Abstract P5-02-25: Transcriptional profiling of CTCs in metastatic breast cancer patients in the course of CDK4/6 inhibition
Authors: Kasimir-Bauer Sabine , Gruber Charlotte , Moukas Stefanos , Tewes Mitra , Kolberg Hans-Christian , Kimmig Rainer , Keup Corinna 
Published: March 2023

Abstract
Background: De novo resistance defined as progression within six months and acquired resistance are one of the major problems in the subset of metastatic (M), hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) patients without visceral crisis receiving CDK4/6 inhibitors (CDK4/6i) plus endocrine therapy (TX). Here, we aim to identify predictive and monitoring markers of CDK4/6i resistance by conducting transcriptional profiling of circulating tumor cells (CTCs) that represent a real-time snapshot of the heterogeneity. Methods: Blood of (A) 60 HR+/HER2- MBC patients drawn at baseline of Palbociclib plus endocrine TX (TX as first line n=31, second or more lines n=29), (B) 19 HR+/HER2- MBC patients drawn before the initiation of endocrine monoTX (control) and matched blood samples of these patients after six months under TX (n=72) and at the time of progression (n=42) were analyzed. To enlarge the global CDK4/6i cohort at baseline, blood of (C) 32 patients before the initiation of Ribociclib plus endocrine TX was also drawn. Patients with progression within six months were defined as non-responders. Isolation of CTCs was conducted using positive immunomagnetic selection (AdnaTest EMT2/StemCell Select) and preamplified cDNA was analyzed by a multimarker qPCR panel utilizing QuantiNova LNA Probe assays targeting 25 genes involved in the DNA damage -, MAPK -, STAT -, Hippo – pathway or cell cycle, chemokine sensing, multidrug resistance and cell adhesion. qPCR data was normalized to CD45 and data of 20 healthy female donor controls to identify BC CTC specific overexpression signals with a specificity of &gt;90% for all targets. Consumables: QIAGEN, Germany. Statistical analysis included log-rank testing and univariate Cox regression. Results: For first line CDK4/6i treated patients at baseline, CETN2 and E2F1 signals correlated significantly with worse progression-free survival (PFS) while CETN2 signals also related significantly to non-response. Furthermore, CETN2 and PCNA signals were significantly associated with worse overall survival (OS). Analyzing the Palbociclib cohort after six months of TX, PCNA signals correlated significantly with a decreased PFS while EpCAM signals showed a significant association with OS. In addition, CETN2, CXCR4, EpCAM, MLH3, WWTR1 signals after six months were shown to correlate significantly with a decreased OS and PFS and MAPK1 signals were only found in the non-responders. While non-response was related to appearing (from baseline to six months under TX) ABCC2, JUN and MAPK1 signals, disappearing ABCC2 signals were only found in the responders. Dynamics of ABCC2, CXCR4, EpCAM, JUN, MAPK1, MLH3, STAT1 and WWTR1 signals from baseline to six months under TX correlated significantly with OS and CXCR4 signal dynamics significantly with a worse PFS. At the time of progression, the presence of E2F1, JUN, MAPK1 and STAT1 signals correlated significantly with a decreased OS and in comparison to baseline analysis, the prevalence of ABCC2, EpCAM, E2F1, CETN2 and CXCR4 signals increased. Conclusion: CTC overexpression signals at baseline of targets involved in the cell cycle (CETN2 and E2F1) might be predictive markers for de novo resistance to CDK4/6i as first line TX, while ABCC2 (multidrug resistance), EpCAM (cell adhesion), E2F1, CETN2 and CXCR4 (chemokine sensing) signals could indicate acquired resistance to Palbociclib. In case of disease progression, E2F1, JUN (cell cycle) and targets of the MAPK- and STAT- pathway could be relevant targets for therapeutic strategies beyond Palbociclib TX. Monitoring and prognostic value was shown for single and repeated measurement of signals under TX in genes involved in resistance, cell cycle progression, DNA damage response (MLH3, PCNA), chemokine sensing, cell adhesion and the MAPK-, STAT- and Hippo (WWTR1) pathway.
Citation Format: Sabine Kasimir-Bauer, Charlotte Gruber, Stefanos Moukas, Mitra Tewes, Hans-Christian Kolberg, Rainer Kimmig, Corinna Keup. Transcriptional profiling of CTCs in metastatic breast cancer patients in the course of CDK4/6 inhibition [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-25.