Sensitive and specific detection of lung cancer DNA in bronchial washing by a novel methylation-specific droplet digital PCR

Rationale: In patients with suspected lung cancer fibrobronchoscopy (FBS) has a key role in the diagnostic process, but its performance is variable (from 90% in the presence of visible lesions to less than 30% for distal ones). DNA methylation analysis emerged as a promising approach for cancer detection. Aberrantly methylated DNA sequences are present in tumour cells and represent cancer specific biomarkers that can be detected in biological fluids. Our study attempts to improve the diagnostic power of BW through the use of cancer specific DNA methylation biomarkers. METHODS: We performed a retrospective case-control study in which we investigated the methylation status of a panel of genes (RASSF1A, CDH1, DLC1 and PRPH) in BWs samples from 91 lung cancer patients and 31 non-cancer controls (current or former smokers). We analysed the methylation status of DNAs from BWs fluids by using a novel highly sensitive two-colour droplet digital methylation-specific PCR. Results: The 4-gene panel exhibited a significant diagnostic power, with a 97% sensitivity and 74% specificity (overall diagnostic accuracy = 0.88), conferring a relative risk of 7.3, at an odds ratio of 76.1 (95% CI from 12.7 to 127). The ROC curve analysis (AUC=0.93) confirmed the excellent diagnostic power of the 4-genes panel. In addition the methylation assay was positive in 35 of the 36 BW samples with negative cytological analyses. CONCLUSIONS. Our results indicate that the aberrant methylation of this panel of genes could represent a helpful support to current diagnostic strategies for lung cancer detection.