Phenotypic Plasticity in Circulating Tumor Cells Is Associated with Poor Response to Therapy in Metastatic Breast Cancer Patients

Simple Summary Circulating tumor cells (CTCs) have served as an independent prognostic factor in the management of metastatic breast cancer (MBC). Through the enrichment of CTCs from peripheral blood, tumor cells can be acquired multiple times during therapy and provide a broad sample of tumor heterogeneity, thereby offering a complementary approach to tissue biopsy. Traditionally, CTCs have been enriched from blood based on the expression of epithelial-specific surface proteins. However, this approach might miss the migratory cells that lack epithelial features and favor the expression of more mesenchymal features. Therefore, enrichment of CTCs based on size and deformability may capture a wider range of tumor cells in circulation. Here we present a longitudinal study using a novel microcavity array to enrich CTCs and find that a shift from epithelial CTCs to those with a mesenchymal expression pattern is associated with inferior clinical outcomes. Circulating tumor cells (CTCs) are indicators of metastatic spread and progression. In a longitudinal, single-center trial of patients with metastatic breast cancer starting a new line of treatment, a microcavity array was used to enrich CTCs from 184 patients at up to 9 timepoints at 3-month intervals. CTCs were analyzed in parallel samples from the same blood draw by imaging and by gene expression profiling to capture CTC phenotypic plasticity. Enumeration of CTCs by image analysis relying primarily on epithelial markers from samples obtained before therapy or at 3-month follow-up identified the patients at the highest risk of progression. CTC counts decreased with therapy, and progressors had higher CTC counts than non-progressors. CTC count was prognostic primarily at the start of therapy in univariate and multivariate analyses but had less prognostic utility at 6 months to 1 year later. In contrast, gene expression, including both epithelial and mesenchymal markers, identified high-risk patients after 6-9 months of treatment, and progressors had a shift towards mesenchymal CTC gene expression on therapy. Cross-sectional analysis showed higher CTC-related gene expression in progressors 6-15 months after baseline. Furthermore, patients with higher CTC counts and CTC gene expression experienced more progression events. Longitudinal time-dependent multivariate analysis indicated that CTC count, triple-negative status, and CTC expression of FGFR1 significantly correlated with inferior progression-free survival while CTC count and triple-negative status correlated with inferior overall survival. This highlights the utility of protein-agnostic CTC enrichment and multimodality analysis to capture the heterogeneity of CTCs.