Detection and Characterization of Circulating Tumor Associated Cells in Metastatic Breast Cancer

Abstract

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.

Keywords: cancer associated macrophage-like cells (CAMLs); circulating tumor associated cells; circulating tumor cell clusters (CTC clusters); circulating tumor cells (CTCs); epithelial–mesenchymal transition (EMT); metastatic breast cancer (MBC); size-exclusion technology.

Figure 1

Detection of circulating cancer associated cells in metastatic breast cancer (MBC): (A) Representative immunostaining images from three patient samples with identifications (N-S-001-87, M-V-002-129, P-I-005-44) showing circulating tumor cells (CTCs) and CTC clusters with cytokeratin (CK) and 4′,6-diamidino-2-phenylindole (DAPI) positive and CD45 negative staining; (B) Immunostaining of epithelial–mesenchymal transition (EMT) CTCs showed mesenchymal markers Vimentin or N-Cadherin positive; (C) Representative images of cancer associated macrophage-like cells (CAMLs) from MGG (May-Grunwald Giemsa) and immunostaining, cytological phenotypes as: spindle-shaped (a,d); round (b,e); oblong (c); cytokeratin positive (f); CD45 positive (g); DAPI (h); and composite image of CK and DAPI (i). The images were acquired on microscope at 40× magnification.

Figure 2

Overall processing workflow of blood sample on: ScreenCell^® Cyto device (A); and DEPArray™ platform (B). Step 1: 3 mL of blood was processed; Step 2: Cell suspension was recovered from the IS and transferred into 1.5 mL of Lo-bind eppendorf tube; Step 3: Enriched cells were stained by a manual staining procedure; Step 4: Cells were resuspended in a final volume of 14 µL and loaded in a DEPArray cartridge; Step 5: Sorting procedures were performed and single cells or pools of cells were isolated on DEPArray™ platform; Step 6: Isolated cells were subjected to WGA using the Ampli1 WGA kit and mutation analysis.

Figure 3

Genomic characterization of isolated CTCs. (A) Composite images visualized on the DEPArray™ platform after ScreenCell Cyto enrichment showing single CTCs (CK-PE⁺/DAPI⁺) and WBCs (CD45-APC⁺/DAPI⁺); (B) Gel images of Ampli1 quality control (QC) PCR products from CTCs and WBC showing the genome integrity with the presence of short, medium and long DNA fragments; (C) The TP53 exon 6 R248W mutations were detected in single CTCs and pools of CTCs, wild-type sequences in single WBC; (D) Wild-type ESR1 sequences were detected in all isolated CTCs.