Detection and Characterization of Circulating Tumor Associated Cells in Metastatic Breast Cancer
The availability of blood-based diagnostic testing using a non-invasive technique holds promise for real-time monitoring of disease progression and treatment selection. Circulating tumor cells (CTCs) have been used as a prognostic biomarker for the metastatic breast cancer (MBC). The molecular characterization of CTCs is fundamental to the phenotypic identification of malignant cells and description of the relevant genetic alterations that may change according to disease progression and therapy resistance. However, the molecular characterization of CTCs remains a challenge because of the rarity and heterogeneity of CTCs and technological difficulties in the enrichment, isolation and molecular characterization of CTCs. In this pilot study, we evaluated circulating tumor associated cells in one blood draw by size exclusion technology and cytological analysis. Among 30 prospectively enrolled MBC patients, CTCs, circulating tumor cell clusters (CTC clusters), CTCs of epithelial-mesenchymal transition (EMT) and cancer associated macrophage-like cells (CAMLs) were detected and analyzed. For molecular characterization of CTCs, size-exclusion method for CTC enrichment was tested in combination with DEPArray™ technology, which allows the recovery of single CTCs or pools of CTCs as a pure CTC sample for mutation analysis. Genomic mutations of TP53 and ESR1 were analyzed by targeted sequencing on isolated 7 CTCs from a patient with MBC. The results of genomic analysis showed heterozygous TP53 R248W mutation from one single CTC and pools of three CTCs, and homozygous TP53 R248W mutation from one single CTC and pools of two CTCs. Wild-type ESR1 was detected in the same isolated CTCs. The results of this study reveal that size-exclusion method can be used to enrich and identify circulating tumor associated cells, and enriched CTCs were characterized for genetic alterations in MBC patients, respectively.
Did circulating tumor cells tell us all they could? The missed circulating tumor cell message in breast cancer
Purpose: To compare circulating tumor cell (CTC) detection rates in patients with early (M0) and metastatic (M+) breast cancer using 2 positive-selection methods or size-based unbiased enrichment.
Methods: Blood collected at baseline and at different times during treatment from M0 patients undergoing neoadjuvant therapy and from M+ women starting a new line of treatment was processed in parallel using AdnaTest EMT-1/ and EMT-2/Stem CellSelect/Detect kits or ScreenCell Cyto devices. CTC positivity was defined according to the suggested cutoffs and cytological parameters, respectively.
Results: Higher CTC detection rates were obtained with the AdnaTest approach when using for CTC-enrichment antibodies against ERBB2 and EGFR in addition to MUC1 and the classical epithelial surface marker EPCAM (13% vs. 48%). In M0 patients mainly, CTC positivity rates further increased when EMT- and stemness-related marker expression (PIK3CA, AKT2 and ALDH1) was evaluated in addition to EPCAM, MUC1 and ERBB2. When the physical properties of tumor cells were exploited, CTCs were detected at higher percentages than with positive-selection-based methods, without any difference between clinical stages (78% in M0 vs. 72% in M+ cases at baseline). Circulating tumor microemboli (CTMs) were detected in addition to single CTCs with significantly higher frequency in M0 than M+ samples (78% vs. 27%, p = 0.0002).
Conclusions: Different approaches for CTC detection probably identify distinct tumor cell subpopulations, but need technical standardization before their clinical validity and biological specificity may be adequately investigated. The distinct role of CTMs compared with CTCs as prognostic and predictive biomarkers represents a further challenge.