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Abstract
Abstract Analysis of circulating cell-free DNA (cfDNA) is a promising approach for non-invasive detection of human cancer. The approach has wide-spread applications including early detection of disease in asymptomatic individuals, residual disease detection and disease monitoring. A challenge for the clinical utilization of cfDNA is the small fragment size of cfDNA and the limited amount of cfDNA present in blood. For detection of circulating tumor DNA (ctDNA) highly sensitive methods for cfDNA detection e.g. targeted sequencing and digital droplet PCR (ddPCR) target tumor specific mutations or DNA methylation alterations. Detection of methylation-based biomarkers requires that cfDNA is bisulfite converted prior to biomarker detection, which is known to be associated with some loss of cfDNA. Yet, surprisingly few efforts have focused on ensuring high cfDNA bisulfite conversion (BSC) efficiency and -recovery, and few commercial kits are especially designed for this purpose. In this study we aimed to find a BSC method suited for cfDNA methylation biomarker research, with high BS conversion efficiency and minimum loss of cfDNA, in order to increase the amount of cfDNA template available for downstream analysis. We studied 13 different methods for BSC and examined both the recovery and the BSC efficiency using quantitative real-time PCR (qPCR), polyacrylamide gel electrophoresis (PAGE) and ddPCR. A significant variation in DNA recovery was observed between the 13 methods tested using both high and low molecular weight DNA templates. Especially five kits performed consistently well on all DNA templates tested, as evaluated by qPCR and PAGE. As the lengths of cfDNA is 146-167 base pairs, only methods that are able to recover fragments of this lengths is suitable for cfDNA pipelines. Results showed that few methods enabled recovery of DNA fragments ≤ 150 base pairs. However, three kits showed a higher amount of 150 base pair fragments compared to the other methods tested. In a clinical setting it is expected that the target cfDNA compose only a minor fraction of the total cfDNA in plasma. Thus, the five best performing kits were tested using ddPCR on serial dilutions of leucocyte DNA fragmented to ~ 150bp to mimic cfDNA. The best performing kit had a recovery of 69% of the initial input compared to only 13% for the poorest performing kit. Collectively, our comprehensive comparison hereby guide which BS conversion methods are suited for cfDNA methylation-based biomarker pipelines. Citation Format: Sarah Ø. Jensen, Mai-Britt W. Ørntoft, Jesper B. Bramsen, Torben F. Ørntoft, Claus L. Andersen. Comprehensive comparison of bisulfite conversion kits: A guide for optimal sensitivity and specificity of circulating cell-free DNA methylation-based biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3804. doi:10.1158/1538-7445.AM2017-3804
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