Abstract P5-13-33: Longitudinal transcriptional profiling of CTCs in metastatic breast cancer patients receiving the CDK4/6 inhibitor Palbociclib to predict therapy response
Authors: Kasimir-Bauer Sabine , Gruber Charlotte , Hoffmann Oliver , Kimmig Rainer , Keup Corinna 
Published: March 2022

Abstract
Background: CDK4/6 inhibitors represent a new treatment option for metastatic (M), hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) patients. However, predictive markers have not been defined to decide who will benefit from CDK4/6 inhibitors. Circulating tumor cells (CTCs) represent the tumoral heterogeneity in real time and are available for molecular characterization via minimal-invasive sequential blood sampling. Here, we aim to identify biomarkers of resistance to Palbociclib by conducting transcriptional profiling of CTCs before therapy initiation (baseline) and after six months under treatment. Methods: Blood of 50 HR+/HER2-MBC patients drawn at baseline of Palbociclib plus endocrine treatment and blood of 20 HR+/HER2-MBC patients drawn before initiation of endocrine monotherapy, as well as blood samples of these 70 patients after six months under treatment will be analyzed. Patients with progressive disease within the first six months of treatment were defined as non-responders. Isolation of CTCs was conducted using positive immunomagnetic selection targeting EpCAM, EGFR and HER2 (AdnaTest EMT2/StemCell Select). cDNA was analyzed by a new multimarker qPCR panel utilizing QuantiNova LNA Probe assays targeting 25 genes. qPCR data was normalized to CD45 to substract the effect of contaminating leukocytes. Additionally, data was normalized to 20 healthy female donor controls to identify BC specific overexpression signals with a specificity of &gt;90% for all targets. Consumables: QIAGEN, Germany. Results: We successfully established the multimarker qPCR according to the MIQE guidelines. Up to now, the binary mRNA overexpression signals of 25 targets in CTCs isolated at baseline were analyzed in 40 of the total 70 planned MBC patients. In detail, 24 of those 40 patients were treated with Palbociclib plus endocrine therapy, including seven responders and 15 non-resonders, while the 16 other patients were treated with endocrine therapy alone (seven non-responders and nine responders). In the entire cohort, STAT1 signals were significantly related to a decreased overall survival (p=0.0034) and NFKB1 signals correlated significantly with an increased progression free survival (PFS; p=0.027). In the Palbociclib group, CDK2, WWTR1 and YAP1 overexpression signals were more common in non-responders compared with responders. MLH1 and NFKB1 signals were more common in responders than in non-responders. In this cohort, MLH3 overexpression at baseline was significantly correlated with prolonged PFS (p=0.047) and the patient with the longest PFS (35 months) was the only patient with detectable ERBB4, JUN, CETN2 and TEAD2 signals. In the control group treated with endocrine therapy alone, signal prevalence of CXCR4 and STAT1 were increased in the non-responders versus responders and ABCC2, ESR1, FAT4 and FGFR1 signals were more frequently detected in the responders. Conclusion: Transcriptional profiling of CTCs represents a real-time snapshot of the disease complexity. Characterization of the CTCs isolated from blood drawn at baseline is promising for the identification of urgently needed predictive markers (here MLH3 overexpression), while characterization of CTCs isolated under treatment can elucidate the transcriptional dynamics and might be used as monitoring marker by differentiation of non-responders and responders. We will finalize CTC analysis of the entire cohort and will continue blood sampling of additional Palbociclib treated patients, also in the follow-up of the disease, to validate CTC MLH3 overexpression as predictive biomarker for CDK4/6 inhibitor treatment and moreover, to find further overexpression signals in CTCs relevant for MBC therapy management.
Citation Format: Sabine Kasimir-Bauer, Charlotte Gruber, Oliver Hoffmann, Rainer Kimmig, Corinna Keup. Longitudinal transcriptional profiling of CTCs in metastatic breast cancer patients receiving the CDK4/6 inhibitor Palbociclib to predict therapy response [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-33.