Purpose: The resistance to the EGFR tyrosine kinase inhibitors (TKI) is a major concern in non–small cell lung cancer (NSCLC) treatment. T790M mutation in EGFR accounts for nearly 50% of the acquired resistance to EGFR-TKIs. Earlier studies suggested that T790M mutation was also detected in TKI-naïve NSCLCs in a small cohort. Here, we use an ultra-sensitive droplet digital PCR (ddPCR) technique to address the incidence and clinical significance of pretreatment T790M in a larger cohort. Experimental Design: ddPCR was established as follows: wild-type or T790M mutation-containing DNA fragments were cloned into plasmids. Candidate threshold was identified using wild-type plasmid, normal human genomic DNA, and human A549 cell line DNA, which expresses wild type. Surgically resected tumor tissues from 373 NSCLC patients with EGFR-activating mutations were then examined for the presence of T790M using ddPCR. Results: Our data revealed a linear performance for this ddPCR method (R² = 0.998) with an analytical sensitivity of approximately 0.001%. The overall incidence of the pretreatment T790M mutation was 79.9% (298/373), and the frequency ranged from 0.009% to 26.9%. The T790M mutation was detected more frequently in patients with a larger tumor size (P = 0.019) and those with common EGFR-activating mutations (P = 0.022), as compared with the others. Conclusions: The ultra-sensitive ddPCR assay revealed that pretreatment T790M was found in the majority of NSCLC patients with EGFR-activating mutations. ddPCR should be utilized for detailed assessment of the impact of the low frequency pretreatment T790M mutation on treatment with EGFR-TKIs. Clin Cancer Res; 21(15); 3552–60. ©2015 AACR.

Development of a reliable platform and workflow to detect and capture a small number of mutation-bearing circulating tumor cells (CTCs) from a blood sample is necessary for the development of noninvasive cancer diagnosis. In this preclinical study, we aimed to develop a capture system for molecular characterization of single CTCs based on high-density dielectrophoretic microwell array technology. Spike-in experiments using lung cancer cell lines were conducted. The microwell array was used to capture spiked cancer cells, and captured single cells were subjected to whole genome amplification followed by sequencing. A high detection rate (70.2%–90.0%) and excellent linear performance (R² = 0.8189–0.9999) were noted between the observed and expected numbers of tumor cells. The detection rate was markedly higher than that obtained using the CellSearch system in a blinded manner, suggesting the superior sensitivity of our system in detecting EpCAM− tumor cells. Isolation of single captured tumor cells, followed by detection of EGFR mutations, was achieved using Sanger sequencing. Using a microwell array, we established an efficient and convenient platform for the capture and characterization of single CTCs. The results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.

We retrospectively investigated the impact of the tumor microenvironment (TME) on the efficacy of epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitors (TKIs) as first‐line treatment in 70 patients with advanced EGFR‐mutant non‐small cell lung cancer and who were seen at Osaka City University Hospital (Osaka, Japan) between August 2013 and December 2017. Using immunohistochemical staining with 28‐8 and D7U8C Abs, the tumor proportion score was assessed for programmed cell death‐1 ligand‐1 (PD‐L1), as high (50% or more) or low (less than 50%), and ligand‐2 (PD‐L2) expression, respectively. The extent of CD8⁺ tumor‐infiltrating lymphocytes was evaluated on a scale of 0‐3, with 0‐1 as low and 2‐3 as high. The TME of the 52 evaluable pretreatment specimens was categorized into 4 subtypes, according to the respective PD‐L1 tumor proportion and CD8⁺ scores, as follows: (a) high/high (13.5%, n = 7); (b) low/low (42.3%, n = 22); (c) high/low (17.3%, n = 9); and (d) low/high (26.9%, n = 14). Expression of PD‐L2 was significantly the highest in type 1 (57.1% vs 4.5% vs 11.1% vs 7.1%, respectively; P = .0090). Response rate was significantly the lowest in type 1 (14.3% vs 81.8% vs 66.7% vs 78.6%, respectively; P = .0085). Progression‐free survival was the shortest in type 1 and the longest in type 4 (median, 2.4 vs 11.3 vs 8.4 vs 17.5 months, respectively; P = .00000077). The efficacy of EGFR‐TKIs differed according to the TME, and the phenotype with high PD‐L1 and CD8⁺ expression might be the subset that would poorly benefit from such treatment.

Blockade of the programmed death receptor-1 (PD-1) pathway is effective against solid tumors including lung cancer. PD-ligand 1 (PD-L1) expression on tumor tissue serves as a predictive biomarker for the efficacy of PD-1 pathway blockade. Here, we evaluated the expression of PD-L1 on circulating tumor cells (CTCs) in patients with lung cancer. Peripheral whole blood (3 mL) was collected from patients, and CTCs and PD-L1 expression were detected using a microcavity array (MCA) system. Immunohistochemistry for PD-L1 detection was also performed using matched tumor tissues. Sixty-seven patients with lung cancer were enrolled in the study between July 2015 and April 2016 at Wakayama Medical University Hospital. The characteristics of the patients were as follows: median age, 71 years (range, 39-86 years); male, 72%; stage II to III/IV, 14%/85%; non-small-cell lung cancer/small-cell lung cancer/other, 73%/21%/6%. CTCs were detected in 66 of 67 patients (median, 19; range, 0-115), and more than 5 CTCs were detected in 78% of patients. PD-L1-expressing CTCs were detected in 73% of patients, and the proportion score of PD-L1-expressing CTCs ranged from 3% to 100%, suggesting intra-patient heterogeneity of PD-L1 expression on CTCs. Tumor tissues were available from 27 patients and were immunostained for PD-L1, and no correlation was observed between tumor tissues and CTCs based on the proportion score (R² = 0.0103). PD-L1 expression was detectable on CTCs in patients with lung cancer, and intra-patient heterogeneity was observed. No correlation was observed between PD-L1 expression in tumor tissues and CTCs.

Circulating tumor cells (CTCs), defined as tumor cells circulating in the peripheral blood of patients with solid tumors, are relatively rare. Diagnosis using CTCs is expected to help in the decision-making for precision cancer medicine. We have developed an automated microcavity array (MCA) system to detect CTCs based on the differences in size and deformability between tumor cells and normal blood cells. Herein, we evaluated the system using blood samples from non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) patients. To evaluate the recovery of CTCs, preclinical experiments were performed by spiking NSCLC cell lines (NCI-H820, A549, NCI-H23 and NCI-H441) into peripheral whole blood samples from healthy volunteers. The recovery rates were 70% or more in all cell lines. For clinical evaluation, 6 mL of peripheral blood was collected from 50 patients with advanced lung cancer and from 10 healthy donors. Cells recovered on the filter were stained. We defined CTCs as DAPI-positive, cytokeratin-positive, and CD45-negative cells under the fluorescence microscope. The 50 lung cancer patients had a median age of 72 years (range, 48–85 years); 76% had NSCLC and 20% had SCLC, and 14% were at stage III disease whereas 86% were at stage IV. One or more CTCs were detected in 80% of the lung cancer patients (median 2.5). A comparison of the CellSearch system with our MCA system, using the samples from NSCLC patients, confirmed the superiority of our system (median CTC count, 0 versus 11 for CellSearch versus MCA; p = 0.0001, n = 17). The study results suggest that our MCA system has good clinical potential for diagnosing CTCs in lung cancer.