AbstractLung cancer remains the leading cause of cancer deaths worldwide. Although low-dose spiral computed tomography (LDCT) screening is used for the detection of lung cancer in a high-risk population, false-positive results of LDCT remain a clinical problem. Here, we developed a blood test of a novel panel of three established lung cancer methylation biomarkers for lung cancer detection. Short stature homeobox 2 gene (SHOX2), ras association domain family 1A gene (RASSF1A), and prostaglandin E receptor 4 gene (PTGER4) methylation was analyzed in a training cohort of 351 individuals (197 controls, 154 cases) and validated from an independent cohort of 149 subjects (89 controls, 60 cases). The novel panel biomarkers distinguished between malignant and benign lung disease at high sensitivity and specificity: 87.0% sensitivity [95% CI 80.2–91.5%], 98.0% specificity [95% CI 94.9–99.4%]. Sensitivity in adenocarcinoma, squamous cell carcinoma, small cell lung cancer, and other lung cancer was 89.0%, 87.5%, 85.7%, and 77.8%, respectively. Notably, cancer patients in stage I and II showed high diagnostic sensitivity at 82.5% and 90.5%, respectively. Moreover, the diagnostic efficiency did not show bias toward age, gender, smoking, and the presence of other (nonlung) cancers. The performance of the panel in the validation cohort confirmed the diagnostic value. These findings clearly showed that this panel of DNA methylation biomarkers was effective in detecting lung cancer noninvasively and may provide clinical utility in stand-alone or in combination with current imaging techniques to improve the diagnosis of lung cancer.