When the AF is below the cutoff, anti-tumor agents such as first- or second-generation EGFR-TKIs can be selected mainly because the re-challenge treatment. of a patient who underwent four tumor biopsies over the treatment program. Despite no detection of this mutation after developing resistance to the first-line EGFR-TKI, in the fourth biopsy specimen, which was collected after progression following EGFR-TKI re-challenge, T790M mutation was recognized using the cobas? EGFR mutation test (Roche Molecular Systems, Pleasanton, CA, USA). Additionally, we analyzed the T790M allele rate of recurrence (AF) in longitudinal biopsy samples acquired at four different time points using droplet digital PCR (ddPCR) (PrimePCR?, Cyhalofop Bio-Rad Laboratories, Inc., Hercules, CA, USA). We discuss the clinical usefulness of longitudinal assessment of the T790M AF, especially for deciding the appropriate treatment (e.g., osimertinib) in individuals with mutations. Case demonstration A 54-year-old Japanese female who had by no means smoked and had no recent medical history was diagnosed at Kanazawa University or college Hospital (Kanazawa, Ishikawa, Japan) with stage IV mutation-positive (a deletion mutation in exon 19, Ex lover19del) lung adenocarcinoma on September 22, 2014. She experienced a main nodule in the top 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 experienced worked well like a school cook, an profession seemingly unrelated to her analysis. Open in a separate window Fig.?1 a Clinical course of this case. b Changes in chest CT images and T790M status. The top and lower images show the top and lower lobes, respectively. The thin black arrow shows the nodule in the top lobe of the right lung at analysis; the first biopsy was performed with this nodule in September 2014, which was diagnosed as EGFR Ex lover19del-positive and T790M-bad adenocarcinoma according to the cobas? test. The nodule in the top lobe of the right lung shrank after gefitinib treatment and then increased again. The second biopsy was performed in August 2015 in the nodule indicated from the solid reddish arrow, which was diagnosed as EGFR Ex lover19del-positive and T790M-bad adenocarcinoma according to the cobas? test. A new nodule appeared like a resistant tumor in the lower lobe of the GNG7 remaining during gefitinib treatment (blue arrow), and this nodule improved after CDDP?+?PEM?+?BEV treatment. The third biopsy was performed in January 2016 in the nodule indicated from the orange arrow, Cyhalofop which was diagnosed as EGFR Ex lover19del-positive and T790M-bad adenocarcinoma according to the cobas? test. The mass in the lower lobe of the remaining lung had improved and multiple small nodules Cyhalofop had appeared after EGFR-TKI re-challenge with E?+?BEV. The fourth biopsy was performed in the mass indicated from the green arrow, which was diagnosed as adenocarcinoma positive for both EGFR Ex lover19del and T790M according to the cobas? test. The lesions improved after initiation of osimertinib treatment. The T790M AFs are demonstrated in the graph below. T790M#, WT20#, Ex lover19del#, and WT19# refer to the copy numbers of the EGFR T790M mutation, no T790M in EGFR exon 20, deletion in EGFR exon 19, and no deletion in EGFR exon 19, respectively. computed tomography, epidermal growth element receptor, cisplatin, pemetrexed, bevacizumab, tyrosine kinase inhibitor, erlotinib, allele rate of recurrence A tumor specimen was collected on the top lobe of her right lung (Fig.?1b, 1st column). The patient 1st underwent treatment with gefitinib (250?mg daily, Iressa?, AstraZeneca, Cambridge, UK) starting on October 16, 2014, and accomplished 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?weeks 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 Cyhalofop lower lobe of the remaining lung and multiple mind metastases had developed (Fig.?1a, b, third column and Additional file 1: Number S1). Computed tomography-guided transthoracic needle biopsy of the regrowth nodule in the top lobe of the right lung was performed on October 13, Cyhalofop 2015. Adenocarcinoma was diagnosed, and the mutation status was Ex lover19del (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,.