Author of

• 12441

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  P3.06 - Poster Session 3 - Prognostic and Predictive Biomarkers (ID 178)

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Biology
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/30/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 12453

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    P3.06-013 - ICE COLD-PCR Combined with Next-Generation Sequencing: Increased Sensitivity for High Throughput Detection of Mutations. (ID 1433)

    09:30 - 09:30  |  Author(s): S. Cherubin
    • Abstract

    Background
    The ICE COLD-PCR (Improved and Complete Enrichment COamplification at Lower Denaturation Temperature PCR) technology is capable of high sensitivity mutation detection. The ICE COLD-PCR (ICP) reaction contains a reference sequence oligonucleotide (RS-oligo) that hybridizes to both alleles but will dissociate from the mutant strand at the critical temperature for all mutation types: point mutations, insertions, deletions and indels. Following ICP, samples with mutations present at 0.1% in the original sample can be enriched and determined using standard Sanger sequencing. Next-Generation Sequencing (NGS) allows high-throughput analysis of somatic mutations in cancer using targeted resequencing panels of genes. Thus a broad mutation signature of tumors can be determined. However, the level of detection is ~4% for mutations unless a higher depth of coverage is used; this comes at the price of reducing the number of samples that can be analyzed on a chip. ICP enrichment of mutations prior to NGS mimics an increased "depth of coverage" without reducing the throughput per chip.

    Methods
    Proof of concept experiments were performed using low level mutations: KRAS G12R (0.5, 0.1, 0.05%) PIK3CA E542K and E545K (1.0, 0.5, 0.1%) and EGFR Exon 19 E746_A750del (0.5, 0.1, 0.05%). In addition, DNA isolated from FFPE tissues and matched plasma were amplified by standard PCR or enriched by ICP and subsequently analyzed for PIK3CA, KRAS, BRAF and EGFR mutations using the Ion AmpliSeq Cancer Hotspot Panel on the Ion Torrent Personal Genome Machine (PGM) with the Variant Caller Plugin.

    Results
    

    ICE COLD-PCR Amplification of FFPE DNA with Sequence Confirmation by Sanger Sequencing or Next Generation Sequencing using the Ion Torrent PGM
    Gene	Mutation in Sample	% Starting Mutant	% Variant Frequency (Sanger)	% Variant Frequency (NGS)	P-value	Coverage	Ref Cov	Var Cov
    EGFR Exon 19	#1 E746_A750_Del	0.50%	30	9.1*	1.00E-10	2,000	1,812	182
    #2 E746_A750_Del	0.10%	10	3*	7.82E-10	2,000	1,940	60
    #3 E746_A750_Del	0.05%	Background	No Variant
    KRAS	#1 G12R	0.50%	70	69.18	1.00E-10	19,174	5,819	13,264
    #2 G12R	0.10%	10	23.58	1.00E-10	10,004	7,513	2,359
    #3 G12R	0.05%	Background	2.86	5.01E-04	11,027	10,603	315
    PIK3CA	#1 E542K	1.0%	15	16.48	1.00E-10	28,899	24,113	4,763
    #1 E545K	1.0%	25	24.23	1.00E-10	30,027	22,700	7,275
    #2 E542K	0.5%	10	9.19	1.00E-10	15,680	14,225	1,441
    #2 E545K	0.5%	10	11.78	1.00E-10	15,267	13,405	1,798
    #3 E542K	0.1%	Background	2.02	1.00E-10	15,358	15,045	310
    #3 E545K	0.1%	Background	3.17	1.00E-10	15,154	14,657	481
    * difficult to detect deletions by NGS

    Conclusion
    ICP is a flexible technology that results in a PCR product enriched for any mutation present in the sample analyzed. This upstream enrichment of low level somatic mutations combined with high throughput analysis by NGS brings NGS one step closer for use in high throughput screening of circulating mutations for patient treatment selection and monitoring of disease.