Molecular biomarker analyses using circulating tumor cells

Abstract

Abstract Background: Evaluation of cancer biomarkers from blood and other accessible tissues could significantly enable biomarker assessment by providing a relatively noninvasive source of representative tumor material. Circulating tumor cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard. Methodology/Principal Findings: Using a tumor-cell spike-in model system we evaluated CTC capture on different CTC technology platforms, including CellSearch® and two biochip platforms, and used the isolated CTCs to develop and optimize assays for molecular characterization of CTCs. We report similar performance for the various platforms tested in capturing CTCs, and find that capture efficiency is dependent on the level of EpCAM expression. We demonstrate that captured CTCs are amenable to biomarker analyses such as HER2 status, qRT-PCR for breast cancer subtype markers, KRAS mutation detection, and EGFR staining by immunofluorescence (IF). We demonstrate that cell surface expression of EGFR can be quantitated in CTCs from metastatic lung cancer patient samples. In addition, we determined HER2 status by IF and FISH in CTCs from metastatic breast cancer patients and in the majority of patients (89%) found concordance with HER2 status from patient tumor tissue, while in a subset of patients (11%), HER2 status had changed from the primary tumor at diagnosis. Surprisingly, we found CTC counts to be higher in ER+ patients in comparison to HER2+ and triple negative patients despite their more aggressive phenotype. This may be explained by our findings that the basal-like molecular subtype of breast cancer has low EpCAM expression and a more mesenchymal phenotype and CTCs will likely not be efficiently captured using EpCAM alone in tumors that arise from this subtype. Conclusions/Significance: Our data suggests that molecular characterization from captured CTCs is possible and can inform us of the patients’ current biomarker status. In this regard, CTCs hold significant promise as a source of tumor material to facilitate clinical biomarker evaluation. However, limitations exist from a purely EpCAM-based capture system and addition of antibodies to mesenchymal markers in addition to EpCAM could further improve CTC capture efficiency to enable routine biomarker analysis from CTCs.