No 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 <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

No abstract

Epidermal growth factor receptor (EGFR) antagonists, such as cetuximab and panitumumab, are therapeutic agents that can be effective in colorectal cancer (CRC) treatment. It has been shown that 40% of CRC tumors have activating K-RAS exon 2 codon 12 and 13 mutations and that these mutations may be associated with a poor response to EGFR antagonists. Very high sensitivity detection of such diagnostic biomarkers is necessary to determine the presence or emergence of drug resistant tumor cell populations. The use of Locked Nucleic Acid (LNA™) containing oligonucleotides (oligos) have been used in microRNA (sample preparation), RNA (in situ hybridization), and DNA (SNP detection using allele specific PCR) applications. The advantage of using LNA-containing oligos is that the denaturation temperature of LNA-oligo:DNA duplex is increased as compared to the DNA:DNA duplex To further the limit of detection of Sanger sequencing, a LNA-based approach has been developed to selectively block the sequencing of the wild-type DNA and allow the sequencing of DNA containing any mutation (BLOC-Sequencing). During cycle sequencing, an additional annealing step is added to hybridize the LNA containing oligo to the template DNA. Then, a denaturing step is performed at a temperature at which the LNA-containing oligo remains annealed to the wild-type sequence; however, the LNA oligo denatures from the mutant sequence. The sequencing primer then anneals to the mutant sequence and subsequently extended. Since LNA-containing oligos block amplification of the wild-type sequence, the mutant-containing DNA will be sequenced in a non-biased manner. Preliminary results using two sequences in a 1:1 ratio differing in only 2 nucleotides within the range where the LNA blocking oligo anneals demonstrated complete blocking of one sequence (sequence 1) where the LNA containing oligo was 100% complementary. The unblocked sequence (sequence 2) sequenced successfully, with minimal background due to sequence 1. Straight Sanger sequencing results of the 1:1 mixture without the LNA blocking oligo gave equal proportion of the two sequences present. To show applicability for use of this methodology in CRC samples, the limit of detection for multiple codon 12 and codon 13 K-RAS exon 2 mutations with and without the addition of the K-RAS exon 2 wild type specific LNA oligo in both the forward and reverse directions will be demonstrated. In addition, we will compare the results of 30 previously genotyped FFPE samples with this technique. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4855. doi:10.1158/1538-7445.AM2011-4855

Introduction: The EGFR Exon 20 T790M mutation confers resistance to the first generation tyrosine kinase inhibitors (TKI), such as Tarceva®. A new generation of treatments, including AZD9291, targets the T790M mutation thus allowing a new approach to cancer therapy for NSCLC patients. Recently EGFR Exon 20 C797S mutations (c.2389T>A and c.2390G>C) have been identified as resistance mutations to the third generation TKI AZD9291. High sensitivity detection of these mutations, as well as the T790M mutation, in patients with acquired resistance to third generation TKI is an important requirement for best practice NSCLC treatment. Materials and Methods: ICE COLD-PCR (Improved & Complete Enrichment CO-amplification at Lower Denaturation temperature PCR) is a technology that preferentially enriches mutant DNA sequences in an excess of wild-type DNA through selective amplification of the mutant DNA population using an oligonucleotide (RS-oligo) complementary to the wild-type sequence. Due to the inherent sequence characteristics of the region between the codons of interest (790 and 797), a RS-oligo focusing on the C797S was developed and is independent of the RS-oligo for T790M, and thus tiling across EGFR Exon 20 can be performed. The RS-oligo in this assay was designed to prevent PCR amplification of wild-type sequences while allowing exponential amplification of any mutations including C797S within the RS-oligo binding region. Serial dilutions of spiked gBlocks® DNA containing C797S mutations mixed with K562 DNA and samples extracted from plasma were amplified with ICE COLD-PCR followed by Sanger sequencing analysis. Result: For mutations c.2389T>A and c.2390G>C, limits of detection as low as 0.05% were achieved. Forty DNA samples extracted from plasma were also tested. These indicated that the assay is suitable for fragmented samples with low quantities of input DNA such as liquid biopsies. Conclusion: The pre-amplification PCR product covers both codons 790 and 797. This allows ICE COLD-PCR to be used for the analysis of both T790M and C797S using the same liquid biopsy sample aliquot. This new ICE COLD-PCR method to detect both C797S mutations c.2389T>A and c.2390G>C may provide a highly sensitive, simple and cost effective assay for monitoring acquired resistance to AZD9291 in the liquid biopsies obtained from patients and clinical trials. Coupled with our existing highly sensitive assay for T790M, ICE COLD-PCR offers a highly sensitive method to determine the emergence of resistance mutations, thus potentially contributing to therapeutic optimization. Citation Format: Grant Wu, Sheena Jensen, Karissa Scott, Erin Montagne, Jason Stoddard, Phil Krzycki, Courtney Schweikart, Benjamin L. Legendre, Philip Eastlake, Katherine A. Richardson. High sensitivity detection of EGFR exon 20 T790M and C797S mutations using ICE COLD-PCR. [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 1390.