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M. Kuebler



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    P2.06 - Poster Session 2 - Prognostic and Predictive Biomarkers (ID 165)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.06-047 - BLOCker Sequencing - An Improvement in Sanger Sequencing which Supercharges Low Level Mutation Detection (ID 3369)

      09:30 - 09:30  |  Author(s): M. Kuebler

      • Abstract

      Background
      Epidermal growth factor receptor (EGFR) antagonists are therapeutic agents that can be effective in colorectal cancer (CRC) treatment. It has been shown that 40% of CRC tumors have activating KRAS exon 2 codon 12 and 13 mutations and that these mutations are associated with a poor response to EGFR antagonists. Recent studies have shown that mutations in KRAS exons 3 and 4 as well as NRAS exons 2, 3, and 4 also predict poor response to EGFR agonists such panitumumab. Sensitive detection (down to 1%) with Sanger sequencing of such diagnostic biomarkers is necessary to determine the presence or emergence of drug resistant tumor cell populations. 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) analysis applications. Incorporation of LNA into oligos has the advantage of increasing the melting temperature of the LNA/complementary template duplex after hybridization as compared to duplexes using standard oligos.

      Methods
      To improve the mutation detection limits of Sanger sequencing, an LNA-based approach has been developed to cycle sequence the mutant allele selectively in the presence of the wild-type allele. During cycle sequencing, an additional annealing step is added to hybridize the LNA-containing oligo (BLOCker-oligo) to the template DNA. A denaturing step is then performed at a temperature at which the BLOCker-oligo remains annealed to the wild-type sequence while the LNA oligo denatures from the mutant sequence. The sequencing primer then anneals to the mutant sequence and is subsequently extended. Both forward and reverse strand BLOCker oligos can be developed enabling sensitive enrichment for bidirectional sequencing.

      Results
      To show applicability of this methodology in CRC samples, the limit of detection in both the forward and reverse sequencing directions for multiple codon 12 and codon 13 KRAS exon 2 mutations with and without the addition of the KRAS exon 2 wild-type specific BLOCker-oligo will be demonstrated. To date, the increase in sensitivity is ~10 fold. In addition, a series of FFPE samples will be analyzed for mutations in NRAS and KRAS exons 2 – 4 with and without BLOCker-oligos to show the increase sensitivity of this approach.

      Conclusion
      BLOCker sequencing is an efficient methodology for the detection of any mutation present in a sample using standard laboratory equipment and Sanger sequencing. The assays being developed at Transgenomic will be offered as CE IVD kits for the detection of all TKI-response associated mutations in KRAS and NRAS Exons 2, 3 and 4.

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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
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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): M. Kuebler

      • 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.