Blood samples were collected within 3 months of death from all enrolled patients, and cfDNA was extracted at initial diagnosis prior to systemic antitumor-treatment (including EGFR-TKIs). Whole blood (12 mL) was collected in ethylenediaminetetraacetic acid tubes and centrifuged at 700 × g (2000 rpm) for 10 min within 2 h of blood collection, as described previously37. cfDNA was extracted immediately from the plasma supernatant (4 mL) using a QIAAMP CIRCULATING NUCLEIC ACID KIT (Qiagen), and germline DNA was extracted from whole blood cells, obtained as the pellet in centrifugation, using a QIAAMP DNA MINI KIT (Qiagen). The extracted DNA was stored at − 20 °C until use. The concentration and quality of cfDNA and germline DNA were measured using the abovementioned methods.

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