OncoTargets and Therapy (Oct 2020)

Highly Sensitive Droplet Digital PCR Method for Detection of de novo EGFR T790M Mutation in Patients with Non-Small Cell Lung Cancer

  • Wang X,
  • Li X,
  • Guo H,
  • Zhu L,
  • Peng Z,
  • Wang J,
  • Yang F,
  • Guo Y

Journal volume & issue
Vol. Volume 13
pp. 10621 – 10630

Abstract

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Xun Wang,1,* Xiao Li,1,* Haifa Guo,1,* Lingxiang Zhu,2 Zhiyong Peng,3 Jun Wang,1 Fan Yang,1 Yong Guo4 1Department of Thoracic Surgery, Peking University People’s Hospital, Peking University, Beijing 100044, People’s Republic of China; 2National Research Institute for Health and Family Planning, Beijing 100081, People’s Republic of China; 3TargetingOne Corporation, Beijing 100190, People’s Republic of China; 4Department of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing 100084, People’s Republic of China*These authors contributed equally to this workCorrespondence: Fan YangDepartment of Thoracic Surgery, Peking University People’s Hospital, Peking University, Beijing 100044, People’s Republic of ChinaEmail [email protected] GuoDepartment of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing 100084, People’s Republic of ChinaEmail [email protected]: The aim of this study was to investigate the allelic relation between de novo T790M and concomitant sensitizing EGFR mutations in EGFR-TKIs naïve NSCLCs and to explore whether the formalin-fixed and paraffin-embedded (FFPE) materials affect the detection of de novo T790M mutation.Methods: Specimens of 300 consecutive EGFR-TKI naïve NSCLCs who received surgical resection between January 2016 and June 2018 were retrospectively investigated. All the snap-frozen tumor tissues from 300 NSCLCs were screened by droplet digital PCR (ddPCR) for the detection of de novo T790M mutation. The allelic relation between de novo T790M mutation and concomitant sensitizing EGFR mutations was also investigated. Furthermore, we assessed de novo T790M mutation in paired FFPE specimens of 50 patients which included tumor tissues and paired normal lung tissues of the pretreatment NSCLCs to investigate whether FFPE materials affect the detection of de novo T790M mutation.Results: The de novo T790M mutation was observed in four patients which included one patient of single de novo T790M mutation and three patients of de novo T790M mutation coexisting with L858R mutation. The incidence of de novo T790M in pretreatment NSCLCs who harboring EGFR mutations was 2.9% (4/139). All the de novo T790M mutations were detected in cis with the concomitant L858R mutations for the three NSCLCs. Our ddPCR method demonstrated that the frequency of de novo T790M mutation was ranging from 0.1% to 0.5% among 90% (45/50) of the FFPE tumor samples and 92% (46/50) of the paired FFPE adjacent normal lung samples. The frequency of de novo T790M mutation in the paired snap-frozen samples was all below 0.1%.Conclusion: Our study demonstrated that most de novo T790M mutations were detected in cis with concomitant sensitizing mutations for pretreatment NSCLCs. Analytical cut-off of ddPCR assay for FFPE specimens should be validated carefully considering the possibility of FFPE-derived artificial gene mutations.Keywords: non-small cell lung cancer, NSCLC, droplet digital PCR, de novo T790M, acquired T790M, EGFR mutation

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