Abstract 5695: Increased sensitivity for detection of oncogenic mutations present in circulating cell-free tumor DNA
Authors: Thorner Aaron R. , Slevin Michael K. , Clapp Andrea , Coleman Haley A. , McShane Suzanne R. , Drinan Samantha D. , Thai Edwin , Wollison Bruce M. , Burns Robert T. , Frieden Alex , Ducar Matthew D. , Nag Anwesha , MacConaill Laura E. , Meyerson Matthew L. 
Published: July 2017

Abstract
Molecular pathways involved in tumor survival and progression are often activated by genetic alterations, and anticancer agents specifically targeting many oncogenic pathways have entered clinical trials. Precision cancer medicine takes advantage of genomic technologies (e.g., massively parallel sequencing, MPS) to perform robust tumor genomic profiling in the clinic, enabling identification of “druggable” mutations. To date, the vast majority of genomic profiling has been performed on DNA extracted from solid tumor biopsies. However, liquid biopsies offer advantages over solid tumor biopsies; chief among them are their non-invasive nature and quick turn-around time when assessed using MPS. Furthermore, liquid biopsies allow for sequential analysis throughout a course of therapy permitting real-time monitoring of treatment effects on tumor expression and subsequent surveillance for development of new oncogenic alterations.
Circulating cell-free tumor DNA (cfDNA) derived from plasma represents a convenient assay material that has been effectively analyzed for oncogenic mutations in specific forms of cancer such as non-small cell lung cancer. However, introduction cfDNA for clinical genotyping is limited by sensitivity requirements. cfDNA is typically present at 20-100 ng/mL of plasma, of which the cancer fraction can be below 1%. Error rates associated with PCR amplification and base calling (Illumina HiSeq platform) are approximately 2%, preventing confidence in rare variants with allele fractions (AF) at or below that percentage. We conducted an evaluation of commercially available kits to determine 1) kit sensitivity and if necessary 2) modify the protocol to achieve sensitivity of &lt;= 1% AF.
To evaluate kit sensitivity gDNA from a translocation positive cell line was mixed at 5% and 20% with normal cell line gDNA. Libraries were prepared for each percent mix using 10 ng of input. Raw mean target coverage (MTC) was similar for evaluated kits (MTC, 250-300X); Percent Duplication, ~34%. Comparison of 20% and 100% translocation samples showed similar levels of unique single nucleotide variant (SNV) detection. However, 5% mixtures showed a ~10 fold reduction in SNV detection compared to both 20% and 100% translocation samples. Furthermore, translocation rearrangement was only detectable in one third of the 5% translocation samples. Increased sensitivity requires unique molecular barcodes coupled with refinements in bioinformatics analysis for clinical implementation of these assays.
Citation Format: Aaron R. Thorner, Michael K. Slevin, Andrea Clapp, Haley A. Coleman, Suzanne R. McShane, Samantha D. Drinan, Edwin Thai, Bruce M. Wollison, Robert T. Burns, Alex Frieden, Matthew D. Ducar, Anwesha Nag, Laura E. MacConaill, Matthew L. Meyerson. Increased sensitivity for detection of oncogenic mutations present in circulating cell-free tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5695. doi:10.1158/1538-7445.AM2017-5695