Abstract 1979: Enhancing cell-free DNA and mutant copy recovery from plasma with novel liquid-liquid extraction
Authors: Janku Filip , Huang Helen J. , Pereira David Y. , Kobayashi Masae , Chiu Chung Hei , Call Steven G. , Woodbury Kristen T. , Chao Felix , Marshak Daniel R. , Chiu Ricky Y. 
Published: September 2020

Abstract
Background: Low yields of extracted cell-free DNA (cfDNA) from plasma limit further development of liquid biopsies especially in early-stage cancer diagnostics and cancer screening applications. We developed a novel liquid-liquid based DNA isolation method (PHASIFY™ MAX; Phase Scientific International Ltd., Kwun Tong, Hong Kong) that leverages aqueous two-phase systems (ATPS) to purify and concentrate circulating cfDNA, which can result in increase of cfDNA recovery and mutant signal detection.
Methods: The PHASIFY™ ATPS consists of unique formulations of polymer and salt components that can intrinsically separate into two distinct phases. The formula can be optimized to drive cfDNA into one phase and contaminants from plasma into the other, achieving simultaneous target isolation and concentration. We compared cfDNA recovery and mutation detection from plasma samples extracted with the industry standard QIAamp Circulating Nucleic Acid kit (QCNA; Qiagen, Hilden, Germany) and PHASIFY™ MAX cfDNA extraction kit. According to the respective kit protocols, the cfDNA was extracted from 1mL of plasma from patients with advanced cancers, including those known to shed low amounts of cfDNA into circulation (e.g., thyroid, pancreatic), with known tissue BRAF, KRAS, or NRAS mutation status. Total DNA recovery was determined with the Quant-iT PicoGreen dsDNA Assay Kit. Mutation status was determined with probe-based (BRAF, KRAS, NRAS) Droplet Digital PCR (ddPCR; Bio-Rad, Hercules, CA).
Results: Plasma samples from 89 patients with diverse advanced cancers (colorectal, N=36; ovarian, N=18; lung N=7; thyroid, N=6; pancreatic, N=6; others, N=16) with known BRAF, KRAS or NRAS mutation status were used for cfDNA extraction with standard QCNA and PHASIFY™ MAX kits. On average, PHASIFY™ MAX demonstrated a 288% increase in total cfDNA recovery compared to the QCNA kit (p-value &lt;0.0001 Wilcoxon Signed-Rank). The ddPCR analysis of samples extracted by PHASIFY™ MAX demonstrated clinical sensitivity and specificity (against tissue genotyping) of 55% (46/82) and 100% (6/6) respectively and 46% (38/82) and 100% (6/6) respectively with samples extracted by QCNA. PHASIFY™ MAX had a sensitivity of 87% (33/38) against QCNA extracted plasma samples as reference. Of the samples for which both methods confirmed mutations, on average, PHASIFY™ MAX achieved a 142% increase in recovery of mutant copies compared to the QCNA kit (p-value 0.0006 Wilcoxon Signed-Rank). In 13 samples, mutations were detected in samples extracted with PHASIFY™ MAX, but not with QCNA. Of the 13, 3 were from pancreatic cancer patients, suggesting that PHASIFY™ MAX may improve mutation detection in low cfDNA shedding tumors.
Conclusion: Our early results indicate that the PHASIFY™ MAX extraction technique offers higher cfDNA recovery resulting in higher sensitivity of cfDNA mutation detection compared to the industry standard.
Citation Format: Filip Janku, Helen J. Huang, David Y. Pereira, Masae Kobayashi, Chung Hei Chiu, Steven G. Call, Kristen T. Woodbury, Felix Chao, Daniel R. Marshak, Ricky Y. Chiu. Enhancing cell-free DNA and mutant copy recovery from plasma with novel liquid-liquid extraction [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1979.