A 54-year-old Japanese woman who had never smoked and had no past medical history was diagnosed at Kanazawa University Hospital (Kanazawa, Ishikawa, Japan) with stage IV EGFR mutation-positive (a deletion mutation in exon 19, Ex19del) lung adenocarcinoma on September 22, 2014. She had a primary nodule in the upper lobe of the right lung (Fig. 1a), multiple small metastases in both lobes, lymph node metastases in the right hilum and mediastinum but no brain metastases at diagnosis. She had worked as a school cook, an occupation seemingly unrelated to her diagnosis.
A tumor specimen was collected on the upper lobe of her right lung (Fig. 1b, first column). The patient first underwent treatment with gefitinib (250 mg daily, Iressa®, AstraZeneca, Cambridge, UK) starting on October 16, 2014, and achieved a partial response by April 8, 2015, according to the Response Evaluation Criteria in Solid Tumors (version 1.1) (Fig. 1b, second column). On August 27, 2015, 10 months after the initiation of gefitinib, the size of a nodule in the right lower lobe had increased and multiple small nodules were detected. On November 11, 2015, a nodule in the lower lobe of the left lung and multiple brain metastases had developed (Fig. 1a, b, third column and Additional file 1: Figure S1). Computed tomography-guided transthoracic needle biopsy of the regrowth nodule in the upper lobe of the right lung was performed on October 13, 2015. Adenocarcinoma was diagnosed, and the EGFR mutation status was Ex19del (no T790M) according to the cobas® test. After radiosurgery was performed on the brain metastases on November 16, 2015, a combination therapy consisting of cisplatin (75 mg/m2, Maruko cisplatin, Yakult, Tokyo, Japan), pemetrexed (500 mg/m2, Alimta®, Eli Lilly, Indianapolis, IN) and bevacizumab (15 mg/kg, Avastin®, Chugai, Tokyo, Japan) (CDDP + PEM + BEV) every 3 weeks, was initiated as the second therapy starting on November 20, 2015. After four cycles of the combination therapy, the number of brain metastases and lung lesions increased on January 19, and January 13, 2016 (Fig. 1a, b, fourth column, Additional file 1: Figure S1). The patient then underwent whole-brain irradiation at a total dose of 40 Gy (2 Gy, 20 times) from February 1, 2016 to March 2, 2016, after which the growing mass in the lower lobe of the left lung was diagnosed as adenocarcinoma positive for Ex19del (no T790M) via transbronchial biopsy on February 25, 2016 (Fig. 1b, arrows). Combination therapy of erlotinib (150 mg daily, Tarceva®, Chugai, Tokyo, Japan) and bevacizumab (15 mg/kg, every 3 weeks) (E + BEV) was initiated as the third-line treatment (EGFR-TKI re-challenge) starting on March 16, 2016. After 2 months, multiple pulmonary nodules were observed to have increased in size, as detected by chest CT on May 23, 2016 (Fig. 1a, b, fifth column), and multiple bone metastases had newly emerged, as detected by positron emission tomography/CT on May 31, 2016. The growing nodule in the lower lobe of the left lung was diagnosed as adenocarcinoma positive for both EGFR Ex19del and T790M at the fourth biopsy via transbronchoscopy on June 8, 2016. The patient received osimertinib (Tagrisso®, 80 mg daily, AstraZeneca) monotherapy as the fourth-line treatment starting on July 1, 2016, and experienced a partial response (Fig. 1b, sixth column).
The T790M AFs in tumor tissues were calculated based on the T790M copy numbers detected by ddPCR, providing a quantitative evaluation of the AF (Fig. 1b). DNA was extracted from tumor samples collected via the four biopsies by macro-dissection. The T790M AFs levels in tumor tissues at baseline, resistance to gefitinib (first EGFR-TKI), resistance to CDDP + PEM + BEV, and resistance to E + BEV (EGFR-TKI re-challenge) were 0, 0.20, 0.23, and 0.47, respectively (Fig. 1b, bottom panel). T790M was detected at a low AF (0.20), even though no mutation was detected by the cobas® test. Additionally, the T790M AF was found to be higher after progression with E + BEV (EGFR re-challenge treatment) than after progression with gefitinib.