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29 Profiling tumor circulating cell-free DNA with an enhanced whole-exome to enable sensitive assessment of somatic mutations
  1. Simo Zhang,
  2. Mengyao Tan,
  3. Fabio CP Navarro,
  4. Josette Northcott,
  5. Shuyuan Ma,
  6. Christopher Nelson,
  7. Devayani Bhave,
  8. L Gordon Bentley,
  9. Manju Chinnappa,
  10. Dan Norton,
  11. Gabor Bartha,
  12. Jason Harris,
  13. John Lyle,
  14. Sean Boyle,
  15. John West and
  16. Richard Chen
  1. Personalis INC, Menlo Park, CA, USA


Background An increasing number of studies have demonstrated the potential use of circulating cell-free DNA (cfDNA) for diagnosis, prognosis, disease progression, and treatment monitoring. However, many of these studies use assays covering a limited set of genes and therefore miss biologically and clinically relevant genetic alterations involving immuno-modulatory pathways which confer treatment resistance, and leading to changes in neoantigen status. To address this, we developed a whole-exome scale cfDNA platform, NeXT Liquid Biopsy™, that enables sensitive detection and tracking of mutations in approximately 20000 genes.

Methods To enable sensitive detection across the exome, our enhanced exome assay and chemistry augments hard-to-sequence genomic regions, such as regions of high GC content, to enable more uniform coverage across the exome. We achieved a high mean sequencing depth of approximately 2000X exome-wide, with additionally boosted depth for 248 clinically relevant oncogenic and tumor suppressor genes to further enhance sensitivity. We developed a computational pipeline for our NeXT Liquid Biopsy assay optimized to lower the noise floor for variant detection, providing sensitive monitoring and de novo detection of variants over multiple time points.

Results We evaluated the sensitivity of our NeXT Liquid Biopsy platform in three ways. First, we evaluated the sensitivity within the coverage boosted regions using the SeraCare reference materials at multiple allele frequency (AF) dilutions. Our platform identified all 8 and 25 Horizon and SeraCare SNV events at 1% AF and above, respectively, and detected 24 out of 25 events at 0.5% for the SeraCare samples. Additionally, to enable sensitivity analysis at the whole-exome scale, we then developed a cell culture media system that models the shedding of tumor DNA fragments seen in human plasma samples and created tumor/normal dilution series in vitro. We achieved >95% sensitivity for variants with AF≥2%, and between 85% to 92% for mutations with AF of 1%-2%. Second, we evaluated false-positive rates on 12 cancer patients using digital droplet PCR. Third, we demonstrated our ability to longitudinally monitor treatment response using a clinical cancer cohort on checkpoint therapy, profiling putative tumor evolution while on therapy.

Conclusions In conclusion, we have developed a whole-exome scale liquid biopsy platform, NeXT Liquid Biopsy, that enables sensitive monitoring and detection of somatic SNVs from cfDNA across ~20000 genes. The platform enables broader monitoring of changes in response to cancer therapy, acquired mechanisms of resistance, and intra- and inter-tumor heterogeneity.

This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See:

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