DNA Methylation Analysis For The Differential Diagnosis Of Pleural Effusion

Objective: The prevalence of malignant pleural effusions(MPEs)is increasing worldwide.The study was designed with the objectives to assess the value of a novel panel of cancer-specific methylated genes for differentiating MPEs from benign pleural effusions(BPEs).Methods: During October 2019 and July 2020,more than 200 patients with pleural effusions admitted to the First Affiliated Hospital of China Medical University and Shenyang Chest Hospital were included.After centrifugation at 4℃,pleural effusion sediment was collected for a RT-PCR-based in vitro diagnostic test,which was developed and characterized to quantify the DNA methylation of 3 genes(SHOX2,RASSF1 A and HOXA9).A total of 115 contiguous pleural effusion samples were eventually collected from 115 patients confirmed by cytological and/or histological examination and clinical evolution.Results: Among the 152 pleural effusion samples,101(66.5%)cases were malignant,including 46(45.5%)males and 55(54.5%)females,aged 40 to 85 years.There were51(33.5%)benign cases,including 37(72.5%)males and 14(27.5%)females,aged 16 to85 years.Among the 101(66.5%)patients with MPEs,there were 68 cases of lung cancer(54 cases of lung adenocarcinoma,6 cases of lung squamous cell carcinoma,4 cases of small cell lung cancer and 4 cases of unclassified lung cancer).33 cases of extrapulmonary metastasis(5 cases of pleural mesothelioma,14 cases of breast cancer,6cases of Gynecological Oncology,4 cases of Gastrointestinal Cancer,2 cases of hemopathy and 2 cases of others.Among the 51 BPE cases,22 were tuberculous,19 were parapneumonic effusion,and 10 was others.The diagnostic yields of the DNA methylation of 3 genes in MPEs were 43.6%(RASSF1A),56.4%(SHOX 2)and 63.4%(HOXA9),respectively.And the specificity were 100%(RASSF1A),96.1%(SHOX 2)and 98.0%(HOXA9),respectively.A two-gene combination of SHOX2 and RASSF1 A has the optimum sensitivity of 74.3%,specificity of 96.1%,positive predictive value(PPV)of 97.4% and negative predictive value(NPV)of 65.3%.The diagnostic yield of combining 3 genes together were 80.2%(81/101),with the specificity of 94.1%(3/51).And the PPV and NPV of 96.4% and 70.6%,respectively.Conclusion: DNA methylation analysis of SHOX2,RASSF1 A and HOXA9 has the potential use as a biomarker for differentiating MPE from BPE.The combined detection of RASSF1 A and SHOX2 gene methylation was identified as an excellent method for the screening and surveillance of MPEs that exhibits high sensitivity and specificity,while HOXA9 might be a good supplement.The sensitivity and specificity of the diagnosis can be significantly improved by the three combined assays.