Multi-laboratory Assessment of a New Reference Material for Quality Assurance of Circulating Tumor DNA Measurements

Abstract The accurate diagnosis and monitoring of cancer, using circulating tumor DNA (ctDNA), is a major challenge, given the low concentration and complexity of the target molecules. Suitable reference materials are required for clinical laboratories to achieve the high levels of measurement assurance and reproducibility. The methods and instruments for ctDNA assays are rapidly evolving to improve the sensitivity and specificity, resulting in a challenge to compare these different assays for the measurement of the diverse classes of ctDNA cancer biomarkers. To address this goal, a new reference material was developed consisting of 40 cancer mutations in a background of wild-type DNA at six different allele fractions (2%, 1%, 0.5%, 0.25%, 0.125, and 0 %). The cancer mutations include single nucleotide variants, insertions, deletions, and two structural variations that were selected for their clinical importance and challenges for next generation sequencing (NGS) methods. The reference material is formulated in a synthetic plasma matrix at a concentration of 25 ng/mL, levels that are consistent with patient samples. The DNA has been processed to accurately simulate the size of patient ctDNA, resulting in a similar conversion rate of the DNA for next generation sequencing methods. The samples were sent to five different laboratories involved in ctDNA measurements. Digital PCR assays for the variants were developed to confirm the allele fractions by several laboratories. When absorbance, fluorescent dye binding, and PCR methods were used to quantify the extracted ctDNA reference material, the results were significantly different, indicating the need for standard methods and reference materials to ensure reproducible results of this critical step. Different NGS methods (including targeted amplicons, hybrid selection, and SiMSen-seq) and instruments were used by the labs to analyze the refence materials at the different allele fractions. The results showed that the assays had significant differences in the ability to detect classes of variants at different concentrations. This new reference material was shown to be useful to benchmark the sensitivity and selectivity of different NGS methods and bioinformatic pipelines. This multi-laboratory assessment clearly demonstrates that the new reference material is highly valuable tool to ensure the reproducibility of ctDNA measurements including the sample extraction, analytical, and bioinformatic steps. Citation Format: Erica Stein, Hua-Jun He, Kenneth D. Cole, Russell K. Garlick, Yves Konigshofer, Tony E. Godfrey, Michael G. Goggins, Michael Borges, Margaret Gulley, Mickey Williams, Chris Karlovich, Corinne Camalier, Lynn Sorbara, Matthew R. Young, Sudhir Srivastava. Multi-laboratory assessment of a new reference material for quality assurance of circulating tumor DNA measurements [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 3657.