Abstract 5438: Multiplexed ICE COLD-PCR coupled to NGS and ddPCR enables enhanced detection of low-level DNA mutations in tissues and liquid biopsies
Authors: Richardson Katherine Anne , Statt Sarah , Wu Grant , Scott Karissa , Montagne Erin , Jensen Sheena , Cubrich Courtney , Krzycki Phil , Stoddard Jason , Kruempel Amy , McCutchen Emily , Veys Stephanie , Baughman Kylee , Cherubin Sarah , Rosendale Vicki , Pope Jaclyn , Bartlett Paula , Eastlake Phil , Peterson Stephanie , Legendre Benjamin 
Published: August 2015

Abstract
Introduction: The use of “liquid biopsies”, where limited or no tumor tissue is available, is increasingly important for molecular demographics, diagnostics and pharmacodynamic monitoring of patients during therapy.
The ICE COLD-PCR (ICP) technique preferentially amplifies sequence alterations in samples having either vast excesses of wild-type sequence or when sample DNA quantity is sub-optimal. ICP delivers unbiased, high-level enrichment of gene regions enabling determination of point mutations and insertions/deletions using Sanger sequencing, Next Generation Sequencing (NGS) or droplet digital PCR (ddPCR). This is especially important when sample DNA, e.g. from circulating free DNA, exosomes and circulating tumor cells (CTCs), is insufficient for multiplexed analysis.
A critical limitation of mutational analysis of such samples is the need for increasing amounts of DNA for detecting very low-level mutations. A range of 100 to330 ng of substrate DNA is usually needed for reliable detection of alterations present at 0.01% in the sample DNA; this is not feasible with the limited quantities of blood/plasma/serum from clinical trials. ICP's ability to enrich alterations can provide a ≥100-fold increase in Sanger, NGS and ddPCR sensitivity.
Materials and Methods: To increase throughput, address the limiting amounts of DNA present in these samples and provide enriched amplification from many different gene regions in a single DNA sample, a multiplex ICP approach has been developed (MX-ICP). This MX-ICP method provides enrichment of any alteration present in all targeted genes from a single sample of DNA. When MX-ICP products are analyzed by Sanger, NGS or ddPCR, lower quantities of sample DNA can be used for detection of mutations at ≤0.01%.
We compared detection of low-level of mutations in limiting amounts of DNA, with or without the use of MX-ICP prior to NGS and ddPCR, using digitally verified chromosomal DNA mixtures from Horizon Diagnostics. The alterations analyzed were from (1) CTC and NSCLC patients’ plasma, (2) longitudinal sampling of melanoma patients and (3) CTCs isolated from NSCLC patients. In all cases, use of MX-ICP, prior to analysis using NGS or ddPCR, enabled very sensitive detection with low amounts of input DNA.
Conclusion: MX-ICP is a key component of procedures for sensitive detection and monitoring of genetic alterations in multiple targets using a single DNA sample. Coupling MX-ICP with platforms such as NGS and ddPCR enables the use of these powerful technologies for high sensitivity detection and monitoring of liquid biopsies from cancer patients. The combination of MX-ICP with NGS and ddPCR platforms means that they can be used efficiently for detection of alterations at
≤0.01% in samples with &lt;100 ng DNA. This enables monitoring and detection of alterations in the low volumes of liquid biopsies obtained from patients and clinical trials.
Citation Format: Katherine Anne Richardson, Sarah Statt, Grant Wu, Karissa Scott, Erin Montagne, Sheena Jensen, Courtney Cubrich, Phil Krzycki, Jason Stoddard, Amy Kruempel, Emily McCutchen, Stephanie Veys, Kylee Baughman, Sarah Cherubin, Vicki Rosendale, Jaclyn Pope, Paula Bartlett, Phil Eastlake, Stephanie Peterson, Benjamin Legendre. Multiplexed ICE COLD-PCR coupled to NGS and ddPCR enables enhanced detection of low-level DNA mutations in tissues and liquid biopsies. [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 5438. doi:10.1158/1538-7445.AM2015-5438