Molecular Biomarker Analyses Using Circulating Tumor Cells

Elizabeth A Punnoose,Jill M Spoerke,Siminder K Atwal,Mark R Lackner,Lukas C Amler,Daniel S Chen,Andrea Pirzkall,Ajay Pandita,Bernard M Fine,Heidi Savage,Ru-Fang Yeh,Janine Santos

doi:10.1371/journal.pone.0012517

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.

Highlights

Oncology drug discovery efforts are increasingly focused on targeted therapies that inhibit major nodes of oncogenic signaling pathways
Epithelial Cell Adhesion Molecule (EpCAM) is well characterized as a marker of epithelial tumor cells [37] and anti-EpCAM antibodies have been widely used in the capture of Circulating Tumor Cells (CTCs) [14]
As a first step in evaluating CellSearchH in comparison with the CTC-Chip platforms, all of which use EpCAM expression as the basis for CTC capture, we looked at the utility of EpCAM as a universal marker expressed on cancer cells by evaluating its expression in cell lines and across diverse tumor types

Summary

Introduction

Oncology drug discovery efforts are increasingly focused on targeted therapies that inhibit major nodes of oncogenic signaling pathways. Obtaining tissue from a fresh biopsy is challenging in this indication as the metastatic lesions are often localized to bone [9,10]. Even if such material can be obtained, it is unclear whether a biopsy from a single site is representative of the majority of metastatic lesions and cases of marked heterogeneity have been reported [10]. Similar considerations are true for metastatic breast cancer, where tumor samples may be limited to tissue from the primary disease site, which again, may be separated from disease recurrence by both time and treatment [11]. Circulating Tumor Cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard

Methods

Results

Conclusion