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K. Stamm

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    MO10 - Molecular Pathology II (ID 127)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Pathology
    • Presentations: 1
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      MO10.04 - High throughput parallel amplicon sequencing of common driver mutations from FFPE lung cancer samples in molecular pathological routine diagnostics for a regional health care provider network (ID 2145)

      16:15 - 17:45  |  Author(s): K. Stamm

      • Abstract
      • Presentation
      • Slides

      Treatment paradigms for non–small-cell lung cancer (NSCLC) have shifted from one based only on histology to one that incorporates molecular subtypes involving particular genetic alterations such as activating mutations in EGFR or translocations of ALK. The list of therapeutically targetable lesions is rapidly increasing including mutations in genes such as EGFR, HER2, KRAS, ALK, BRAF, PIK3CA, AKT1, ROS1, NRAS, FGFR1 and MAP2K1. Analysis of these potential targets is becoming a challenge in terms of work load, tissue availability as well as cost. Within the Network Genomic Medicine Lung Cancer (NGM), a regional molecular screening network of the Center for Integrated Oncology Köln Bonn, we aimed to improve on the sequential analysis of a set of 9 target amplicons by Sanger sequencing using bench top ultra-deep parallel sequencing platforms. We aimed to reduce 1) the time requirement for comprehensive molecular diagnostics, 2) the minimal amount of formalin fixed paraffin embedded (FFPE) derived input DNA, 3) while at the same time increasing the number of target regions analysed.

      We established a multiplex PCR to amplify up to 640 lung cancer relevant target regions from at least 20ng of FFPE derived tumor DNA. The amplicon libraries were ligated to adapters encompassing medical identifier sequences that allowed multiplexing of up to 48 patients. The resulting libraries were sequenced on a benchtop Illumina platform (MiSeq). Mutations identified by parallel sequencing were confirmed by Sanger sequencing.

      330 patients were analyzed both by traditional single PCR based Sanger sequencing of 9 amplicons and the newly established parallel sequencing protocol. We found that the NGS approach worked reliably, was less prone to sequencing analysis errors and that the time needed to complete the mutation screening was significantly reduced to 7 working days from previously 21 days. A total of at least 300ng of DNA was needed to complete the analysis of 9 amplicons by Sanger sequencing compared to 20 to 100ng of DNA needed for up to 640 amplicons analyzed by parallel sequencing.

      Newly multiplex PCR based parallel sequencing allows rapid comprehensive mutation testing in routine molecular pathological diagnostics even on small FFPE embedded transbronchial biopsies.

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