Abstract A63: Examination of ctDNA false positive variants reported from commercial vendors by ultrasensitive orthogonal testing
Authors: Stetson Daniel , Ahmed Ambar , Nuttall Barrett , Lubinski Tristan , Barrett J. Carl , Dougherty Brian 
Published: June 2020

Noninvasive circulating tumor DNA (ctDNA) assays have the potential to revolutionize clinical trial design; however, challenges still remain in using ctDNA for the molecular profiling of cancer with next-generation sequencing (NGS). Our previous work has defined some of the technical and biologic factors that contribute to these challenges, in particular for variants below 1% allele frequency (AF). To continue to examine these factors, we have designed a study to investigate tumor-plasma discordant variants reported from commercial vendors with an orthogonal ultrasensitive NGS assay focusing primarily on false positive variant calls. The study used 24 commercially purchased matched FFPE tumor-plasma samples from breast, prostate, lung, and ovarian cancer. The FFPE tumor-normal pairs were sent to Foundation Medicine (FMI) for sequencing, while replicate sets of plasma were shipped to three commercial vendors for ctDNA sequencing using the currently available panel at the time of shipment. An additional set of samples, which included tumor, normal, and plasma samples, was sent to a fourth vendor for ultrasensitive NGS assay design and sequencing. The returned variants from three commercial vendors were compared to the tissue variants to define truth. True positive variants were defined as an alteration in the plasma that matches either the tissue or another vendors plasma result, while a false positive is a reported variant identified by only one vendor. The comparative analysis identified that Vendor A had the highest sensitivity at 87%, while Vendor B had the highest PPV at 91%. It should be noted that Vendor B had the smallest targeted panel. Vendors A and C contributed the majority of reported false positive variants with Vendor B only having two false positive variants. A total of 54 false positive variant calls were identified, collated along with several driver mutations and germline SNPs, and sent to Vendor D in a blinded fashion for assay design and sequencing. The orthogonal NGS data returned from Vendor D found that two false positive variants had read support to be tissue false negatives. The tissue AF of both variants was found to be just below the standard cutoff for FMI of 5% AF. Both variants were from Vendor A, which improved the sensitivity of Vendor A to 88%. The additional false positive calls from Vendor A were found in the plasma only, with no supporting evidence in the tissue. Vendor B’s false positive calls had no support from the orthogonal assay in either plasma or tissue. Vendor C had variants that had no support in either the tissue or plasma and variants that have support in the plasma. Overall, the false positive variants tested with the ultrasensitive NGS assay could be grouped into three general categories: variant was not found in either the tissue or plasma, variant was found in only the plasma, and variant had support in both the tissue and plasma. The false positive variants found in plasma or in both tissue and plasma may represent true positive variants and need to be investigated further.

Citation Format: Daniel Stetson, Ambar Ahmed, Barrett Nuttall, Tristan Lubinski, J. Carl Barrett, Brian Dougherty. Examination of ctDNA false positive variants reported from commercial vendors by ultrasensitive orthogonal testing [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A63.