31PD DNA methylation of SHOX2 and PTGER4 as a plasma-based tool to differentiate between patients with malignant and benign lung disease.

Today, low-dose computed tomography (LDCT) is a guideline recommended screening method for lung cancer (LC) in high risk patients in the US, but not in Europe. Definition of high risk groups and impact of the substantial rate of LDCT false positive findings has led to significant dispute. Non-invasive diagnostic tools may provide a means to improve this situation. Recently, a DNA methylation panel of the genes SHOX2 and PTGER4 has been evaluated in three independent case-control studies comprising a total of 330 plasma specimens from LC patients and healthy individuals with promising results (AUC = 91 to 95%). Here, we report on evaluation of this marker panel in patients with malignant or benign lung disease (BLD). For this case-control study 172 plasma specimen were collected from 50 LC patients, 50 BLD patients and 72 healthy subjects. A triplex real-time PCR assay for methylated DNA of SHOX2 and PTGER4 and ACTB as control assay was developed. Total DNA was extracted from 3.5 ml plasma samples and bisulfite converted utilizing a commercially available kit. After purification DNA was assayed in PCR triplicates. Cycle threshold values were aggregated utilizing a predefined algorithm. Receiver operating characteristic and the area under the curve (AUC) were analyzed. The marker panel showed significant discriminatory power to distinguish LC cases from the remaining subjects (AUC = 0.88), including BLD patients (AUC = 0.85) and healthy subjects (AUC = 0.89). At specificity fixed at 95% the sensitivity for LC was 60%, at fixed sensitivity of 94% specificity was 56%. BLD and healthy subjects were similar with respect to the methylation markers (AUC = 0.55). The clinically important group of COPD patients (18 cases) was significantly different from the LC group (AUC = 0.79). Combination of DNA methylation markers SHOX2 and PTGER4 can be used to distinguish lung cancer patients from patients with non-malignant diseases with high sensitivity at a reasonable false positive rate. The marker panel will be developed into a plasma-based lung cancer diagnostic tool that may ultimately show clinical utility in combination with current imaging techniques to improve lung cancer risk stratification.