br Analysis of cfDNA quantification br The serum samples

2.2. Analysis of cfDNA quantification
The serum samples were harvested in 10-mL serum separator tubes. Following centrifugation for 10 min at 2000 × g at 4 °C, the super-natants were transferred and again centrifuged for 5 min at 16,000 × g and 4 °C. The resulting serum was frozen at −80 °C until use. The cfDNA was extracted from serum aliquots (500 μL) using the QIAamp circulating nucleic Cisplatin kit (Qiagen, Hilden, Germany). The extraction efficiency of cfDNA was measured using an Agilent High Sensitivity  Lung Cancer 134 (2019) 158–166
DNA kit and the Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, CA). Additional purification was performed using Agencourt AMPure XP (BeckMan Coulter, Brea, CA). The isolated DNA was quantified with the Agilent High Sensitivity DNA kit and measured in a Qubit 2.0 Fluorometer.
2.3. Analysis of PET/CT images
All patients underwent combined PET/CT scanning under a routine clinical protocol with Gemini TF/16 channel PET/CT scanners (Philips Medical Systems, Cleveland, OH). After fasting for at least 6 h, the radiotracer (5–6 MBq/kg) FDG was administrated intravenously and scanning was performed after 60 min. Unenhanced CT scans were ac-quired first for attenuation correction, and then PET scans (1 min per bed) were acquired.
The SUV, MTV, and TLG were extracted using MIRADA XD3 soft-ware (MIRADA Medical, Oxford, UK) by drawing a VOI on the PET image [18]. A fixed SUV cut-off value of 2.5 was used for the semi-automated contouring system to exclude physiologic FDG uptake of the non-tumor tissue. The manual correction of the boundaries of the au-tomated VOI was performed by nuclear medicine physicists who were blinded to the cfDNA levels of the subjects. The MTV was defined as the total tumor volume inside the boundaries of the manually corrected VOI. TLG was calculated as the product of MTV and the SUV mean.
The cfDNA quantification data in all the NSCLC and stage IV ADC patients were expressed as median and IQR ranges. The correlation between the cfDNA levels and MTV/TLG was assessed by Spearman’s rank correlation test. The clinical outcomes were assessed by progres-sion free survival (PFS) and overall survival (OS). The Kaplan-Meier survival curve for PFS and OS was constructed, and the statistical dif-ferences were measured by the log-rank test. The optimal cut-off values for cfDNA quantification, MTV, and TLG were determined using X-tile software. X-tile software is plate tectonics specialized for the analysis of survival data, which is divided into matched training and validation sets (1:1 ratio). Then, X-tile plots show the χ2 log-rank values with a Kaplan-Meier curve and identify a minimal p-value with histogram. Since the small number of events favored the use of the OS, which is closer to the PFS, the OS cut-off was determined as the optimal cut-off [19]. Multivariate Cox proportional hazard model was used to investigate the hazard ra-tios (HR) for the impact of cfDNA levels on survival outcomes by ad-justed covariates including age, sex, performance status, Charlson co-morbidity index, clinical stage, and computable MTV/TLG levels. The statistical analyses were performed using X-tile software v 3.6.1 (Yale University, New Haven, CT), SPSS v 24.0 (SPSS Inc., Chicago, IL), and R software v 3.2.2 (R Core Team, Vienna, Austria).
3. Results
3.1. Baseline characteristics according to patients’ cfDNA quantification
This study was conducted according to the guidelines of the REporting recommendations for tumor MARKer prognostic studies (REMARK) (Supplementary Table S1) [20]. The detailed baseline clin-icopathological characteristics and PET/CT parameters according to the cfDNA concentration are listed in Table 1. The median cfDNA levels of the entire cohort, stage III, stage IV, and stage IV ADC only patients were 70 ng/mL (IQR = 40–161 ng/mL), 64 ng/mL (IQR = 28–87 ng/ mL), 72 ng/mL (IQR = 41–165 ng/mL), and 65 ng/mL (IQR = 38–158 ng/mL), respectively. There was no significant difference in cfDNA level between stage III and IV patients (p = 0.273) (data not shown). Overall, the baseline characteristics between patients with low- and high-cfDNA levels were similar (Table 1). However, patients with high-cfDNA levels showed significantly higher MTV and TLG levels