Molecular Biomarker Analyses Using Circulating Tumor Cells

Abstract

BackgroundEvaluation of cancer biomarkers from blood could significantly enable biomarker assessment by providing a relatively non-invasive source of representative tumor material. Circulating Tumor Cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard.Methodology/Principal FindingsUsing spiked tumor-cells 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 quantify cell surface expression of EGFR in metastatic lung cancer patient samples. In addition, we determined HER2 status by IF and FISH in CTCs from metastatic breast cancer patients. In the majority of patients (89%) we found concordance with HER2 status from patient tumor tissue, though in a subset of patients (11%), HER2 status in CTCs differed from that observed in the primary tumor. Surprisingly, we found CTC counts to be higher in ER+ patients in comparison to HER2+ and triple negative patients, which could be explained by low EpCAM expression and a more mesenchymal phenotype of tumors belonging to the basal-like molecular subtype of breast cancer.Conclusions/SignificanceOur data suggests that molecular characterization from captured CTCs is possible and can potentially provide real-time information on 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 could further improve CTC capture efficiency to enable routine biomarker analysis from CTCs.

Analysing the Mutational Status of PIK3CA in Circulating Tumor Cells from Metastatic Breast Cancer Patients

“…Assessing the presence of target antigens on CTCs could be considered as a real-time biopsy allowing the evaluation of changes in tumour phenotype during the clinical course of the disease. A combination of highly sensitive multi-parametric molecular methods and imaging has been evaluated very recently for the molecular characterization of CTCs (Punnoose et al, 2010). However, this has been hindered by the very limited sample amount available.…”

Minimal Residual Disease and Circulating Tumor Cells in Breast Cancer

“…An example of a microfl uidic platform is the 'CTC-chip' , which is capable of effi cient and selective separation of viable CTCs from peripheral whole blood samples, mediated by the interaction of target CTCs with EpCAM-coated microposts under precisely controlled laminar fl ow conditions [29]. A direct comparison between CellSearch® and two commercially available CTC-chips showed that these platforms provided similar sensitivity and yield in patient samples [33]. Stott and colleagues [34] recently reported improved sensitivity of the CTC-chip for CTC detection in patients with localized prostate cancer.…”

“…As an example, some available technologies detect only EpCAM+ CTCs (Table 1). However, it has been shown that BC cell lines with low EpCAM expression and high expression of mesenchymal markers cannot be effi ciently captured using a purely EpCAM-based mechanism [33,43,44]. Some other technologies are using enrichment based on red blood cell lysis or leukocyte depletion (CD45-negative depletion) aiming at a less biased CTC enrichment (Table 1).…”

“…Because of the availability of anti-HER2 agents, HER2 expression was studied on DTCs [71][72][73] and CTCs [33,42,55,59,66,[74][75][76][77][78] (Table 2) and was correlated with HER2 expression on the primary tumor. In most studies, HER2 expression on DTCs/CTCs is more prevalent in women with HER2-positive BC than in women with HER2-negative BC in both non-metastatic and metastatic settings.…”

Novel Tissue Types for the Development of Genomic Biomarkers

“…Recently, CTCs have been the target of multiple molecular profiling studies (Bosma et al, 2002;Punnoose et al, 2010;Smirnov et al, 2005;Tewes et al, 2009). mRNA expression and DNA mutations can be measured from captured CTCs.…”