Abstract 3385: A pipeline for mRNA analysis on circulating tumor cells (CTCs) collected and stored in preservative tubes
Authors: Nicolo Eleonora , Manai Marwa , Zhang Youbin , Arcos Laura Munoz , Kaylan Strickland , D'Amico Paolo , Liu Huiping , Curigliano Giuseppe , Reduzzi Carolina , Cristofanilli Massimo 
Published: June 2023

Abstract
Introduction: Circulating tumor cells (CTCs) are associated with prognosis in breast cancer. In addition to enumeration, there is great interest in the molecular characterization of CTCs. Nonetheless, this is technically challenging, especially with regards to RNA analysis, which requires immediate CTC processing to avoid RNA degradation. The purpose of this study was to evaluate the performance of the CellRescueTM preservative tubes in preserving RNA, and to optimize a method for mRNA expression analysis on CTCs up to 4 days after blood collection.
Methods: Ten blood sample from one healthy donor (10 mL each) were collected into CellRescueTM tubes and spiked-in with 5 (n=4) or 500 (n=4) MCF7; two tubes were used for white blood cell (WBC) collection on day (d) 1, 2, 4, and 8 (4 mL used for each timepoint). The spiked-in samples were processed for CTC enrichment on d2 and d4 using CellSearch® (cs5d2, cs500d2, cs5d4, cs500d4) or Parsortix® (p5d2, p500d2, p5d4, p500d4). RNA was extracted, quantified, and subjected to reverse transcription to cDNA. Half cDNA from each sample was analyzed through TaqMan real-time PCR (in technical duplicates) for the detection of 3 transcripts of interest: ACTB (housekeeping), CD45 (WBC marker), and EPCAM (tumor cell marker). Considering the low amount of RNA, the second half of cDNA for each sample underwent preamplification of the 3 target transcripts, followed by real-time PCR (with technical triplicates) to evaluate whether this could increase sensitivity.
Results: RNA quantification by Qubit was 11.6, 23, 9.9 and 4.8 ng/uL in WBC samples collected on d1, d2, d4 and d8, respectively. Thus, using CellRescueTM tubes, RNA was preserved up to 8 days, with a slight reduction in RNA quantity on d8. For spiked-in samples, RNA was below detection at any timepoint. Real-time PCR analysis of CD45 and ACTB in WBC samples confirmed a similar quantity of transcripts across all timepoints. For spiked-in samples processed with CellSearch®, CTCs were detected only after preamplification and when 500 MCF7 were present. For spiked-in samples processed with Parsortix®, 500 CTCs were detected both at d2 and d4, regardless of preamplification. Preamplification allowed for the detection of 5 CTCs as well, but only at d2. The spiked-in samples confirmed that by using CellRescueTM tubes, RNA is preserved up to 4 days, with slight transcript signal decrease between D2 and D4.
Conclusions: With the developed pipeline, it is possible to detect 500 CTCs after blood storage for up to 4 days, and as low as 5 CTCs after 2-days storage. The enrichment methods can however affect the detection. More tests need to be performed to define the pipeline’s sensitivity using different enrichment methods and with different numbers of cells. Using this pipeline, up to a total of 100 gene expression assays of interest can be evaluated in a single sample.
Citation Format: Eleonora Nicolo, Marwa Manai, Youbin Zhang, Laura Munoz Arcos, Strickland Kaylan, Paolo D'Amico, Huiping Liu, Giuseppe Curigliano, Carolina Reduzzi, Massimo Cristofanilli. A pipeline for mRNA analysis on circulating tumor cells (CTCs) collected and stored in preservative tubes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3385.