From April 2016 to May 2019, consecutive patients screened for EGFR mutations were retrospectively reviewed at the National Center for Global Health and Medicine, Japan. The peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp method  was used to detect the EGFR mutation, using tissue biopsy specimens during the initial diagnosis of non-small non-squamous-cell lung cancer. After EGFR-mutated lung cancer acquired clinical resistance to EGFR-TKIs, the cobas® EGFR Mutation Test (Version 2; Roche Molecular Systems)  was repeatedly performed to detect T790M mutation status through tissue or liquid biopsy. Clinical resistance was defined as an increase in monitoring of tumor markers, disease progression through radiological imaging, or clinical disease progression.
Rebiopsy and genetic analysis
All types of clinical rebiopsies were repeated when patients were suspected to be clinically resistant to EGFR-TKIs. If patients were likely to provide tumor tissue through a clinical procedure (e.g. bronchoscopy or CT-guided biopsy) at the time of radiographic disease progression, they underwent tissue biopsy numerous times. Otherwise, liquid biopsy was performed. After each rebiopsy, the cobas® version 2 was used. When a new T790M substitution was detected, patients were administered osimertinib; if not, they were administered treatment other than osimertinib, such as cytotoxic chemotherapy or other molecular-targeted therapy. Among patients not harboring the T790M substitution, tissue or liquid rebiopsies were repeated numerous times until the T790M substitution was detected. Cobas® version 2 is a single plexus real-time PCR procedure to detect EGFR mutations, which potentially uses unstained 5-μm-thick sections obtained from a formalin-fixed paraffin-embed block and mounted on slides or whole blood samples, as previously reported . Mutations were analyzed at the central laboratory of LSI Medience Corporation (Tokyo, Japan).
The following data were obtained from each patient’s medical records: patient characteristics including age, sex, smoking index, smoking status, comorbidities, and Eastern Cooperative Oncology Group performance status (PS) at diagnosis, as well as oncological data including histologic type, staging in accordance with the 8th edition of the TNM Classification of Malignant Tumors , tumor size of biopsy site, number of tumor lesions, metastatic organ, EGFR mutation sites detected via the PNA-LNA PCR clamp method or cobas® version 2, treatment data including surgical treatment, radiotherapy including radical or palliative radiation, pharmacotherapy (gefitinib, erlotinib, afatinib, and osimertinib) at EGFR-TKI naïve line, subsequent systemic therapies including cytotoxic chemotherapy regimens, immunotherapy, or other molecular-targeted therapy, data on the best supportive care, and tumor markers for CEA (ng/mL). Computed tomography (CT), positron-emission CT, and magnetic resolution imaging were performed within 1 month of each biopsy for corresponding biopsy specimens. Patients harboring the T790M substitution were defined under the category of “at least one detection of T790M using single-plexus PCR through any type of clinically available biopsy.”
The study was conducted in accordance with the tenets of the Declaration of Helsinki. The study protocol was approved by the certified review board of the National Center for Global Health and Medicine (NCGM-G-003361-00). In accordance with the Japanese Ethical Guidelines for Medical and Health Research Involving Human Subjects, we used the opt-out method. We informed the participants about this study and obtained informed consent from subjects by displaying the disclosure document in the hospital as per the approval data by the appropriate review board until 31st January 2020.
The primary outcome was the identification of EGFR-mutated lung cancer patients harboring the T790M substitution with acquired clinical resistance to EGFR-TKIs. Secondary outcomes were the identification of factors inducing the T790M substitution through any type of rebiopsy among patients harboring the T790M substitution and factors inducing the T790M substitution through liquid rebiopsy. Fisher’s exact test was performed to compare the proportion of subjects with dichotomous outcomes in both groups. For continuous variables, receiver-operator characteristics (ROC) curves were analyzed using SigmaPlot version 14 software (Systat Software, Inc., San Jose CA, USA). At a p-value of < 0.05 (p < 0.05), the optimal cutoff values of these continuous variables were set on the basis of a pre-test probability of 0.5 and cost ratio of 1.0.
Logistic regression analysis was performed to assess the aforementioned three factors among patients with EGFR-mutated lung cancer with acquired clinical resistance to EGFR-TKIs, as previously described . To select a model for multivariate analysis, we identified variables with a p-value less than 0.15 based on univariate analysis. Spearman’s rank test and clinically clarified dependent variables were used to exclude dependent variables from the aforementioned selected variables. A correlation coefficient (ρ) of more than 0.3 as the absolute value based on Spearman’s rank test indicated a significant association. Some models were constructed with only independent variables as candidates. ROC curves were used to select the best model among candidate models. In the final multivariate analysis using the simultaneous method, statistical significance was determined at a p-value < 0.05 through a two-sided test. All analyses were performed using SPSS Statistics software version 25 (IBM, Armonk NY, USA) or Stata version 15.1 (StataCorp LLC, College Station TX, USA).