Abstract 4920: The detection of circulating tumor DNA using Quantstudio 3D digital PCR

Abstract

Abstract Purpose: The purpose of this study is to employ the Quantstudio 3D (Q3D) system to detect circulating tumor DNA in cancer patients’ plasma. Background: Circulating tumor DNA (ctDNA) as a non-invasive biomarker plays roles in monitoring solid tumor progression, metastasis, and therapeutic effects. The identification of ctDNA depends on mutant determination. The digital PCR technology is a powerful tool to identify ctDNA. The Q3D platform is a relatively new digital PCR systemwhcih has not been extensively tested in the detection of ctDNA. In this study, we tested the capacity of Q3D in detecting ctDNA. Methods: Genomic DNA was extracted from tumor tissue of patients. Variants were identified in tumor tissue DNA by Illumina's TruSeq Amplicon Cancer Panel. To detect the circulating tumor DNA, DNA was extracted from the plasma of the patient with known variants identified by targeted-sequencing. The circulating DNA was isolated from 400μl of plasma from 12 patients using QIAamp DNA Blood kit. The DNA concentration was measured using the Qubit 2.0. The quality and size of the circulating DNA was determined using the Agilent 2100. The circulating DNA size was approximately 170-180 nucleotides in length, as reported in literatures. The variant determination by Q3D was tested with custom TagMan SNP Genotyping Assays. The absolute copy number of variant was determined with QuantStudio® 3D AnalysisSuite™ Software. Results: We first tested several assays with known-variant tumor tissue DNA samples in Q3D. The mutants identified by assays are BRAF V600E, KRAS G12D, KRAS G12V, and PIK3CA E542K. The results showed that data points of mutant call, wild type call were well separated from non-signal data points. We examined the sensitivity of these assays using serial dilution of tumor DNA sample in non-tumor human tissue DNA. 0.1% to 1% of mutants were detectable depending specific assays. We collected circulating DNA from XX Patients’ plasma. The DNA concentrations ranged from 5 ng/ml to 30 ng/ml of plasma. KRAS G12D and G12V was detected with circulating DNA from patients carrying KRAS mutant. The mutant frequency of KRAS G12D and G12V determined by Q3D is 3.2% and 3.7%, respectively. The absolute mutant copy number for KRAS G12D is 9 and for KRAS G12V is 8. Conclusions: Q3D with validated custom TagMan SNP Genotyping Assays is applicable in detecting ctDNA. We will expect to utilize the technique to evaluate ctDNA. Citation Format: Adam H. Greer, Glenn Mills, Hong Yin. The detection of circulating tumor DNA using Quantstudio 3D digital PCR. [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 4920. doi:10.1158/1538-7445.AM2015-4920