Diagnostic Performance of Plasma DNA Methylation Profiles in Lung Cancer, Pulmonary Fibrosis and COPD

• The multiplexed MSRE enrichment strategy allowed a highly parallel assessment the cfDNA methylation status based on 400 μl of patient serum.
• The multivariate classification of the discovered biomarkers, thus, stabilized the prediction and allowed for differential diagnosis.
• This method including the biomarker set may be used to monitor the lung cancer risk in COPD and fibrotic ILD patients.Research in ContextWe developed a multiplexed DNA methylation profiling strategy to track disease specific DNA methylation changes prevailing in 400 μl serum of lung cancer, fibrotic ILD and COPD patients. Plasma or serum samples, often referred to as ‘liquid biopsies’ have several advantages over tissue sampling: (a) they are easily accessible, (b) are not subject to biopsy bias and (c) can be repeatedly drawn from the same patient. The differential diagnosis attempt performed in the study on patients suffering from different lung diseases suggests that multiplexed cfDNA methylation profiling allows for the capture of these interconnected disease phenotypes, stabilizes the prediction and enhances diagnostic accuracy.

Disease-specific alterations of the cell-free DNA methylation status are frequently found in serum samples and are currently considered to be suitable biomarkers.Candidate markers were identified by bisulfite conversion-based genome-wide methylation screening of lung tissue from lung cancer, fibrotic ILD, and COPD. cfDNA from 400 μl serum (n = 204) served to test the diagnostic performance of these markers. Following methylation-sensitive restriction enzyme digestion and enrichment of methylated DNA via targeted amplification (multiplexed MSRE enrichment), a total of 96 markers were addressed by highly parallel qPCR.Lung cancer was efficiently separated from non-cancer and controls with a sensitivity of 87.8%, (95%CI: 0.67–0.97) and specificity 90.2%, (95%CI: 0.65–0.98). Cancer was distinguished from ILD with a specificity of 88%, (95%CI: 0.57–1), and COPD from cancer with a specificity of 88% (95%CI: 0.64–0.97). Separation of ILD from COPD and controls was possible with a sensitivity of 63.1% (95%CI: 0.4–0.78) and a specificity of 70% (95%CI: 0.54–0.81). The results were confirmed using an independent sample set (n = 46) by use of the four top markers discovered in the study (HOXD10, PAX9, PTPRN2, and STAG3) yielding an AUC of 0.85 (95%CI: 0.72–0.95).This technique was capable of distinguishing interrelated complex pulmonary diseases suggesting that multiplexed MSRE enrichment might be useful for simple and reliable diagnosis of diverse multifactorial disease states.