Objectives. The differential diagnosis of pleural effusion (PE) is a common but major challenge in clinical practice. This study aimed to establish a strategy based on a PE-cell-free DNA (cfDNA) methylation detection system for the differential diagnosis of malignant pleural effusion (MPE) and benign pleural effusion (BPE). Methods. A total of 104 patients with PE were enrolled in this study, among which 50 patients had MPE, 9 malignant tumor patients had PE of indefinite causes, and the other 45 patients were classified as benign controls. The methylation status of short stature homeobox 2 (SHOX2) and RAS association domain family 1, isoform A (RASSF1A) was detected using PE-cfDNA specimens by real-time fluorescence quantitative PCR. Total methylation (TM) was defined as the combination of the methylation levels of SHOX2 and RASSF1A. The electrochemiluminescence immunoassay was applied to evaluate the levels of multiple serum tumor markers. Results. The PE-cfDNA methylation status of either SHOX2 or RASSF1A was much higher in MPE samples than in benign controls. The combination of SHOX2 and RASSF1A methylation in PE yielded a diagnostic sensitivity of 96% and a specificity of 100%, respectively. When compared with the corresponding serum tumor marker detection results, TM showed the highest diagnostic efficiency (AUC = 0.985). Furthermore, the combination of the SHOX2 and RASSF1A methylation panels using PE-cfDNA could apparently improve the differential diagnostic efficacy of BPE and MPE and could help compensate for the deficiency of cytology. Conclusions. Our results indicated that SHOX2 and RASSF1A methylation panel detection could accurately classify BPE and MPE diseases and showed better diagnostic performance than traditional serum parameters. The SHOX2 and RASSF1A methylation detection of PE-cfDNA could be a potentially effective complementary tool for cytology in the process of differential diagnosis. In summary, PE-cfDNA could be used as a promising non-invasive analyte for the auxiliary diagnosis of MPE.

Background:The plasma sample has emerged as a promising surrogate sample for EGFR mutation detection in advanced non-small cell lung cancer (NSCLC). In clinical practice, whether EGFR variants in baseline plasma ctDNA of advanced NSCLC can predict prognosis in addition to guiding targeted therapy remains to be further explored. Material and Methods:In total, 315 NSCLC patients were retrospectively enrolled. EGFR mutation data from tissue detected by ARMS-PCR and paired plasma samples within 1 month of admission detected by SuperARMS or ARMS-PCR were collected. The correlation between baseline plasma ctDNA EGFR mutation status and survival was compared.Results: EGFR mutation detection rates in tumor samples and plasma samples were 65.1% (205/315) and 43.8% (138/315). Referred to tissue results, the consistent rate of test ctDNA EGFR alteration by SuperARMS was higher than that detected by ARMS (79.5% vs. 69.0%, p = 0.04), either in stage I-IIIA patients (85.7% vs. 50.0%, p = 0.4) or stage IIIB-IV patients (79.1% vs. 69.4%, p = 0.04).Patients' treatment status and pathological subtype were the two factors that affected plasma ctDNA EGFR alteration detection accuracy. The concordance in non-adenocarcinoma patients was obviously higher than that in adenocarcinoma (p = 0.02), and the concordance in treatment naïve patients was significantly higher than that in relapse patients (p = 0.047). In treatment naïve patients, the median PFS (mPFS) in plasma ctDNA EGFR-positive patients was shorter than that in plasma ctDNA EGFR negative patients (7.0 vs. 10.0 months, p = 0.01). In relapsed patients, the mPFS in plasma ctDNA EGFR-positive patients was 9.0 months versus 11.0 months in plasma ctDNA EGFR negative patients (p = 0.1).