Abstract 2402: A research approach for the detection of somatic mutations at 0.5% frequency from cfDNA and cTc DNA using a multiplex sequencing assay targeting 2000 tumor mutations
Authors: Brinza Dumitru , Dhingra Dalia , Scafe Charles , Chien Richard , Hyland Fiona 
Published: August 2015

Abstract
Background:
Availability of effective blood screening for tracking of recurrence and resistance of tumors may improve outcomes. Research studies suggest that virtually all tumors carry somatic DNA mutations, and these may serve as biomarkers that may be tracked from blood. The two well-characterized sources of tumor DNA in blood are circulating tumor cells (cTc) and cell-free tumor DNA (ctDNA). The abundance of cTc and/or ctDNA in blood may be very low at critical stages such as early recurrence or development of resistance. Hence there is great interest in being able to detect biomarkers at very low frequency from blood, and in characterizing the relationship between somatic mutations present in the tumor and those in cTc or ctDNA.
Method:
We present a research use only analysis workflow that facilitates researchers in detecting low frequency variants in blood. We developed an analysis algorithm, using statistical modeling of next generation sequencing reads, and optimizing parameters and filters to enable sensitive and specific detection of somatic mutations to 0.5% allele ratio.
We demonstrate the analysis on a blood sample split into 3 sub-samples comprising germline blood cells, cTc, and ctDNA.
We barcode the 3 sub-samples and run them on a single Ion PGM 318 sequencing chip using Ion AmpliSeq CHPv2 cancer hot-spot panel, that enables very deep (∼10,000x coverage) and accurate sequencing.
This panel allows interrogation of ∼2000 relevant biomarkers from COSMIC and FDA actionable databases, and de-novo variant detection at ∼20,000 genomic positions. Mutations were annotated using the Oncomine database in Ion Reporter software.
The assay requires a small amount of input DNA (∼10ng), and has a fast turn around time from sample to variants of less than 24 hr.
Results:
We tested the limits of variant detection in a dilution series; in cTc; and in ctDNA.
First, we diluted an engineered DNA sample from Acrometrix into a control sample (NA12878) down to 0.5% frequency. The Acrometrix sample contains ∼500 common cancer mutations from COSMIC and FDA actionable databases. We achieved &gt;99% sensitivity and specificity for variants present at frequency above 0.5%.
Next, we spiked cTc cells from cancer cell lines into normal blood samples at ratio 1:1,000,000, obtaining 40% purity of cTc after enrichment, and demonstrated &gt; 99% sensitivity and specificity of variant detection.
Finally, we performed analytical validation of variant detection performance in ctDNA using a dilution series of two normal blood samples, and we were able to detect in all 20 variants present in either sample in ctDNA when their frequency was above 0.5%.
Conclusions:
The analysis workflow may facilitate researchers to study biomarkers in DNA from germline, cTc, and ctDNA, all available from a single blood sample, and to explore the relationship between biomarkers observed in solid tumor and those in blood.
Citation Format: Dumitru Brinza, Dalia Dhingra, Charles Scafe, Richard Chien, Fiona Hyland. A research approach for the detection of somatic mutations at 0.5% frequency from cfDNA and cTc DNA using a multiplex sequencing assay targeting 2000 tumor mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2402. doi:10.1158/1538-7445.AM2015-2402