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J.J. Yang



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    ORAL 16 - Clinical Care of Lung Cancer and Advanced Biopsies (ID 115)

    • Event: WCLC 2015
    • Type: Oral Session
    • Track: Treatment of Advanced Diseases - NSCLC
    • Presentations: 1
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      ORAL16.07 - Intratumor Heterogeneity of EGFR Activating Mutations Analyzed in Single Cancer Cells in Advanced NSCLC Patients (ID 2311)

      10:45 - 12:15  |  Author(s): J.J. Yang

      • Abstract
      • Presentation
      • Slides

      Background:
      Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) can achieve dramatic response in EGFR activating mutation positive lung cancer patients. However, the duration of treatment is quite different. Some patients experienced longer progression-free survival (PFS) of more than 1 year, whereas some had PFS of shorter than 6 months. Our previous study showed that the relative EGFR mutation abundance in tumor tissues could predict benefit from EGFR-TKIs treatment. However, it still remains controversial whether the intratumor heterogeneity of EGFR activating mutation exists. This study explored the intratumor heterogeneity of EGFR activating mutation at the level of single cancer cell.

      Methods:
      Single H1975 cells which harbor EGFR exon 21 L858R mutation were isolated by flow cytometry (FCM). The whole DNA extracted from a single cell was submitted to perform nested polymerase chain reaction (PCR) amplification of EGFR exon 21. The amplified products from nested PCR were sequenced to evaluate the feasibility of single-cell analysis for EGFR exon 21. Then, six patients diagnosed with lung adenocarcinoma whose fresh frozen specimens harbored EGFR exon 21 mutation tested by direct sequencing were chosen. All of them received gefitnib treatment and the PFS of three patients was longer than 14 months (Group A) while the PFS of other three patients was shorter than 6 months (Group B). By using the established method based on single H1975 cells, EGFR exon 21 mutational status was analyzed in single tumor cells which were captured from tumor sample by Laser Capture Microdissection (LCM). At least 20 tumor cells were captured from each tumor sample. X[2] test was used to compare the amplification rate of nested PCR and EGFR mutational rate between the two groups.

      Results:
      A total of 104 individual H1975 cells were obtained to detect EGFR exon 21 mutational status through the application of single-cell nested PCR. The amplification rate and allele drop-out rate were 96.2% and 7.0%. A total of 135 tumor cells from six samples were captured. The amplification rate of nested PCR was 84.3% (59/70) in Group A and 93.8% (61/65) in Group B. There was no statistical difference between the two groups (X[2] =3.119, P=0.077). The mutational rate of EGFR exon 21 L858R was 89.5% (17/19), 89.5% (17/19), and 81.0% (17/21) in the three patients in Group A and 72.2% (13/18), 68.4% (15/22), and 66.7% (14/21) in the three patients in Group B respectively. The total mutational rate was 86.4%(51/59)in Group A, which was significantly higher than the total mutational rate 68.9%(42/61)in Group B (X[2] =5.321, P=0.021).

      Conclusion:
      It is feasible to perform EGFR mutation detection in single cancer cells. The intratumoral heterogeneity of EGFR activating mutation in lung adenocarcinoma does exist based on the analysis in single cancer cells and the abundance of EGFR activating mutation is relevant to the benefit from EGFR-TKIs treatment.

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