Abstract 1989: Enhanced whole exome profiling of tumor circulating cell-free DNA enables sensitive assessment of tumor mutations
Authors: Zhang Simo V. , Tan Mengyao , Northcott Josette M. , Ma Shuyuan , Nelson Christopher S. , Bentley L. Gordon , Chinnappa Manju , Bhave Devayani P. , Norton Dan , Harris Jason , Boyle Sean M. , West John , Chen Richard 
Published: September 2020

An increasing number of studies have demonstrated the potential use of circulating cell-free DNA (cfDNA) for diagnosis, prognosis, and disease progression monitoring. However, many of these studies utilize assays covering a limited set of genes, typically tens to a few hundred genes, and therefore can miss biologically and clinically important genetic alterations such as DNA repair pathways, immuno-modulatory pathways, mechanisms of resistance and changes in neoantigen status. To address this, we have developed a whole-exome scale cfDNA platform, NeXT Liquid Biopsy, that enables sensitive detection and tracking of mutations in approximately 20000 genes.

To enable sensitive detection across the exome, we developed an enhanced exome assay and chemistry that augments hard to sequence genomic regions such as regions of high GC content, to enable more uniform coverage across the exome. Additionally, we achieve a high average depth of approximately 2000X for the entire exome, with additional boosted depth for 248 clinically relevant oncogenic and tumor suppressor genes to further enhance sensitivity. For analysis, we developed a computational pipeline for our NeXT Liquid Biopsy assay optimized to lower the noise floor for variant detection, enabling sensitive monitoring and de novo detection of variants over multiple time points.

We have evaluated the sensitivity of our NeXT Liquid Biopsy platform using three approaches. First, we evaluated the sensitivity using both Horizon and Seracare reference materials at multiple allele frequency (AF) dilutions. Our platform identified all 25 Seracare SNV events at 1% AF, all 25 events at 2% AF, and 24 out of 25 events at 0.5% AF. We detected no variants from the negative control. Next, we expanded our sensitivity evaluation using a much larger reference panel of 555 SNVs from the Acrometric Oncology HotspotControl. Similarly, we were able to achieve high sensitivity at our detection limit. Further, to enable sensitivity analysis at the whole exome scale, we developed a cell culture media system that models the shed and degraded tumor DNA fragments seen in human plasma samples. Finally, we demonstrated our ability to capture mutations across the exome using a head and neck cancer cohort on checkpoint therapy, observing a large fraction of mutations in genes not covered by commercially available targeted panels.

In conclusion, we have developed a whole-exome scale NeXT Liquid Biopsy platform that enables sensitive monitoring and detection of somatic SNVs from cfDNA across approximately 20000 genes. The NeXT Liquid Biopsy platform generates a much broader view of the tumor mutational landscape from the plasma than typical liquid biopsy platforms that are focused on a much smaller set of genes. The platform enables broader monitoring of changes in response to cancer therapy, acquired mechanisms of resistance, mechanisms of drug resistance, and intra- and inter-tumor heterogeneity.

Citation Format: Simo V. Zhang, Mengyao Tan, Josette M. Northcott, Shuyuan Ma, Christopher S. Nelson, L. Gordon Bentley, Manju Chinnappa, Devayani P. Bhave, Dan Norton, Jason Harris, Sean M. Boyle, John West, Richard Chen. Enhanced whole exome profiling of tumor circulating cell-free DNA enables sensitive assessment of tumor mutations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1989.