Abstract 3655: DNA methylation analysis in liquid biopsy: A detailed study on quality control, sample storage and whole genome amplification procedures for downstream applications

Abstract

Abstract Background: Studies on epigenetic alterations and especially DNA methylation in liquid biopsy samples like plasma circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) are of growing interest nowadays. To ensure the reliability of DNA methylation results prior to any clinical applications in liquid biopsy, there is an urgent need to standardize a plethora of parameters. The aim of our study was to evaluate: a) the stability of DNA methylation in plasma, b) the stability of sodium bisulfite (SB) converted DNA samples under different storage temperatures, and c) the accuracy and precision of whole genome amplification (WGA) protocols for downstream DNA methylation analysis. Methods: a) Stability of DNA methylation in plasma: Plasma from healthy donors was pooled and aliquoted in six tubes and spiked with genomic DNA (gDNA) isolated from 106 MCF-7 cells. Five aliquots were kept at -700C, while one was immediately processed for ctDNA isolation and SB treatment. Stored aliquots were processed in the same way after storage for a period of 3 months. All SB-treated DNA samples were further evaluated by real time methylation specific PCR (MSP) for ACTB and SOX17. b) Stability of SB-treated DNA: gDNA was isolated from 106 SKBR3 cells and processed for downstream SB-treatment. SB-treated DNA was split into aliquots and stored at -20°C and -70°C. One aliquot, used as control, was immediately processed for SB treatment followed by real time MSP for ACTB and BRMS1 amplification. The remaining aliquots were processed in exactly the same way after storage for different time points with a maximum period of one year. Additionally, a sample that was subjected to repeated freeze-thaw cycles was added in each run. c) Evaluation of WGA for SB-treated DNA: we evaluated two different SB conversion kits, EZ DNA Methylation-Gold (Zymo), and Epitect Fast Bisulfite, (Qiagen) in combination with downstream WGA (EpiTect Whole Bisulfitome Kit, Qiagen), using 0%, 100% methylated DNA standards and MCF-7, SKBR3 cell lines. Results: a) DNA methylation in plasma is stable at -700C for at least 3 months, since the average Cqs detected, varied from 1 to 2 cycles for ACTB and SOX17, respectively, b) Quality of SB-treated DNA is not affected by repeated freezing-thawing cycles; SB-treated DNA samples are stable when kept for up to one year both at -200C and -700C. c) Identical results by real time MSP were obtained before and after WGA amplification (Qiagen), after using either the Zymo or Qiagen kits for SB conversion. Conclusions: Plasma and SB-treated DNA samples are stable and can be safely used for multiple real time MSP assays over time when kept at -70°C. Downstream WGA of SB-treated DNA could bypass the limited amount of available sample for liquid biopsy applications. In any case, quality control checking should be included in each step of analysis. Citation Format: Sofia Mastoraki, Maria Chimonidou, Eleni Tzanikou, Evi S. Lianidou. DNA methylation analysis in liquid biopsy: A detailed study on quality control, sample storage and whole genome amplification procedures for downstream applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3655.