has been considered as surrogate marker to improve treatment decisions in metastatic breast cancer (MBC). In addition, cell-free tumor DNA (ctDNA) released by tumor cells and harboring tumor-associated variants is further discussed to give additional information for therapeutic options. Thus, CTC and ctDNA analysis from the same blood tube is desired. To test usability of plasma, generated after CTC isolation from whole blood for ctDNA analysis, we analyzed ctDNA from 42 hormone receptor-positive/HER2-negative MBC patients (pts) for the detection of tumor-associated variants (plasma isolated straight from whole blood) and compared the results for similarities and differences of the detected variants in a subgroup of these pts to those, obtained from plasma generated after CTC selection (taken from a separate tube).
Methods: 4 ml plasma of all MBC pts and 4 ml plasma obtained after immunomagnetic isolation of CTCs from 2x5ml blood [AdnaTest EMT-2/Stem Cell Select (n=17pts) followed by multimarker qPCR] were used for the analysis of cell-free DNA (cfDNA) applying the QIAamp MinElute ccfDNA Kit. A total of 30ng - 60ng cfDNA was applied for library construction using the QIAseq Targeted DNA Panel for Illumina with integrated unique molecular identifiers. Sequencing was executed on the NextSeq® 500 platform (Illumina, US). Data were analyzed using the QIAseq Targeted Sequencing Data Analysis Portal, the Biomedical Genomics Workbench and the Ingenuity Variant Analysis. All materials used were manufactured by QIAGEN, Germany.
Results: In the total cohort of 42 pts, most variants of all analyzed genes were detected in the MUC16 gene (31.2%). ERBB2, EGFR and AR (androgen receptor) also showed high numbers of variants (11.6%, 11.0% and 8.9%, respectively) with a majority detected pathogenic variants (47.7%) in AR. 92% of all detected variants showed an allele frequency of &lt;5% and some of the detected MUC16, ERBB2 and AR mutations significantly correlated with overall survival. Comparing the plasma results from a separate blood draw with the results from plasma samples after CTC selection in a subgroup of 17/42 pts, no significant difference was found for cfDNA concentration but variability within the cohort. Whereas the variant comparison of ctDNA isolated from both plasma sources showed great concordance, additional variants (around 15%) were exclusively found in one of the two matched samples. Interestingly, in the variant population exclusively found in ctDNA isolated after CTC isolation, the relative amount of pathogenic variants was increased compared to the variant fraction only found in ctDNA from plasma of a separate blood tube. Results obtained for frequently overexpressed CTC transcripts in this subgroup included genes involved in the PI3K signaling pathway as well as ERBB2 and ERBB3 in about 30% of the pts.
Conclusion: We here present a feasible workflow for CTC and ctDNA evaluation for expression and mutation analysis from the same blood sample. These data emphasize that the use of different liquid biopsy analytes can empower treatment decisions of MBC pts in the future.
Citation Format: Kasimir-Bauer S, Bittner A-K, Hoffmann O, Hauch S, Sprenger-Haussels M, Storbeck M, Benyaa K, Hahn P, Mach P, Tewes M, Kimmig R, and Keup C. The analysis of cell-free DNA and circulating tumor cells from one blood tube might empower treatment decisions in metastatic breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-10.