Despite recent advances in the treatment of non-small cell lung cancer (NSCLC), less than 10% of patients survive the first five years when the disease has already spread at primary diagnosis. Methods: Blood samples were taken from 118 NSCLC patients at primary diagnosis or at progression of the disease before the start of a new treatment line and enriched for circulating tumor cells (CTCs) by microfluidic Parsortix™ (Angle plc, Guildford GU2 7AF, UK) technology. The gene expression of epithelial cancer stem cell (CSC), epithelial to mesenchymal (EMT), and lung-related markers was assessed by qPCR, and the association of each marker with overall survival (OS) was evaluated using log-rank tests. Results: EpCAM was the most prevalent transcript, with 53.7% positive samples at primary diagnosis and 25.6% at recurrence. EpCAM and CK19, as well as NANOG, PROM1, TERT, CDH5, FAM83A, and PTHLH transcripts, were associated with worse OS. However, only the CSC-specific NANOG and PROM1 were related to the outcome both at primary diagnosis (NANOG: HR 3.21, 95%CI 1.02–10.14, p = 0.016; PROM1: HR 4.23, 95% CI 0.65–27.56, p = 0.007) and disease progression (NANOG: HR 4.17, 95%CI 0.72–24.14, p = 0.025; PROM1: HR 4.77, 95% CI 0.29–78.94, p = 0.032). Conclusions: The present study further underlines the relevance of the molecular characterization of CTCs. Our multi-marker analysis highlighted the prognostic value of cancer stem cell-related transcripts at primary diagnosis and disease progression.

Objectives The stability of gene transcripts associated with the presence of circulating tumor cells (CTCs) has been predominantly studied in cultured cancer cell lines added to blood samples under artificial conditions. In the present study the effect of storage on CTC-related transcripts was assessed in blood samples taken from patients with non-small lung cancer (n=58). Methods The blood samples were split in two equal parts to compare the gene expression with and without storage for 24 h at ambient temperature without preservative added. After enrichment using the microfluidic Parsortix® technology, the expression levels of selected genes were assessed using quantitative PCR following a gene-specific pre-amplification. The prognostic relevance of each gene in fresh and stored blood samples was evaluated using the R-package Survminer. Results Some genes were either not affected (TWIST1, CDH5, CK19) or upregulated upon storage (NANOG, MET, UCHL1) but still associated with poor prognosis. In contrast, ERBB3, PTHLH, EpCAM, and TERT were no longer associated with the overall survival of the patients. Conclusions The study demonstrates the surprising stability of CTC-related transcripts, which makes overnight shipping of native blood samples possible. Careful verification is required when using model systems – such as normal blood spiked with tumor cells – or other CTC-related markers, as individual transcripts may respond differently to storage.