Abstract 5366: Metastatic breast cancer subgroup specific mRNA profiles of circulating tumor cells and extracellular vesicles
Authors: Keup Corinna , Hauch Siegfried , Tewes Mitra , Kolberg Hans-Christian , Hoffmann Oliver , Kimmig Rainer , Kasimir-Bauer Sabine 
Published: September 2020

Abstract
Background: We recently demonstrated great differences in the RNA profiles of matched circulating tumor cells (CTCs) and extracellular vesicles (EVs) in HER2 negative (HER2-) hormone receptor positive (HR+) metastatic breast cancer (MBC) patients. To elucidate the clinical utility of CTC and EV RNA profiling, we here a) tested the RNA profiles of another MBC subgroup, namely the HER2 positive (HER2+) patients, and b) compared the profiles in the two cohorts.
Patients and methods: Blood was collected from 25 HER2+ (HR+ and HR-) and 35 HER2- (only HR+) MBC patients at the time of disease progression and at two consecutive staging time points. CTCs were isolated from 5 ml blood by positive immunomagnetic selection targeting EpCAM, EGFR and HER2 (AdnaTest EMT2/StemCell Select). EVs were isolated from 4 ml pre-filtered plasma by affinity-based binding to a spin column (exoRNeasy). The mRNA was purified by Oligo-dT beads and reverse transcribed (AdnaTest EMT2/StemCell Detect). Pre-amplified cDNA was analyzed by multimarker qPCR assays (AdnaTest TNBC Panel prototype) for 18 genes (including AKT2, ALK, AR, AURKA, BRCA1, KIT, MET, EGFR, ERCC1, ERBB2, ERBB3, KRT5, mTOR, NOTCH1, PARP1, PIK3CA, SRC, GAPDH). Consumables: QIAGEN, Germany.
Results: In the HER2+ cohort, transcripts involved in the PI3K pathway were significantly more prevalent in CTCs compared to EVs and were found in more than 50% of these patients. In contrast, NOTCH1 and PARP1 were significantly more prominent in EVs as compared to their matched CTCs and EV PARP1 and EV AURKA overexpression signals were detected in more than 50% of patients. ERCC1 and SRC signals were present in CTCs as well as EVs in the majority of patients. ERCC1 signals in CTCs and EVs showed a significantly higher frequency in the HER2+ compared to the HER2-HR+ cohort. However, ERCC1 signals in CTCs of the HER2-HR+ cohort correlated with shorter survival time after first diagnosis of metastasis (p=0.011). In general, the overexpression signal prevalence was similar in both cohorts and both blood analytes. A small overlap of identical signals in CTCs and EVs of 15%/5% in the HER2+/HER2-HR+ cohort was observed. Interestingly, ERBB2 signals were only found in CTCs, not EVs, and in both cohorts with a similar prevalence (36%/40%). ERBB3 overexpression signals in CTCs significantly correlated with non-response only in the HER2-HR+ cohort (p=0.021). However, in HER2+ patients ERBB3 signals were only present at the time of progressive disease, but not at the time point of stable disease.
Conclusions: RNA profiling indicated great differences in CTCs and EVs in both subgroups. Moreover, data emphasize the immense diversity between HER2+ versus HER2-HR+ MBC patients, revealing the need for blood analyte specific and subgroup specific multimarker panel for therapy management based on liquid biopsies in the future.
Citation Format: Corinna Keup, Siegfried Hauch, Mitra Tewes, Hans-Christian Kolberg, Oliver Hoffmann, Rainer Kimmig, Sabine Kasimir-Bauer. Metastatic breast cancer subgroup specific mRNA profiles of circulating tumor cells and extracellular vesicles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5366.