Abstract
Circulating tumour cells (CTCs) are an emerging resource for monitoring cancer biomarkers. New technologies for CTC isolation and biomarker detection are increasingly sensitive, however, the ideal blood storage conditions to preserve CTC-specific mRNA biomarkers remains undetermined. Here we tested the preservation of tumour cells and CTC-mRNA over time in common anticoagulant ethylene-diamine-tetra-acetic acid (EDTA) and acid citrate dextrose solution B (Citrate) blood tubes compared to preservative-containing blood tubes. Blood samples spiked with prostate cancer cells were processed after 0, 24, 30, and 48 h storage at room temperature. The tumour cell isolation efficiency and the mRNA levels of the prostate cancer biomarkers androgen receptor variant 7 (AR-V7) and total AR, as well as epithelial cell adhesion molecule (EpCAM) were measured. Spiked cells were recovered across all storage tube types and times. Surprisingly, tumour mRNA biomarkers were readily detectable after 48 h storage in EDTA and Citrate tubes, but not in preservative-containing tubes. Notably, AR-V7 expression was detected in prostate cancer patient blood samples after 48 h storage in EDTA tubes at room temperature. This important finding presents opportunities for measuring AR-V7 expression from clinical trial patient samples processed within 48 h—a much more feasible timeframe compared to previous recommendations.
Highlights
Circulating tumour cells (CTCs) are cells shed from tumours into the peripheral blood, and are believed to be the mechanism for metastasis [1,2]
CTC counts have been associated with prognosis and importantly, isolated CTCs can function as a surrogate tumour samples to detect therapy-determining biomarkers, including the mRNA based androgen receptor variant androgen receptor variant 7 (AR-V7) in CTCs [5,6,7,8]
Increased Proteinase K Treatment We investigated the effect of increased proteinase K digestion on the cellular RNA recovery as per manufacturer’s suggestions for the BCTs. 22Rv1 cells were spiked into a new set of blood tubes (EDTA, citrate dextrose solution B (Citrate), DNA blood collection tubes (DNA BCT), and RNA blood collection tubes (RNA BCT)), followed by enrichment after 48 h of storage
Summary
Circulating tumour cells (CTCs) are cells shed from tumours into the peripheral blood, and are believed to be the mechanism for metastasis [1,2]. A major technical challenge for CTC analysis has been efficient recovery of rare CTCs from a background of approximately 109 erythrocytes and 107 leukocytes per mL of blood, and this has spurred the development of improved technologies for CTC isolation [13,14,15]. Despite these advances, feasible CTC analysis for clinical trials involving multiple sites are challenging, due to strict requirements for pre-analytical conditions of blood samples in transport and storage, and time restrictions to ensure CTC integrity and biomarker detectability is maintained. CTCs should be protected from apoptosis or cell lysis and, importantly, preserve relevant tumour biomarkers, an issue that is considered especially challenging for mRNA-based biomarkers
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