The present prospective study included patients with newly diagnosed NSCLC prior to receiving treatment, which were admitted to the Department of Oncology of Guangdong Provincial Hospital of Chinese Medicine (Guangzhou, China) between October 2014 and May 2015. A total of 51 patients participated in the current study, 31 of which were male and 20 were female with a mean age of 60.89±1.48 years. Those with uncontrolled other malignant tumor types, uncontrolled infection or tubercle bacillus, underlying diseases that were severe or life threatening, or severe mental disease were excluded from the current study. The clinical characteristics were recorded. If the EGFR gene status in the cancerous tissue of patients had been determined in other qualified hospitals, only the results were recorded. Otherwise, the EGFR gene mutation in formalin-fixed paraffin-embedded specimens were detected using ARMS at the Department of Pathology of Guangdong Provincial Hospital of Chinese Medicine, using the human EGFR gene mutation fluorescence polymerase chain reaction diagnostic kit (YQ Biomed, Shanghai, China), according to the manufacturer's protocol. The PCR was performed at 94°C for 3 min to activate the DNA polymerase, followed by 40 cycles at 94°C for 15 sec and 40 cycles at 60°C for 1 min, in a ViiA™ 7 instrument (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The treatments were decided upon by the doctors and patients together. Briefly, stage III/IV patients with sensitive EGFR gene mutation took EGFR-TKIs as a first line therapy. Those without EGFR gene mutation received platinum-based chemotherapy. Image examination was followed up every 2 months if possible, with a maximum interval of 3 months. Written informed consent for the study and EGFR gene tests were provided by all patients. The present study was approved by the Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine.

For each patient, baseline plasma was collected prior to first-line therapy. Additional follow-up plasma was collected every 2 months for those who received EGFR-TKIs if possible, with a maximum interval of 3 months. A total of 6–10 ml whole venous blood was collected into EDTA-containing vacutainers, stored at 4°C prior to centrifugation, and centrifuged for 10 min at 1,800 × g and 4°C within 6 h of collection. The plasma was frozen at −80°C until use. Prior to DNA extraction, the plasma was further cleared through centrifugation for 10 min at 3,000 × g and 4°C. Circulating cfDNA was isolated using the QIAmp circulating nucleic acid kit and eluted in 100 µl AVE buffer (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. The DNA was stored at −80°C until use in subsequent experiments.

The ddPCR workflow was performed at WuXi AppTec, Co., Ltd. (Shanghai, China). The investigator for ddPCR was blinded to the tissue results. TaqMan PCR reaction mixtures were assembled from a 2X ddPCR Master Mix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and custom-made 40X TaqMan probes/primers (Thermo Fisher Scientific, Inc.) specific for each assay. A total of 4 µl of template DNA, 16 µl of assembled ddPCR reaction mixture and pure distilled water was loaded into sample wells of an eight-channel disposable droplet generator cartridge (Bio-Rad Laboratories, Inc.). An additional 70 µl of droplet generation oil (Bio-Rad Laboratories, Inc.) was loaded into the oil well of each channel. Following droplet generation, the cartridge was removed and manually transferred with a multichannel pipette to a 96-well PCR plate. The plate was heat-sealed, placed on a conventional thermocycler, and amplified to the end-point. Following PCR, the 96-well PCR plate was read on the QX-100 droplet reader (Bio-Rad Laboratories, Inc.). Analysis of the ddPCR data was performed with QuantaSoft analysis software (version 1.7.4; Bio-Rad Laboratories, Inc.) that accompanied the droplet reader.

Droplet digital PCR reagents were ordered from Bio-Rad Laboratories, Inc., and primer/probe mix for EGFR T790M (according to GRCh38:7:55181273:55181483), EGFR L858R (according to GRCh38:7:55191717:55191927) and EGFR ex19del (according to GRCh38:7:55174671:55174889) were custom-made by Thermo Fisher Scientific, Inc. The allele-specific MGB probes were labeled with either VIC or FAM at the 5′ end and a nonfluorescent quencher (NFQ) at the 3′ end. For the EGFR L858R assay the following primer sequences were used: Forward, 5′-GCAGCATGTCAAGATCACAGATT-3′ and reverse, 5′-CCTCCTTCTGCATGGTATTCTTTCT-3′; the probe sequences were: 5′-VIC-AGT TTG GCC AGC CCA A-MGB-NFQ-3′ and 5′-FAM-AGT TTG GCC CGC CCAA-MGB-NFQ-3′. For the EGFR ex19del ddPCR assay, the following primer sequences were used: Forward, 5′-GTGAGAAAGTTAAAATTCCCGTC-3′ and reverse, 5′-CACACAGCAAAGCAGAAAC-3′; the probe sequences were: 5′-VIC-ATC GAG GAT TTC CTT GTTG-MGB-NFQ-3′ and 5′-FAM-AGG AAT TAA GAG AAG CAA CATC-MGB-NFQ-3′ (ex19 deletion hotspot). For the EGFR T790M assay, the following primer sequences were used: Forward, 5′-GCCTGCTGGGCATCTG-3′ and reverse, 5′-TCTTTGTGTTCCCGGACATAGTC-3′; the probe sequences were: 5′-VIC-ATGAGCTGCGTGATGAG-MGB-NFQ-3′ and 5′-FAM-ATGAGCTGCATGATGAG-MGB-NFQ-3′. The thermocycling conditions for L858R and T790M were as follows: 95°C × 10 min (1 cycle), 40 cycles of 94°C × 30 sec and 58°C × 1 min, followed by 10°C hold. The thermocycling conditions for ex19del were as follows: 95°C × 10 min (1 cycle), 40 cycles of 94°C × 30 sec and 55°C × 1 min, followed by 10°C hold.

Continuous data are presented as the mean ± standard deviation for normal distribution, or the median and 25–75% percentile for other distributions. Numerical data are expressed as proportions. Statistical differences were evaluated using the unpaired student's t-test, one-way analysis of variance, Wilcoxon rank sum test, or Kruskal-Wallis H test for continuous data, and the χ² or Fisher's exact test for numerical data with a threshold of α<0.05. OS of patients treated with EGFR-TKIs was considered the primary end point for assessing the association between plasma mutant EGFR concentration and treatment efficacy. However, as of July 2015, the censor date for the present report, only 1 patient had succumbed to pneumonia, and 2 patients had suffered local progression. Therefore, only the percentage decrease in tumor burden following ≥3 months of EGFR-TKIs treatment as compared with the baseline was reported. The tumor burden was measured as the sum of the longest diameters of the target lesions, according to the Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1) (15). The association between plasma mutant EGFR concentration and baseline tumor burden, baseline number of metastatic organs, and tumor burden percentage decrease was assessed with Spearman's rho and linear regression with a threshold of α<0.05. Data was documented using EpiData software (version 3.1; The EpiData Association, Odense, Denmark) and analyzed using Stata software (version 11.0; StataCorp LP, College Station, USA). P<0.05 was considered to indicate a statistically significant difference.

A total of 51 pathologically diagnosed patients with NSCLC were enrolled in the present study, including 48 newly diagnosed patients, 2 with progression following the first line of chemotherapy and 1 following radical resection of lung cancer. Tumor tissue samples from 42 patients were tested at Guangdong Provincial Hospital of Chinese Medicine, 2 in Guangdong General Hospital, 3 in Sun Yat-Sen University Cancer Center and 3 in the First Affiliated Hospital of Guangzhou Medical University (Guangzhou, China). The tissue from 50 patients qualified for EGFR gene testing. The tissue sample from the last patient was insufficient for gene testing. The demographical and clinical characteristics are presented in Table I. Certain data were not available for all patients. There were no significant differences in demographical or clinical characteristics among patients with different tissue EGFR status, except for smoking history and brain metastasis. All of the patients with EGFR mutation in tumor tissues (30 patients) accepted EGFR-TKIs for therapy, except the patient who received radical resection and another patient with stage I NSCLC. The patient with an unknown EGFR status in the tumor tissue sample was treated with gefitinib as the L858R mutation was detected in the plasma. The EGFR-TKIs types were similar between the L858R mutation and ex19del patients.

According to our previous study, the ddPCR technology used for L858R and ex19del assays possessed a 0% technical background level, while the T790M assay exhibited a 0.03% technical background level (10). Therefore, mutant EGFR concentration >0 copies/ml plasma was defined as positive for L858R and ex19del. According to preliminary experimental data performed by Wuxi AppTec Co., Ltd. (Shanghai, China), the threshold for T790M was 20 copies/ml plasma (data not shown). In the present study, compared with tumor tissue samples, the sensitivity and specificity of ddPCR for mutant L858R were 76.19% (16/21) and 96.55% (28/29), respectively (Table II). A low concentration of mutant L858R in the plasma was detected in the one patient who tested false positive (5.8 copies/ml). Two patients with mutant L858R in the tumor tissue samples received EGFR-TKIs for 1 week prior to plasma sampling, and 1 patient had gone 1 year following radical resection without recurrence at the time of plasma sampling. Therefore, the modified sensitivity for L858R was 88.89% (16/18; Table II). A 79-year-old male patient with an unknown EGFR status in the tumor tissue sample had 380 copies/ml of mutant L858R in plasma detected. The patient started receiving gefitinib therapy, however 16 days later the patient succumbed to pneumonia. The computerized tomography scan indicated tumor response to gefitinib although with severe infection.

Compared with tumor tissue samples, the sensitivity and specificity of ddPCR for ex19del were 88.89% (8/9) and 100% (41/41), respectively (Table II). No ex19del was detected following ddPCR in the patient with the unknown EGFR status in the tumor tissue. T790M mutation was not identified to be positive in any plasma samples nor detected in the tumor tissue of any patients, although 2 patients with wild-type tissue EGFR exhibited low concentrations of mutant T790M in the plasma (1.3 and 7.8 copies/ml, respectively).

The baseline median cfDNA concentration detected in 51 patients, median mutant L858R concentration detected in 18 patients, and median ex19del concentration detected in 8 patients were 0.4 ng/µl (range, 0.14–4.36; 25–75% percentile, 0.26–0.64 ng/µl), 261.95 copies/ml (range, 4.6–4490; 25–75% percentile, 40.8–625.7 copies/ml) and 262.35 copies/ml (range, 3.8–1790; 25–75% percentile, 41–693.55 copies/ml), respectively (Table III). No significant associations between plasma cfDNA or mutant EGFR concentration and demographical or clinical characteristics were identified. However, patients with larger compared with those with smaller baseline tumor burden exhibited higher cfDNA concentrations, patients with higher numbers of metastatic organs exhibited higher ex19del concentrations, and those with liver metastasis exhibited lower mutant L858R concentrations compared with those without (Table III).

In total, 5 patients with L858R mutation and 4 patients with ex19del in plasma and tumor tissue samples were followed up with image examination for ≥3 months following EGFR-TKIs treatment. The baseline plasma mutant EGFR concentrations in 9 patients were significantly and positively correlated with the tumor burden reduction following ≥3 months of EGFR-TKIs treatment as compared with the baseline. (Spearman's r=0.7000, P=0.0358; Fig. 1) When analyzed separately, pre-treatment ex19del concentrations in 4 patients were also significantly and positively correlated with the tumor burden reduction (Spearman's r=1.0000, P<0.0001; Fig. 2 and Table III), while the correlation between mutant L858R concentrations and tumor burden reduction in 5 patients was not significant (Spearman's r=0.7000, P=0.1881; Fig. 3 and Table III). The pre-treatment plasma cfDNA concentration in 9 patients (Spearman's r=0.5167, P=0.1544) were not associated with the reduction in tumor burden, either (Table III).