Abstract P2-02-07: Unbiased liquid biopsy in breast cancer patients utilizing novel light sheet microscopy of circulating tumor cells. Proof of concept in 10 patients and 13 controls
Authors: Tannenbaum Susan , Hsu Emily , Reddy Ashwanth , Keilich Spencer , Tafas Triantafyllos 
Published: March 2022

Abstract
Background It is widely expected that circulating tumor cells (CTCs) in cancer patients shall turn out to be good prognosticators for outcomes. However, enumeration and monitoring of CTCs continues to pose serious hurdles. Current methods to assess CTCs utilize various approaches to enrich for CTCs. The only FDA-approved system detects CTCs via enrichment for EpCAM. Cancer heterogeneity requires an enrichment-free characterization of all CTCs to better identify this ever-changing landscape for biologic understanding and clinical management. Methods CLINBREAC is a single site, prospective, longitudinal trial for detecting and characterizing all CTCs in breast cancer patients. We present results from 8 metastatic and 2 neoadjuvant patients followed over time. Collection of 18ml blood samples occurred at enrollment and at change of treatment, or at 3-month intervals. Using the RareScope™ Fluorescence Light Sheet Microscope (QCDx LLC, Farmington CT), an aliquot of all morphologically intact, un-enriched nucleated cells placed in immobilized suspensions were analyzed. All cells were stained with immunofluorescent markers against CD45, Vimentin (Vim), EpCAM and Cytokeratin (CK) for characterization of epithelial or mesenchymal CTC phenotypes and trophoblast surface antigen 2 (TROP-2), ER-α and HER2 for treatment-specific characterization. Results No CTCs were observed in blood samples from 13 healthy volunteers. All patient samples contained CTCs collected over all time-points from 10 patients. In all, 1261 CTCs were identified, range, 18-85 CTCs per 1.7K to 2.7K nucleated cells analyzed. Each sample had a unique pattern of epithelial and mesenchymal markers that changed with time and treatment and mirrored clinical course. Of the CTC populations seen, 239 (19%) stained EpCAM+. Of the remaining CTCs, 798 CK+ (63%) and 842 Vim+ (67%) . An example: Patient 1: progression of metastatic triple negative breast cancer on nab-paclitaxel and atezolizumab after which anti-TROP-2 directed therapy with sacituzumab govitecan was initiated. In 39 CTCs present in sample 1, EpCAM-5%, CK-36%, and Vim-92% alone and co-expressed with EpCAM and CK. In subsequent samples, CTCs decreased from 39 to 23 to 6 consistent with response to treatment. TROP2+ CTCs decreased from 14/25 cells to 1/10 with treatment. CTCs co-expressed TROP2, HER2 and ER-α. As TROP2+ CTCs decreased from Sample 1 to 3 (56 to 10%) HER2+ CTCs increased (12 to 70%). The correlation of CTCs with clinical course of disease was observed in all 8 metastatic patients. Phenotypic changes occurred in all patients’ CTCs followed over time. Conclusions RareScope technology, utilized here, is a novel CTC-detection methodology that does not rely on prior enrichment of blood for CTCs. In patients analyzed here, all samples contained CTCs in higher numbers than described previously; a minority of CTCs contain EpCAM+ CTCs and may account for lower numbers in prior studies. Continuing refinement of this technology with addition of multiplex testing on single cells with a larger panel of antibodies will enhance targeted analysis and shall be useful in detection and detailed characterization of CTCs with un-precedented resolution and in real time.
Citation Format: Susan Tannenbaum, Emily Hsu, Ashwanth Reddy, Spencer Keilich, Triantafyllos Tafas. Unbiased liquid biopsy in breast cancer patients utilizing novel light sheet microscopy of circulating tumor cells. Proof of concept in 10 patients and 13 controls [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 P2-02-07.