Abstract P1-01-11: Something from nothing? The case for quality control in liquid biopsy studies
Authors: Porras TB , Bains PK , Ring A , Carrasco S , Forte V , Punj V , Lang JE 
Published: May 2019

Abstract
Background: Circulating tumor cells (CTCs) as a liquid biopsy strategy are currently being studied as a surrogate biomarker that may reflect metastatic tumor biology. Given the rarity of CTCs, target enrichment is commonly used to profile the gene signatures of valuable clinical samples to evaluate for multiplexed gene panels of interest. The aim of our study was to evaluate if the NanoString PAM50 could be used for accurate gene expression profiling of CTCs and controls using the research-use-only probeset.
Methods: We collected two 7.5mL EDTA tubes of blood from 12 healthy female volunteers. CTC assays were performed using the ANGLE Parsortix system as a microfluidics filter that separates cells based on size and deformability. The cell lines Hs578T (basal-like) and SK-BR-3 (HER2 amplified) were used to spike 20 cells into n=6 blood tubes per cell line (termed spiked samples). N=12 7.5mL tubes of blood (termed unspiked samples) and n=12 spiked samples were processed using Parsortix for CTC harvesting and lysis using a 10 micron cassette. From each lysate, 5uL was taken for cDNA amplification, multiple target enrichment for 14 cycles, followed by NanoString PAM50 assays. From each of the 12 peripheral blood (PB) samples, we extracted RNA and used 100ng for NanoString PAM50 assays. For cell line controls, 100ng of Hs578T or SK-BR-3 were subjected to NanoString Assays.
Results: Low PAM50 gene expression was observed in all 12 PB samples. Unspiked PB harvested from the CTC assay showed a higher level of PAM50 gene expression compared to PB, suggesting that the target enrichment amplification produces false positive detection of expected breast cancer related transcripts. On ANOVA testing, 10/12 (83%) of unspiked, sorted, target enriched samples had significant differential expression (p&lt;0.0001) of the mean log normalized counts for the PAM50 genes compared to PB.
In spiked experiments using n=20 cells in 7.5mL of PB, sorted Hs578T were found to be triple negative in only 3/6 (50%) while sorted SK-BR-3 were found to be HER2 positive in only 3/6 (50%). On ANOVA testing, the spiked/sorted and bulk were found to have a difference among the mean log normalized counts for the PAM50 genes across all samples for both cell lines (p&lt;0.0001). However, 3/6 (50%) samples had a difference in mean PAM50 gene expression when compared to bulk Hs578t on multiple comparison testing while 2/6 (33%) were statistically significantly different when comparing spiked, sorted SK-BR-3 versus bulk cell line.
Conclusions: Unspiked blood processed via a CTC assay and subjected to target enrichment showed high expression of genes in the NanoString PAM50 assay, likely due to amplification bias. When working with enriched but not ultra-pure CTC samples, amplified gene expression of background leukocytes may influence read counts. This is important to consider in assays that enrich for CTCs but retain a leukocyte background. Further studies will address the effect of the CTC assay procedure and number of leukocytes on accuracy of gene expression of rare CTC mimics. This study emphasizes the importance of selecting genes that are not expressed in PB or performing background subtraction or normalization as strategies for accurate gene expression profiling of CTCs.
Citation Format: Porras TB, Bains PK, Ring A, Carrasco S, Forte V, Punj V, Lang JE. Something from nothing? The case for quality control in liquid biopsy studies [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-11.