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P. Meldgaard



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    MO21 - Prognostic and Predictive Biomarkers V - EGFR (ID 98)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 2
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      MO21.07 - Monitoring of EGFR TKI sensitizing and resistance mutations in plasma DNA of advanced adenocarcinoma of NSCLC during erlotinib treatment. (ID 2168)

      11:10 - 11:15  |  Author(s): P. Meldgaard

      • Abstract
      • Presentation
      • Slides

      Background
      EGFR TKI sensitizing mutations in plasma DNA isolated prior to treatment were shown to be a potent predictor for survival outcome of advanced NSCLC (T. Mok, ASCO 2013). Levels of EGFR mutations (pEGFRmut) in plasma during treatment, including sensitizing and resistance mutations, may offer an opportunity to monitor patient’s response to therapy and disease progression. In this study, we measured the levels of pEGFRmut at every 4 weeks during erlotinib treatment and investigated the emergence of the T790M mutation in relationship to disease progression.

      Methods
      Retrospective EGFR mutation testing of plasma samples from an unselected cohort of 227 patients with adenocarcinoma with an allele-specific PCR assay, cobas® EGFR_blood test (in development at Roche Molecular Systems, Inc.). The test is designed to detect 42 mutations in exon 18-21 of the EGFR gene including TKI sensitizing mutations (Exon 19 deletions, L858R, G719X and L861Q), resistance mutation (T790M) and atypical mutations (S768I and Exon 20 Insertions). 2 ml plasma of each patient was used for EGFR_blood PCR test. The genomic equivalent copy number of plasma DNA was determined by comparing to a standard curve of genomic DNA.

      Results
      25 (11%) of 227 unselected patients with adenocarcinoma had a sensitizing EGFR mutation in their plasma prior to the erlotinib treatment. Sequential plasma samples were retrieved for 23 of the 25 pEGFRmut+ patients. 22 (96%) of the 23 pEGFRmut+ patients had lower TKI sensitizing mutations after the first cycle (4 weeks) of erlotinib treatment. The mutated DNA was reduced below the limit of detection for 13 (57%) of the 23 pEGFRmut+ patients during the course of erlotinib treatment. At the time of progression, 6/23 had the same EGFR sensitizing mutations, 9/23 developed T790M mutation with the original mutation and 6/23 patients had no detectable mutation. T790M mutation was not detected in 227 plasma samples taken prior to erlotinib treatment. The figure shows the time course of two representative patients where a T790M resistance mutation emerges. In the 9 patients with T790M mutation, it can be detected in the blood between 15 and 344 days (mean of 98 days) before progression is clinically evident.Figure 1

      Conclusion
      The amounts of EGFR TKI sensitizing mutant DNA in plasma change during the erlotinib treatment. T790M mutation was not detected prior to erlotinib treatment and is detected between 15 and 344 days before disease progression is evident.

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      MO21.08 - Detection of EGFR mutations in plasma and diagnosis biopsies from non-small cell lung cancer patients using allele-specific PCR assays. (ID 2248)

      11:15 - 11:20  |  Author(s): P. Meldgaard

      • Abstract
      • Presentation
      • Slides

      Background
      EGFR TKI sensitizing mutations from plasma prior to treatment were shown to be a potent predictor for survival outcome of advanced NSCLC (T. Mok, ASCO 2013). In this study, we tested EGFR mutations in the archived plasma from 199 advanced adenocarcinoma. The plasma samples were taken when they progressed on their chemotherapy and before their 2nd erlotinib treatment a mean of 10.5 months after the diagnostic biopsy was obtained. EGFR mutations detected in plasma after chemotherapy and in the tumor DNA from their original diagnostic biopsies were also compared.

      Methods
      Plasma DNA and tumor DNA were tested with two allele-specific PCR assays, cobas® EGFR_ FFPET tissue test and cobas® EGFR_blood test (in development at Roche Molecular Systems, Inc.). Both allele-specific PCR assays detect 41 mutations in exon 18-21 of the EGFR gene including TKI sensitizing mutations (Exon 19 deletions, L858R and G719X), resistance mutation (T790M) and atypical mutations (S768I and Exon 20 Insertions). cobas® EGFR_blood test also detects L861Q. Plasma samples of all 199 adenocarcinoma were collected immediately (less than 2 days) prior to the patient’s erlotinib treatment and stored at -80°C. From 197 (99%) of 199 of the patients tumor DNA was extracted from the diagnostic biopsy.

      Results
      Among 199 advanced adenocarcinoma patients, 24/199 (12%) were EGFR mutation positive in plasma. 28/196 (14%) were EGFR mutation positive in tumor DNA. The comparison of EGFR mutation in plasma and tumor DNA is shown in the table 1. The overall concordance of EGFR mutation status in plasma and tumor biopsy was 91% (179/196). 17/196 (9%) patients had the same EGFR mutations in plasma as in their original diagnosis biopsy and 162/196 (82%) patients were mutation negative in both samples. In this study, different EGFR mutation status in plasma and original biopsy was observed in 17 of 196 (9%) patients. 6 of 17 were EGFR mutation positive in plasma only and 11 of 17 were EGFR positive in tumor DNA only. These differences could reflect alterations in the tumor cells between sampling of biopsy and blood (average of 10.5 months) where the patients are treated with chemotherapy. Another possibility is limitations of assay technology with circulating cell-free DNA in plasma or heterogeneity of tumor.

      Conclusion
      Tumor mutations in the patient’s original diagnostic biopsy can be detected in their plasma when they progress on chemotherapy which may provide another opportunity for mutation testing. Table 1. Comparison of EGFR mutations detected in plasma and diagnostic biopsy. MND=Mutation-Not-Detected.Figure 1

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    P2.17 - Poster Session 2 - Bronchoscopy, Endoscopy (ID 183)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pulmonology + Endoscopy/Pulmonary
    • Presentations: 1
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      P2.17-008 - A new procedure for sampling fine needle aspirations enables isolation of high quality mRNA (ID 2748)

      09:30 - 09:30  |  Author(s): P. Meldgaard

      • Abstract

      Background
      Fine needle aspirates (FNA) are widely used in the diagnostics of non-small cell lung cancer (NSCLC). As relevant oncogenic drivers have emerged, the demand for molecular tests is increasing. To investigate mRNA expression fresh frozen tissue is required. In an everyday setting isolation of snap-frozen tissue is rarely a possibility and new procedures are needed to allow use of FNA material for mRNA expression studies.

      Methods
      Tumour samples from patients under suspicion of having lung cancer were isolated. Routine diagnostic procedures of FNA and/or transbronchial needle aspiration (TBNA) were performed and smears used for routine diagnostics were prepared. The remaining sample in the needle and syringe was mixed with 10 ml Cytolyt©. 9.5 ml was used for routine diagnostics, while 0.5 ml was mixed with 4.5 ml RNAprotect©. mRNA was isolated, transcribed into cDNA and quantified with quantitative reverse transcription quantitative PCR (RT-qPCR).

      Results
      Analysis of four reference genes in the first 50 samples consecutively collected showed that the most stable reference gene was β-actin. 211 tissue samples from 81 patients were then examined for β-actin mRNA expression. 92 % of the samples contained sufficient material for mRNA quantification containing mRNA corresponding to 100 HCC827 NSCLC cells or more. Furthermore 100 HCC827 NSCLC cells were sufficient to quantify the mRNA splice variant of BIM conferring erlotinib sensitivity.

      Conclusion
      We describe a novel procedure that optimizes the use of diagnostic FNA and TBNA and demonstrate that these samples contain enough material for isolation of high quality mRNA without influencing routine procedures.