Abstract 1989: Heterogeneity of epithelial, mesenchymal, progenitor, and stem cell phenotypes on CTCs correlates to clinical outcomes in metastatic cancer patients

Abstract Circulating tumor cells (CTCs) are cancer cells that detach from tumors, enter the blood and seed distant sites. CTCs spread as single cells, or cluster aggregates, and can be heterogenous with differing biological phenotypes, i.e. epithelial, mesenchymal, progenitor, & stem cell. Three clinically relevant CTC subtypes are often described in patients (pts) 1) CTCs with normal epithelial traits (strong filamentous cytokeratin (CK+ & CD45-/CD14-), 2) Epithelial-mesenchymal CTCs (EMTs), cancer cells with downregulated epithelial & upregulated mesenchymal traits (low diffused CK+, CD45-/CD14-) and 3) tumor-macrophage fusion cells (TMFCs), cancer cells that fuse with macrophages (polynucleated DAPI+, diffuse CK+ and CD14+/CD45+). To better elucidate the biological and clinical importance of the 3 CTC subtypes, we isolated CTCs by a marker agnostic method from the blood of n=49 metastatic cancer pts. We then enumerated and phenotypically subtyped CTCs comparing clinical utility of clustering and CTC heterogeneity over 2 years. Peripheral blood samples were collected from 49 metastatic cancer pts, breast (n=38), pancreatic (n=9), colon (n=3), lung (n=1), and prostate (n=1). Whole blood (7.5mL) was filtered by size exclusion using CellSieve microfilters and fluorescently stained for CD45, CD14, CK, and DAPI to identify CTCs, EMTs and TMFCs. Cells were imaged, enumerated and fluors quenched by the QUAS-R technique; followed by re-stained for CD44, CD24, CD133, CD144, N-Cadherin, EpCAM, and Vimentin. Markers were categorized as negative, low, medium, or high. A heterogeneity biomarker score was developed as high, 2-3 log deviations between marker expression, or low 0-1. Pt progressive free survival (PFS) and overall survival (OS) were analyzed over 2 years. CTCs were found in 40% of pts, EMTs in 51%, & TMFCs in ∼93%. For subtyping, CD44 was in CTCs 88%, EMTs 59%, & TMFCs 91%; CD24 in CTCs 40%, EMTs 41%, & TMFCs 6%; CD133 in CTCs 100%, EMTs 8%, & TMFCs 100%; CD144 in CTCs 100%, EMTs 77%, & TMFCs 82%; N-cadherin in CTCs 100%, EMTs 0%, & TMFCs 45%; Vimentin in CTCs 51%, EMTs 100%, & TMFCs 83%; EpCAM in CTCs 74%, EMTs 12%, & TMFCs 20%. CTCs and EMTs were found to have high heterogeneity in 30% of samples in both individual cells and clusters, with a high biomarker heterogeneity significantly correlated with poorer outcomes (PFS: HR=15.4, p=0.0014, CI=3.4-68.9; OS: HR=12.4, p=0.0056, CI=2.6-59.1). TMFCs were found to have high heterogeneity, 40% of samples, but this was not correlated with outcomes. In this pilot study, it appeared that isolated CTC, EMTs and TMFCs subtypes have differing heterogenous expressions of epithelial, mesenchymal, progenitor, and stem cell markers. Further, high rates biomarker heterogeneity in CTCs and EMTs, but not TMFCs, correlated with faster rates of progression & death. Larger validation experiments with biomarker stratification is ongoing. Citation Format: Alexis B. Duffy, Massimo Cristofanilli, Carolina Reduzzi, Cha-Mei Tang, Saranya Chumsri, Susan Tsai, Mohammed Aldakkak, Daniel L. Adams. Heterogeneity of epithelial, mesenchymal, progenitor, and stem cell phenotypes on CTCs correlates to clinical outcomes in metastatic cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1989.