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

Abstract

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.

Keywords: EMT; biomarkers; biopsy; blood; breast cancer; breast neoplasms/pathology; circulating; circulating tumor cells (CTCs); circulating/pathology; liquid biopsy; metastatic process; neoplasms/diagnosis; neoplastic cells; tumor.

Figure 1

CTC count distribution. (a) Range of CTC counts per ~9.5 mL of blood across time points. (b) Swimmer plots of CTC counts demonstrate that enumeration is prognostic. (c) Swimmer plots of CTC count from cases that progressed and those that did not progress demonstrate the effect of therapy. (d) CTC enumeration at 3 months confirms ≥5 CTCs/9.5 mL of blood as the most relevant cutoff. Median PFS for patients above (green) and below (red) each CTC threshold (left y axis) and associated hazard ratio (blue, right y axis, dashed blue line HR = 1). CTC, circulating tumor cell; HR, hazard ratio; PFS, progression-free survival.

Figure 2

CTC count ≥5 is prognostic at the start of therapy for MBC. (a) Forest plots showing Cox proportional hazard for ≥5 CTCs/~9.5 mL of blood at each timepoint for PFS and OS. (b) Kaplan-Meier plots for PFS and OS stratified by ≥5 CTCs ~9.5 mL of blood. (c) Kaplan-Meier plots stratified by clearance of ≥5 baseline CTCs by visits 2 and 3. (d) Mean cumulative progression events for ≥5 CTCs vs. <5 CTCs at baseline and progression and death rates per 100 person-years. CTC, circulating tumor cell; OS, overall survival; PFS, progression-free survival. *** p < 0.0001.

Figure 3

CTC gene expression. (a) Heat map of gene expression Z-score normalized to HD samples. (b) Mean sum of CTC-related gene expression (epithelial, mesenchymal, and total) stratified by progression. (c) Mean expression of 5 individual genes stratified by progression. (d) Waterfall plot showing a change in CTC-related gene expression from baseline for the same 5 genes. CTC, circulating tumor cell. CR, complete response; HR, hormone receptor (ER or PR); PD, progressive disease; PR, partial response; SD, stable disease; TNBC, triple negative (for ER, PR, and HER2).

Figure 4

Expression of at least 4 CTC-related genes is prognostic for evaluation of response to therapy. (A,B) Forest plots showing Cox proportional hazard for PFS and OS stratified by ≥4 CTC-related genes at each timepoint. (C,D) Kaplan-Meier plots for PFS and OS stratified by ≥4 CTC-related genes. CTC, circulating tumor cell; PFS, progression-free survival; OS, overall survival.

Figure 5

CTC clusters and EMT plasticity (a) Example of CTC cluster. (b) CTC clusters are more frequent in patients who experience progression at visits 3 (6 months) and 5 (1 year). (c) PFS by detection of clusters. (d) Median EM score stratified by patients with and without CTC clusters and in terms of their disease condition; size proportional to total CTC count. (e) Median EM scores across timepoints stratified by patients with and without progression where positive has greater mesenchymal gene expression and negative has greater epithelial gene expression, irrespective of the presence of clusters. (f) PFS by a change in EM scores from baseline. CK: pan-cytokeratin antibody; E: epithelial; M: mesenchymal; PFS, progression-free survival.