The supernatants were collected and centrifuged at 3000 rpm

for 5 minutes. The final supernatants were transferred to Eppendorf tubes and stored at −70°C until DNA extraction. DNA was extracted from 1–2 ml of supernatant with a DNeasy blood kit (Qiagen) according to the this website manufacturer’s instructions. The final elution volume for DNA extraction was 60 μl and the amount of plasma DNA used for mutation testing was 30 ng. PNA-mediated real-time PCR clamping method to detect deletions in EGFR exon 19 and L858R point mutations in EGFR exon 21 Plasma DNA was analyzed using the PNAClampTM EGFR Mutation Detection kit (PANAGENE, Inc., Daejeon, Korea) as described in a previous retrospective study [34]. All reactions were conducted in a 20-μl volume using template DNA, primers Pinometostat ic50 and PNA probe set, and SYBR Green PCR master mix. All reagents were included in the kit. Real-time PCR reactions were performed using a CFX 96 instrument (Bio-Rad, USA). PCR cycling commenced with a 5 min hold

at 94°C followed by 40 cycles at 94°C for 30 s, MLN2238 70°C for 20 s, 63°C for 30 s, and 72°C for 30 s. Two EGFR mutation types were detected using PNA-mediated real-time PCR. The efficiency of PCR clamping was determined by measuring the cycle threshold (Ct) value. Ct values for the control and mutation assays were obtained by observing the SYBR Green amplification plots. The delta Ct (∆Ct) value was calculated (control Ct − sample Ct), ensuring that the sample and control Ct values were from the test and wild-type control samples. The cut-off ∆Ct was defined as 2 for both the G746_A750 deletion and the L858R point mutation. Tumor mutation data At time of blood collection, we reviewed the EGFR mutation status in patient matched tumor tissue. By the direct sequencing used in routine practice

at each Terminal deoxynucleotidyl transferase institution to established EGFR mutation status in tumor tissue, forty tumor specimens were analyzed for EGFR mutations before gefitinib. Statistical analyses The relationship between EGFR mutations and demographic and clinical features, including age, gender, histological type, performance status (PS), smoking status, TNM stage and response to gefitinib, was analyzed using Pearson’s chi-square test or Fisher’s exact test. Two-sided P values <0.05 were considered statistically significant. All analyses were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Results Patient characteristics The clinical characteristics of the 60 patients are shown in Table 1. The median age was 62.5 years (range: 38–84 years). Thirty-nine (65.0%) of the patients were female and 21 (35.0%) were male. Forty-three patients (71.7%) were non-smokers. Fifty patients (83.3%) had good PS. The most common histological subtype was adenocarcinoma (53 patients, 88.3%) and the majority of patients (88.3%) had stage IV disease.