Abstract 5569: Donor-derived circulating cell-free DNA (ccfDNA) reference materials for concordance studies
Authors: Konigshofer Yves , Butler Matthew G. , Dickens Jessica , Bianco Katherine , Sylvester Karl G. , Anekella Bharathi , Garlick Russell K. 
Published: August 2018

Abstract
The limited quantities of ccfDNA in plasma can make it difficult to assess the sensitivities and specificities of circulating tumor DNA (ctDNA) assays because there is often insufficient material to confirm the presence or absence of particular mutations by orthogonal assays. Additionally, there have been reports of discordance between different ctDNA assays with patient samples at lower variant allele frequencies (VAF), but the limited amount of material is a bottleneck to investigating the causes of discordance. To overcome this, we developed a method to amplify nanograms of ccfDNA from donors to microgram quantities. As an initial proof of concept, we amplified ccfDNA from pregnant female donors that were known to be carrying either a normal fetus or one with chromosomal abnormalities. These samples were analyzed externally at commercial Non-Invasive Prenatal Testing (NIPT) laboratories and were found to be compatible with both chromosome counting and SNP-based testing, and fetal fraction could also be assessed. In order to evaluate the suitability of amplified ccfDNA for ctDNA assays, we split ccfDNA samples so that part was analyzed as-is by a given ctDNA assay and part was analyzed after amplification. The same amplified material was also analyzed with four different ctDNA assays on different NGS platforms in order to assess concordance between assays in shared regions. Good concordance was seen between variants and their VAFs identified in native and amplified ccfDNA, although increasing amounts of discordant background errors were observed at lower VAF. With the Archer Reveal ctDNA 28 assay, obtaining more unique fragments was associated with obtaining more low VAF background errors that could be offset by obtaining more replicate reads of a given unique fragment. Discordant variants were often C&gt;T (or the corresponding G&gt;A) that can be generated by cytosine deamination, which can occur by heating the sample during PCR for library construction. In conclusion, we have developed a method for amplifying limited amounts of ccfDNA that generates sufficient donor ccfDNA-like material for analysis by multiple assays. This should enable the development, improvement and validation of assays that analyze ccfDNA - and especially ctDNA - because sufficient material can be generated in order to assess sensitivity and specificity using orthogonal assays. Additionally, this should also allow for concordance studies that attempt to analyze the same material at multiple sites and with multiple assays.
Citation Format: Yves Konigshofer, Matthew G. Butler, Jessica Dickens, Katherine Bianco, Karl G. Sylvester, Bharathi Anekella, Russell K. Garlick. Donor-derived circulating cell-free DNA (ccfDNA) reference materials for concordance studies [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 5569.