2020 AACR: Enhanced Whole Exome Profiling of Tumor Circulating Cell-Free DNA Enables Sensitive Assessment of Tumor Mutations

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 well known genes, and therefore can miss biologically and clinically important genetic alterations in 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. The NeXT LB platform sequences a triplet of tumor, normal, and plasma samples from a patient, allowing 1) discovery of novel genetic mutations, 2) non-invasively close monitoring of clonal and subclonal evolution in response to treatment of curative intent, and 3) broad interrogation of inter- and intra-tumor heterogeneity.

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 (5000X) for 248 clinically relevant oncogenic and tumor suppressor genes to further enhance sensitivity. For analysis, we developed a computational algorithm, Silencer, that enables accurate somatic mutation detection without compromising sensitivity in the plasma. Silencer is empowered by an error suppression model estimated from a panel of normal individual plasma samples, and ad hoc filters including a dedicated blacklist that is tailored to our NeXT LB technology.