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Donor-Derived Circulating Cell-Free DNA (ccfDNA) Reference Materials for Concordance Studies

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American Association for Cancer Research (AACR) | Chicago, Illinois 2018

Yves Konigshofer, Matthew G. Butler, Jessica Dickens, Katherine Bianco, Karl G. Sylvester, Bharathi Anekella, Russell K. Garlick

The limited quantities of ccfDNA in plasma and the limited amount of ccfDNA that can therefore be obtained from a single donor 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 (VAFs). The limited and ephemeral nature of patient samples has been a bottleneck to investigating and resolving the causes of discordance. To overcome this, we developed a method to amplify nanograms of ccfDNA from donors to microgram quantities.

In order to evaluate the suitability of amplified ccfDNA for ctDNA concordance and other studies, we amplified ccfDNA from pregnant female donors and assessed reported variants and compatibility with non-invasive prenatal tests (NIPT) and various NGS-based tumor assays. In such samples, the presence of a fetus is expected to contribute some paternal SNPs at lower – but similar – frequencies that can be used to simulate somatic mutations. Additionally, the donors are expected to be free from cancer, so variants present at frequencies below those of the paternal SNPs are either from the sample (e.g., due to clonal hematopoiesis or the amplification method) or introduced by the assay.

In order to assess the impact of the amplification method on the apparent variants in a sample, we split ccfDNA samples into two parts – one for analysis in their native state, and the other for amplification and analysis thereafter.

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