Challenges in using liquid biopsies for gene expression profiling
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Tania B. Porras1,3, Pushpinder Kaur1,3, Alexander Ring1,3, Naomi Schechter2,3 and Julie E. Lang1,3
1Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
2Department of Radiation Oncology, Keck Medical Center, University of Southern California, Los Angeles, CA, United States
3University of Southern California Norris Comprehensive Cancer Center, Los Angeles, CA, United States
Julie E. Lang, email: Julie.Lang@med.usc.edu
Keywords: circulating tumor cells (CTCs); NanoString PAM50; gene expression; target pre-amplification; PCR
Received: June 20, 2017 Accepted: December 26, 2017 Published: January 11, 2018
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.
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