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M. Skronski



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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-044 - Diagnostic validation of PNA-LNA PCR clamp assay for detection of EGFR exon 19 and 21 mutations in non-small cell lung cancer specimens. (ID 2729)

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

      • Abstract

      Background
      PNA-LNA PCR clamp is highly sensitive, real-time PCR-based laboratory technique developed to enable reliable detection of EGFR gene mutations in wide spectrum of tissue/biopsy samples from NSCLC patients. The aim of the study was to assess diagnostic reliability of PNA-LNA PCR clamp assay in EGFR mutations detection in different NSCLC samples.

      Methods
      Evaluation was performed: (i) in reference NSCLC tissue FFPE samples (n=10), (ii) in comparison to direct sequencing in resected NSCLC tissue (n=199) and biopsy material specimens (n=179) characterized by different tumor cells content (TCC) and fixation [Table 1].

      [Table 1.] NSCLC samples
      Resected tissue Biopsy material
      fresh -frozen FFPE FFPE Cytology smear
      84 115 115 64

      Results
      (i) PNA-LNA PCR clamp correctly detected all exon 19 deletions and L858R mutations in the reference FFPE materials, including those with meager TCC (5% and 10%). (ii) In total of 378 samples analyzed with PNA-LNA PCR clamp method EGFR mutations were detected in 36 (9.5%). No significant differences in detection efficiency were observed in reference to material (resected tissue vs biopsy, p=0,3972; OR=0,7405; CI=0,3694-1,4844) and fixation procedure (FFPE vs fresh-frozen tissue, p=0,5459; OR=0,7304; CI=0,2635-2,0248; biopsy material FFPE vs cytology smear, p=0,4366, OR=0,6908; CI=0,272-2,7544). (iii) PNA-LNA PCR clamp method and direct sequencing presented high conformity (overall percent agreement, OPA=99%; Cohen’s Kappa score of 0.94 (95% CI=0.9, 0.99) in n=100 samples with >50% TCC. (iv) PNA-LNA PCR clamp presented higher sensitivity in samples with TCC <50% (p=0.004). Reevaluation with direct sequencing proved positive only in 24 out of 36 (67%) mutation positive samples [Table 2].

      [Table 2. ]Comparison of PNA-LNA PCR clamp vs direct sequencing EGFR mutation detection sensitivity in 36 EGFR mutation positive materials with different %TCC.
      PNA-LNA PCR clamp direct sequencing
      ≥50% 25/25 (100%) 22/25 (88%)
      20<50% 6/6 (100%) 2/6 (33%)
      ≤20% 5/5 (100%) 0/5 (0%)
      total 36/36 (100%) 24/36 (67%)

      Conclusion
      PNA-LNA PCR clamp method is characterized by high sensitivity of EGFR exon 19 and 21 mutations detection in tissue and biopsy material, particularly in samples with TCC lower than 50%. Fixation procedures did not affect PNA-LNA PCR clamp method mutation detection effectiveness.