Author of

• 11543

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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
  • Moderators:
  • Coordinates: 10/29/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 11590

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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): P. Krzycki
    • 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.