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

  • 11565

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    P2.06-022 - Use of the Real-Time PCR and Multiplex Ligation-dependant Probe Amplification (MLPA) Assay in the Molecular Diagnosis for NSCLC biomarker detection. (ID 1971)

    09:30 - 09:30  |  Author(s): K. Czubak
    • Abstract

    Background
    Variety of molecular methods for somatic mutations detection in NSCLC demonstrate discrepancies in sensitivity, cost and performance. Presence of activating mutation in TK domain of EGFR gene qualifies NSCLC patients to targeted therapy with TK inhibitors. Sanger sequencing is still a 'gold standard' for EGFR point mutation analysis and FISH is the primary method for detecting copy number of specific genes in tissue sections. Alternative methods like qRT-PCR, CISH or MLPA are more and more commonly used to detect EGFR point mutations or MET or HER2 amplification, yet results on their accuracy remain inconclusive.

    Methods
    We compared EGFR mutation analysis performed on 278 adenocarcinoma ( NSCLC FFPE and cytology) samples using Real-Time PCR technology with modified taqman probes to detect 29 EGFR mutations in exon 18, 19, 20 and 21 and Multiplex Ligation-dependent Probe Amplification (MLPA) to detect the most common deletion in exon 19, substitutions in exon 21 (L858R), 20 (T790M) and 18 (G719X), and insertion in exon 20 (S768I). Secondly, we evaluated MET and HER2 amplification in all 278 samples by MLPA only.

    Results
    EGFR mutation analysis demonstrated identical results in 97.5% cases between qRT-PCR and MLPA; 2.5% of discrepancy came from limit detection between those two methods (1% Limit of Detection for qRT-PCR; and 10-15% for MLPA). The fluorescent signals to cross the threshold for those 2.5% cases were detected in late cycles and positively correlated with small percentage of tumour cells in the sample. Both methods are more sensitive than traditional Sanger Sequencing which limit of detection is in a range 15%-25%. Additional MET and HER2 amplification analysis indicated that 10% of NSCLC samples carried MET and 1.8% - HER2 amplification.

    Conclusion
    Both Real-Time PCR and MLPA are accurate tools to detect point mutations or chromosomal aberration, respectively - as an alternative method to time consuming Sanger sequencing and FISH analysis for NSCLC biomarker detection. As MET amplification is known negative prognostic factor, in particular for patient treated with TK inhibitors, we suggest to use both technologies in NSCLC diagnostics: qRT-PCR for EGFR somatic mutation analysis and MLPA to detect MET amplification.