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C.A. De Paula

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    OA 07 - Biomarker for Lung Cancer (ID 659)

    • Event: WCLC 2017
    • Type: Oral
    • Track: Biology/Pathology
    • Presentations: 1
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      OA 07.08 - Clinical Potential of Sputum in Detecting Driver Mutations in Patients with Non-Small Cell Lung Cancer: A Preliminary Study (ID 7540)

      15:45 - 17:30  |  Author(s): C.A. De Paula

      • Abstract
      • Presentation
      • Slides

      The incidence of lung cancer has significantly increased over the last century and remains the most common cause of cancer deaths worldwide. Our better understanding of the tumor microenvironment and the systemic actions of tumors, combined with the recent advent of the liquid biopsy, may allow molecular diagnostics to be done with non-invasive method for detection and monitoring of patient tumors. Sputum has been the target for the discovery of non-invasive biomarkers for lung cancer because it contains airway epithelial cells, and molecular alterations identified in sputum are most likely to reflect tumor-associated changes. Since January of 2017, sputum samples have been prospectively collected at the time of diagnosis for future evaluation of actionable mutations in EGFR, KRAS, BRAF and NRAS in patients with non-small cell lung cancer in our center. Currently, from 20 sputum samples already collected, 5 are confirmed for driver mutations (one in KRAS and 4 in EGFR) in tissue biopsy, with 2 of the samples being positive for T790M in circulating tumor DNA (ctDNA) isolated from plasma. Our aim is to evaluate whether sputum may be representative in the detection of these mutations.

      DNA was extracted from sputum using QIAamp DNA midi kit (Qiagen). Tumor somatic mutations were investigated by target-sequencing using a custom Ion Ampliseqâ„¢ Panel (ThermoFisher Scientific), containing hotspot regions of 14 genes frequently mutated in solid tumors (including EGFR). Multiplex amplification was performed with 10 ng of DNA using Multiplex PCR Master Mix (Qiagen) and high-throughput sequencing was performed using Ion Proton platform. Somatic mutations were considered if the variant allele was present in more than 0.5% of the reads, considering a minimum coverage depth of 20,000X. A medium coverage of 172,524X was obtained in the five samples.

      We detected mutations in 3 out of 5 sputum samples of patients with previously known driver mutations (two exon 19 deletions and one exon 18 G719A in EGFR). The highest frequency was detected in the only patient with spontaneous sputum collection (23%).The other two mutations were detected in low frequencies (0.5 and 0.6%) in samples derived from sputum induction. We found T790M in one patient positive for T790M in ctDNA isolated from plasma.

      These preliminary findings indicate that driver mutations can be identified in sputum routinely obtained from sputum samples. Thus, the ability to examine sputum might provide a convenient source of sampling and may be adapted for future large-scale screening.

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