Abstract 3623: Comparison of dPCR versus hybridization capture methods for next generation sequencing of ctDNA for a SNV, copy number amplification, and fusion liquid biopsy assay

Abstract

Abstract Circulating tumor DNA can be found in the plasma of cancer patients, and can be utilized for non-invasive cancer detection and genotyping through Next Generation Sequencing. However, unique characteristics of ctDNA pose technical challenges that limit technical sensitivities and robustness. ctDNA are fragmented, short, rare in abundance, and found in high background of normal DNA from leukocytes. Cancer variants range as low as 0.1-1% minor allele fractions, within the noise of NGS methods. Furthermore, clinical interest includes large number of genes and mutation types including single nucleotide variants, copy number amplification, and fusions. We compared two popular target enrichment categories, digital PCR and a hybridization-based capture panel for target enrichment. We assessed the assay performance and detection limitations, using a diverse sample set to test the range of input material that reflects ctDNA found in plasma. We titrated important cancer mutations (BRAF, EGFR, KRAS, PI3KCA, etc.) testing minor allele fractions ranging from 33% down to 0.1%, MET amplification ranges of 14x to 5x, and CCDC6-RET fusions of 50% to 2%. We also assessed input DNA ranges of 1ng and 10ng, well below manufacturer recommend input of library prep methods. Testing the performance of a 29kb amplicon panel and a 114kb hybridization-probes panel, we found superior on-target rates with digital PCR compared to the hybridization capture method across all samples. However, despite higher depths of coverage with droplet PCR, hybridization based methods had superior read mapping, uniformity (1.4x better), error rate (0.16% vs 0.02%), and greater linearity of SNV variants. Hybridization methods had greater accuracy of variant detection and were able to accurately identify the RET fusion. We demonstrated the feasibility of utilizing target enrichment methods for next generation sequencing for ctDNA variant detection. Citation Format: Jonathan Choi. Comparison of dPCR versus hybridization capture methods for next generation sequencing of ctDNA for a SNV, copy number amplification, and fusion liquid biopsy 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 3623.