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

  • 11593

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    P2.06-050 - Validation and Utilization of a Clinical Next Generation Sequencing Assay to identify Mutations and Genomic Copy number changes in Lung Cancer (ID 3419)

    09:30 - 09:30  |  Author(s): A. Fox
    • Abstract

    Background
    Recent advances in next generation sequencing provide us with the ability to simultaneously analyze both mutation status and copy number changes across a large number of cancer related genes. Here we discuss our experience in developing, validating and applying a 47 gene panel across our lung tumor patient population in a clinical laboratory.

    Methods
    Genomic DNA was extracted from 20 Formalin Fixed Paraffin embedded (FFPE) tissue samples from lung tumors for validation and over 80 patient samples upon clinical implementation. Extracted DNA was analyzed for quality and amplifiable quantity with suitable performance criteria established during validation. Between 10 and 250 ng of total genomic DNA was then subjected to the Truseq Amplicon (Illumina) assay for enrichment of the target regions across 47 different genes. Libraries were then sequenced on the Miseq (Illumina) to an average depth of coverage between 2000 and 4000x with 186 basepair, paired end reads. Data was then analyzed using analysis pipelines composed of various in house and open source tools, to detect insertions, deletions, amplifications and single nucleotide variants to an allele frequency of 5%.

    Results
    Across the clinical sample set, 67% of samples were found to have at least one disease associated mutation, 8% had only an unclear variant, 13% were ‘normal’, while 12% had DNA that was not adequate for testing. The average number of mutations seen in samples with disease associated changes was 1.6, with a range from 1 to 4. Half of the samples analyzed were found to have Tp53 mutations, followed by EGFR changes seen in 11%. While only 16 patients had EGFR changes, nearly half (7) of these had co-mutations, including changes at positions 709, 719, 768 and 790. Many of the EGFR changes were complicated insertion/deletions found in exons 19 and 20, which were missed by standard bio-informatic algorithms and only captured by in house tools. KRAS changes were seen in 21% of patients followed by PTEN, MET, STK11 and APC mutations seen in at least 2 patients. Other known somatic mutations were also identified in ATM, BRAF, CTNNB1, FBXW7, FLT3, GNAS, HRAS, NRAS, PIK3CA and ALK in at least one patient. This included the hotspot mutation F1174L in ALK, which is seen with high frequency in neuroblastoma.

    Conclusion
    The mutation status of many clinically relevant genes can be reliably detected in FFPE samples using a single molecular assay followed by high throughput sequencing. Using this approach known somatic mutations seen frequently in other tumor types can be readily identified in lung tumors, and highlights the future benefits of tumor profiling.