Abstract 438: Multicenter evaluation of circulating tumor DNA assays
Authors: Brudzewsky Dan , Lampignano Rita , Weber Sabrina , Sartori Alexander , Mohan Sumitra , Konigshofer Yves , Davis Lorn , Rothwell Dominic , Schuuring Ed , Garlick Russell K. , Schlange Thomas , Heitzer Ellen 
Published: August 2019

Abstract
Measurements of circulating tumor DNA (ctDNA) hold great potential to detect residual disease, therapeutic response monitoring and tumor evolution. However, there are many technical challenges including trace levels of circulating cell free DNA 1-10 ng/ml in plasma (300-3,000 genome equivalents/ml), DNA fragment size 170 bps found in the circulation, low variant allelic fractions ~ 0.1% and the need to detect all variant types SNVs, insertions, deletions, CNVs and structural variants. CANCER-ID partners comprise a consortium of experts, companies and institutions for blood -based biomarker validation, assay development, clinical sciences and bioinformatics. This CANCER-ID ring trial was established to help standardize complex ctDNA diagnostic assays by testing a highly complex reference material. The trial looks to improve concordance and to evaluate analytical performance by collecting metrics covering the entire workflow from sample preparation through variant reporting. The common reference material, Seraseq ctDNA Complete, used in the trial is human genomic DNA background material from the Genome-in-a-bottle consortium mixed with synthetic DNA sized to ~170 bp and formulated in artificial plasma. The material contains 25 variants per sample including 12 SNVs (8 genes), 5 deletions (3 genes), 2 insertions (2 genes), 3 CNVs (3 genes) and 3 fusion pairs (3 genes). The variant allelic fractions range from 0.1% to 5% for the SNVs, INDELS and ~ 1 -6 additional copy number for ERBB2, MET and MYC genes. Interlab and intralab testing was done on Roche AVENIO targeted Assay, Agilent SureSelect XT custom assay, QiaSeq Targeted DNA panel, Qiagen GeneRead QIAAct Lung Assay, one clinically validated digital droplet assay and one research digital droplet PCR assays and Agena Bioscience’s UltraSEEK Lung Panel. Data analysis, peer comparison and reporting of common and unique QC metrics was accomplished through the cloud based, analytical QC software iQ NGS QC Management. The results demonstrate that a common full process reference material can be used to compare distinctly different assays formats at different levels of input DNA by mutations. For the 1% AF material, the BRAF V600 variant was detected by all assay formats and the cv’s ranged from 12%-52% or on average for all variants measured by the assay ranged from 11% to 46%. The study furthermore shows that peer review analytical QC software is useful in establishing standardization of ctDNA assays. This work is supported by IMI JU & EFPIA (grand no. 115749, CANCER-ID).
Citation Format: Dan Brudzewsky, Rita Lampignano, Sabrina Weber, Alexander Sartori, Sumitra Mohan, Yves Konigshofer, Lorn Davis, Dominic Rothwell, Ed Schuuring, Russell K. Garlick, Thomas Schlange, Ellen Heitzer. Multicenter evaluation of circulating tumor DNA assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 438.