Effect of short-term storage of blood samples on gene expression in lung cancer patients

Abstract

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.

Gene expression markers in peripheral blood and outcome in patients with platinum‐resistant ovarian cancer: A study of the European GANNET53 consortium

“…Since the GANNET53 study used blood collection tubes with a cell preservative, a higher yield is plausible because the preservative leads to a higher stiffness of cells and, thus, to a higher capture rate by the microfluidic system. This assumption is also based on our recent study comparing blood collection tubes with and without preservatives, showing an about 1.5‐fold increased recovery in CellFree DNA BCT tubes 45 …”

“…This assumption is also based on our recent study comparing blood collection tubes with and without preservatives, showing an about 1.5-fold increased recovery in CellFree DNA BCT tubes. 45 An important issue in molecular analyses is the use of a reference gene to normalize the data, and the presence of non-detects, here defined as mean C t values ≥35.0. In the present study, the transcript levels were not normalized to reference genes because contaminating leukocytes would have introduced a bias by restraining positive findings in samples containing just a few CTCs and a relatively large number of leukocytes.…”

Integrated transcriptomics, proteomics and metabolomics-based analysis uncover TAM2-associated glycolysis and pyruvate metabolic remodeling in pancreatic cancer

“…Kaplan-Meier survival curves were created to explore variations in survival (overall survival [OS], disease-specific survival [DSS], progression-free survival [PFS]) among various metabolic subtypes utilizing the R tools Survival and Survminer ( 20 ). The clinical characteristics of the different metabolic subtypes were also examined by chi-square or Fisher’s exact test.…”