p16 Hypermethylation Detected by Fluorescent Methylation- specific PCR in Plasmas from Non-Small Cell Lung Cancer

Purpose: The p16 tumor suppressor gene is inactivated in many solid tumors, including non-small cell lung cancers (NSCLCs), through promoter hypermethylation. Presence of p16 hypermethylation in precursor lesions of NSCLC and in body fluids of individuals at risk makes it a potential candidate for early disease detection. However, the current low sensitivity of p16 hypermethylation detection in plasma limits its consideration in a diagnostic grid. Experimental Design: A fluorescent methylationspecific PCR assay (F-MSP) was established to evaluate p16 promoter hypermethylation in 35 NSCLC and paired plasma samples and in 15 plasmas from healthy donors. F-MSP sensitivity was investigated in combination with microsatellite alterations on 3p (evaluated by fluorescent PCR), K-ras mutations (determined by a mutantenriched PCR), and quantification of circulating DNA. Assay results were analyzed by two-sided 2 or Fisher’s exact tests. Results: p16 promoter hypermethylation, detectable by F-MSP in 22 of 35 NSLCs (63%) and in 12 of 22 (55%) plasmas from patients with methylated tumors, was independent of microsatellite alterations (detectable in 57% of tumors and 50% of paired plasmas), K-ras mutations (detectable in 31% of tumors but in no paired plasma), or amount of circulating DNA. p16 methylation in association with microsatellite alterations identified 62% (18 of 29) of plasma samples from patients presenting the same alteration in their tumors, and its sensitivity increased to 80% when combined with the amount of circulating DNA. Conclusions: The establishment of F-MSP remarkably improved p16 promoter hypermethylation detection in plasmas from NSCLC patients. Microsatellite alterations, circulating DNA quantification, and p16 hypermethylation might contribute to a diagnostic grid for NSCLC.