15: Circulating Tumor Cells Identified in Early-stage Breast Cancer Patients Using Fluorescence in Situ Hybridization Can Predict Therapy Resistance

C. Liu, P.A. Althof, D. Maroni, J.M. Stevens, C.E. Grabow, A.Z. VanDyke, J.D. Price, J.N. Sanmann, S.P. Thayer*, Surgical Oncology, University of Nebraska Medical Center, Omaha, Nebraska, UNITED STATES|

Introduction: Fluorescence in situ hybridization (FISH)-identified circulating tumor cells (CTCs) found in patients on anti-estrogen therapy are the earliest and most specific indicator of anti-estrogen therapy resistance. Molecular characterization of these CTCs and their parent tumor will allow insight into their mechanism of resistance and enable the development of individualized therapy tailored to specific residual tumor burden.

Methods: Primary tumor DNA was analyzed to select tumor-specific FISH probes. Whole blood was collected longitudinally and evaluated pre- and post-treatment. CTCs were captured using ScreenCell® Cyto V2 devices and identified by FISH. A luminal tumor was evaluated by single cell RNA sequencing.

Results: At time of diagnosis, 53% percent of patients were positive for CTCs. The presence of CTCs did not correlate with tumor molecular subtype, stage, grade, or genetic complexity. In patients with early-stage, luminal tumors, 50% were found to be FISH CTC-positive prior to first treatment. Of the patients with FISH-CTCs pre-treatment, 75% were identified with FISH-CTCs post-treatment while on anti-estrogen therapy, suggesting that a significant proportion of patients have residual disease that is anti-estrogen therapy-resistant. In order to better determine the mechanism of resistance, single cell RNA-sequencing of a parent tumor identified three distinct populations, and analysis from one cluster identified 12 functional pathways with altered gene expression, including immune pathways, transcriptional changes, and ribonucleoprotein interactions. The analysis also identified a limited population of cells that expressed ERBB2, ESR1, and FGFR1, genes associated with anti-estrogen therapy resistance, underscoring the necessity to compare expression signatures observed in CTCs with those observed in primary tumors.

Conclusion: FISH-CTCs found in patients on anti-estrogen therapy represent the best and earliest opportunity to evaluate molecular mechanisms of resistance. The molecular characterization of these cells may allow the addition of targeted secondary therapies to decrease clinical recurrence.