Abstract PS4-02-08: Circulating tumor cell (CTC) transcriptional profiling identifies diverse CTC phenotypes in metastatic breast cancer

Abstract Background: Breast cancer is a heterogeneous disease with multiple transcriptionally defined subtypes including luminal A and B hormone receptor (HR) positive subtypes, the HER2 amplified subtype, and the basal subtype most commonly associated with clinically triple negative breast cancer (TNBC). These transcriptional subtypes have differing disease biology, clinical prognosis, and treatment vulnerabilities, and studies utilizing paired tissue biopsies have demonstrated that subtype switching/evolution can occur from primary tumor to metastatic recurrence, as well as in metastatic disease over time on treatment. However, understanding the role of subtype switching in treatment response and resistance has been limited by the challenges of obtaining serial tissue biopsies from patients. Liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), are a source of tumor-derived material shed into the blood of patients with cancer. Because liquid biopsies require only a peripheral blood draw, they are uniquely suited for longitudinal sampling to evaluate for subtype evolution in metastatic breast cancer. Here, we report the development of a targeted gene expression assay for genes associated with estrogen and HER2 signaling as well as luminal and basal identity in breast cancer CTCs, to address this need. Methods: 15mL peripheral blood was collected in EDTA vacutainer tubes from patients with metastatic breast cancer. CTCs were isolated with automated mTAE technology integrating live cell CTC capture using anti-EpCAM-conjugated paramagnetic particles with RNA extraction. CTC RNA underwent pre-amplification followed by qRT-PCR for genes related to epithelial cell identity, estrogen signaling, HER2 and other growth factors, basal differentiation, as well as controls for immune cell content and housekeeping genes. Expression values were calculated as 33-Ct values. Epithelial/CTC content score was calculated by summing the expression of the epithelial cell identity genes (EpCAM, KRT18, KRT19). Samples with epithelial content score 10 were classified as having high CTC content. Results: CTCs were detected in 34/51 (66.6%) of HR+ samples, 3/3 (100%) of TNBC samples, and 2/3 (66.6%) of HER2+ samples. 27/51 (52.9%) of HR+ samples, 2/3 (66.6%) of TNBC samples, and 1/3 (33.3%) of HER2+ samples were classified as having high CTC content. In patients with progressing disease (24 samples from 21 patients), 20/24 (83.3%) had CTCs detected and 17/24 (70.8%) had high CTC content. In patients with stable disease (19 samples from 15 patients) 14/19 (73.7%) samples had CTCs detected and 8/19 (42.1%) samples had high CTC content. Hierarchical clustering of high CTC content samples by expression of the estrogen signaling, HER2/growth factor, and basal genes demonstrated clusters consistent with differing transcriptional subtypes. Among HR+ HER2 non-amplified samples (n=22), higher estrogen signaling gene expression was associated with shorter progression free survival (median 2.2 versus 6.9 months, log rank p0.05) Conclusions: In a pilot cohort of 37 patients with metastatic breast cancer, we demonstrate the feasibility of detecting gene expression relevant to breast cancer transcriptional subtypes via liquid biopsy circulating tumor cell gene expression profiling and relatively high sensitivity even in patients with stable disease, with CTCs detected in 73% of samples including 42% with high CTC content. Interestingly, in HR+ breast cancer CTCs in this cohort, higher estrogen-regulated gene expression was associated with shorter progression free survival, suggesting that this assay could also be utilized to monitor response to anti-estrogen therapy in this patient population. Citation Format: V. Carreno, A. H. Chang, I. G. Fernandez, R. Qamar, M. T. West, K. B. Wisinski, M. N. Sharifi. Circulating tumor cell (CTC) transcriptional profiling identifies diverse CTC phenotypes in metastatic breast cancer. abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-02-08.