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.

Sequential Tracking of PD-L1 Expression and RAD50 Induction in Circulating Tumor and Stromal Cells of Lung Cancer Patients Undergoing Radiotherapy

“…Furthermore, only a small fraction of patients will actually respond to PD-L1/PD-1 therapy, indicating that the reported CTC assays may be overly sensitive for detecting PD-L1 and will unlikely translate to clinical benefit for PD-L1/PD-1 therapies (16,(47)(48)(49)). An alternative issue may be that many CTC technologies do not differentiate the EMTCTC subpopulation from the CellSearch CTC population (i.e., PDCTCs), or make any mention of CStCs (3,4,6,7,16,17,(47)(48)(49). These methods describe a BBB approach that differentiates CAMLs and the PDCTC/EMTCTC subpopulations by well-defined intrinsic biological indicators, that is, EMTCTCs lose EpCAM and downregulate cytokeratin (6,10,11,13,14,43).…”

“…EMTCTCs are CD45 negative with a diffuse cytokeratin signal and a DAPI-positive nucleus with abnormal criteria, as defined previously (1, 6-9, 12, 13). CAMLs are described as enlarged (>30 mm), multinuclear cells with diffuse cytoplasmic cytokeratin staining, and/or CD45 þ /CD14 þ (6,11,14,17,22,43). All three cell types were identified and imaged by a trained CTC cytologist and confirmed by a pathologist.…”

“…11,13,14,17]. CAMLs are defined by their CD45/CD14 positivity and giant size phenotype (11,13,14,17). Although CAMLs appear to be cancer specific and disseminate from the organ sites of malignancy, it remains unknown whether they actually reside at the primary tumor site or whether they possess clinical utility.…”

“…CAMLs are recently defined circulating myeloid-derived stromal cells, found in all the stages of invasive malignancy and in various solid malignancies [e.g., breast, prostate, non-small cell lung carcinoma (NSCLC), and pancreatic; refs. 11,13,14,17]. CAMLs are defined by their CD45/CD14 positivity and giant size phenotype (11,13,14,17).…”

Prospective study of the relevance of circulating tumor cell status and neoadjuvant chemotherapy effectiveness in early breast cancer

“…Breast cancer (BC) is the most common cancer among women worldwide, while distant metastasis remains the major cause of mortality. 1 Among various therapeutic strategies, neoadjuvant chemotherapy (NCT) take a core position in systematic treatment of local advanced BC. 2 Except for its downstage effect, NCT also provides information about the chemosensitivity of tumor cells.…”