Introduction: Variant detection in circulating tumor DNA (ctDNA) provides an unprecedented opportunity to study the mutational landscape of cancers with minimal patient invasiveness. Meanwhile, new variants providing information about drug response and disease prognosis are being rapidly discovered. Therefore, to fully harness the power of ctDNA variant detection it is critical to be able to rapidly modify the set of variants targeted in an assay. Here we present a platform for next generation sequencing-based ctDNA assays that allows researchers to readily customize their panel targets.

Methods: Anchored Multiplex PCR (AMP™) is a molecular biology technique that allows for near infinite modularity of assay targets while maintaining variant detection sensitivity. In order to ensure assay specificity, AMP utilizes molecular barcoded adapters (MBC) to uniquely label input molecules prior to amplification, enabling post-sequencing error correction. This increases analytical sensitivity by reducing background noise. We also developed bioinformatics methods, which allow the characterization of position-specific error levels. By combining molecular barcode-based error correction and error profile characterization, we can reliably detect low frequency variants with high specificity.

Results: We tested the modularity of the AMP ctDNA platform by creating ctDNA panels targeting either 6 kb of cancer variant hotspots or 34 kb of tumor suppressors plus the same 6kb of cancer hotspots (40 kb total target sequence). We then prepared and sequenced libraries using ctDNA-like inputs containing important cancer variants at low allele fractions (AF). We used these data to characterize the noise inherent in AMP library preparation and Illumina sequencing. We then performed a power analysis to measure the lowest AF at which we could detect variants at each target position given the noise level and sequencing coverage. Our results show that we can confidently detect variants with an AF of less than 0.18% in 50% of bases and 0.47% in 95% of bases covered in the 6kb panel. Variant detection sensitivity remained consistent over the 6kb of hotspot targets in the context of the 40kb panel (50% of bases powered to detect 0.17% AF variants and 95% powered to detect 0.45% variants). Across the full 40kb of the larger panel we had statistical power to call 0.23% AF variants at 50% of target bases and to call 0.56% variants at 95% of target bases. These data demonstrate that ctDNA panels can maintain assay performance upon panel modifications.

Conclusions: The modular nature of AMP ctDNA panels allows for endless combinations of targeted genes to maximize detection sensitivity in relevant regions while also maintaining assay performance. Our experience with AMP assays indicates that the range in the number of targets that can be included in a panel is quite large, allowing broad implementation of the assays developed on the AMP platform.

Citation Format: Matt Egleston, Paula Roberts, Ian Hoskins, Aaron Garnett, Laura Griffin, Abel Licon, Ryan Walters. Development of a Highly Customizable Platform for Variant Detection in Circulating Tumor DNA [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 413.