Abstract
Circulating tumor cells (CTCs) have potential utility as a surrogate biomarker of tumor biology via a liquid biopsy. The aim of this study was to evaluate if the nCounter NanoString assay could be used for accurate gene expression profiling of CTCs using the PAM50 research-use-only CodeSet. Analysis was performed on CTCs isolated by the ANGLE Parsortix system from healthy blood spiked with the breast cancer cell lines Hs578T, SkBr3, MDA-MB-231 or MCF7. Using cell lines as gold standard positive controls and Parsortix processed blood without spiking (unspiked) as negative controls, we found an average of 12 significantly differentially expressed genes among spiked samples versus unspiked controls. We validated our findings with the NanoStringDiff differential expression statistical method. The NanoString recommended targeted pre-amplification introduced false positive results due to pre-amplification bias, and the amplification of non-cancer genes from normal leukocytes confounded gene expression profiling of CTCs. Pre-amplification bias is a concern for other similar assays that may be used as discovery tools or target validation of transcripts of interest in gene expression profiling of CTCs. We recommend the use of an unspiked negative control when evaluating CTC technologies regarding gene expression profiling. Given that the molecular profiling of CTCs as a liquid biopsy may have clinical ramifications for potential treatment selection in future clinical trials, our study emphasizes cautious consideration of pre-analytical variables such as amplification bias in the context of liquid biopsy studies.
Highlights
Over the last years, research focused on circulating tumor cells (CTCs) has captured great attention as a potential “liquid biopsy” with recognized potential utility in cancer diagnosis, prognosis, and personalized treatments in patients with a variety of solid tumors [1]
It has been previously shown that the Parsortix system recovers about 4080% of Circulating tumor cells (CTCs) [8, 14], the relatively low number of the identified epithelial cell adhesion molecule (EpCAM)+ captured cells may be explained by heterogeneity in EpCAM expression among SkBr3 cells, as well as cell loss during the antibody staining, washing and FACS process
While we have optimized a workflow for transcriptomic profiling of rare CTCs using the Parsortix system, in this report, our data indicate that the NanoString PAM50 panel has limited utility in differentiating CTC mimics from leukocyte background
Summary
Research focused on circulating tumor cells (CTCs) has captured great attention as a potential “liquid biopsy” with recognized potential utility in cancer diagnosis, prognosis, and personalized treatments in patients with a variety of solid tumors [1]. The CellSearch CTC assay, the only currently United States Food and Drug Administration approved system, enriches CTCs based on the detection and identification of the epithelial cell adhesion molecule (EpCAM) and pan-cytokeratins in the tumor cell membrane and cytoplasm. This system has limited utility enriching CTCs with low EpCAM expression, and CTC populations are recovered along with considerable amounts of contaminating leukocytes [8]. Various studies have shown the Parsortix system’s versatility in capturing and harvesting CTCs suitable for in vitro culture and molecular analysis by techniques such as genomic hybridization, qPCR and RNA-seq [13,14,15,16]
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