Abstract 3959: Detection of somatic mutations at 0.1% frequency from cfDNA in peripheral blood with a multiplex next-generation sequencing assay
Authors: Brinza Dumitru , Mongan Ann , Chen Richard , Dhingra Dalia , Gu Jian , Au-Young Janice , Hyland Fiona , Bramlett Kelli 
Published: July 2016

Abstract
Background:
Effective blood screening for tracking of recurrence and resistance of tumors may improve outcomes in the future. Research studies suggest that virtually all tumors carry somatic DNA mutations, and these may serve as biomarkers that also can be tracked in blood. One of the sources containing tumor DNA in blood is circulating cell-free DNA (cfDNA). Tumor DNA comes from different tumor clones, and its abundance in plasma can be very low at critical stages such as early recurrence or development of resistance. Hence, there is great interest in being able to detect mutation biomarkers at very low frequency from cfDNA for detection and characterization of tumor clones.
Method:
We present a research use only analysis workflow for peripheral monitoring that enables detection of low frequency DNA variants in blood.
We developed an analysis algorithm that models errors accumulated during amplification and sequencing, and accurately reconstructs sequence of original DNA molecules based on multiple next generation sequencing reads. The reads contain genomic sequence and an adaptor that allows identification of reads originated from the same DNA molecule. We then developed a variant calling method that uses accurately reconstructed sequences to enable sensitive and specific detection of somatic mutations to 0.1% allele ratio. We demonstrate the analysis in control and archived cfDNA research samples.
We used a next generation sequencing assay that allows interrogation of ∼150 biomarkers relevant in lung from COSMIC and Oncomine™ databases, and de-novo variant detection at ∼1,700 genomic positions in 11 genes implicated in non-small cell lung cancer (NSCLC).The assay delivers &gt;95% on target reads and highly uniform amplification across targeted cfDNA molecules.
Results:
We tested the limits of variant detection in controlled dilution series and in cfDNA. First, we diluted engineered AcroMetrix™ Oncology Hotspot Control plasmids into background GM24385 genomic DNA down to 0.1% frequency, and then fragmented into fragments with average size of 170bp. The Acrometrix sample contains ∼45 common tumor mutations interrogated by our assay. Next, we used 0.1% Horizon's (HD780) cfDNA reference sample that contains 8 mutations at our hotspot positions including two large insertion and deletion variants of size &gt;10bp. Finally, we performed analytical verification of variant detection performance in cfDNA using a dilution series of normal blood samples.
We achieved &gt;95% sensitivity with &gt;20ng input DNA and &gt;90% sensitivity with ∼20ng input DNA and &lt;1 false call per sample for variants in hotspot positions present at frequency 0.1%.
Conclusions:
This new lung cfDNA analysis workflow may facilitate researchers to study relevant NSCLC biomarkers at 0.1% frequency in cfDNA. Analysis is compatible with lower frequency variant detection, but will require higher input DNA amount and higher sequencing coverage.
Citation Format: Dumitru Brinza, Ann Mongan, Richard Chen, Dalia Dhingra, Jian Gu, Janice Au-Young, Fiona Hyland, Kelli Bramlett. Detection of somatic mutations at 0.1% frequency from cfDNA in peripheral blood with a multiplex next-generation sequencing assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3959.