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A. Szpechcinski



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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): A. Szpechcinski

      • 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.

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    P3.02 - Poster Session 3 - Novel Cancer Genes and Pathways (ID 149)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.02-016 - Proteinase inhibition regulates the anti-tumor activity of PAI-1 towards lung and prostate cancer cells (ID 2706)

      09:30 - 09:30  |  Author(s): A. Szpechcinski

      • Abstract

      Background
      Plasminogen activator inhibitor type 1 (PAI-1) plays an important role in tumor growth and metastasis formation, directly via specific urokinase complexing or indirectly due to its affinity to vitronectin.

      Methods
      The aim of this study was to analyze the impact of the mutant forms of PAI-1: very long half-life (VLHL PAI-1) or devoid of affinity towards vitronectin (Vn neg PAI-1) and wild form (wPAI -1) on proliferation of lung cancer (A549 and H1299 ) and prostate cancer (LNCaP and DU145) cells characterized by different proteinase (urokinase) production.

      Results
      The dose- and time-dependent inhibition of cell proliferation in the presence of VLHL PAI-1 was evident in A549 and LNCaP cultures. In H1299 cells inhibitory effect was only dose-depended (p<0.001), while in DU145 only 100 mg/ml of VLHL PAI-1 in 72 hrs cultures suppresed prostate cancer cells proliferative activity (p <0.001). No significant effect of Vn neg PAI-1 on the proliferation of A549 and H1299 was observed while in prostate cancer lines (DU145, LNCaP) only the inhibitory effect of the highest Vn neg PAI-1 concentration (100μg/ml) was evident (p <0.001) but not time-dependent. wPAI-1 did’t affect A549 and LNCaP proliferation while in highest concentration it had the stimulating effect on H1299 and Du145 (24,48 hrs cultures).

      Conclusion
      PAI-1 is a negative regulator of cancer cells proliferation due to its anti-proteinase activity. Its biological effect on lung cancer cells is time and dose-dependent.