p16(INK4A) Hypermethylation detected by fluorescent methylation-specific PCR in plasmas from non-small cell lung cancer.
OBJECTIVE
The p16(INK4A) tumor suppressor gene is inactivated in many solid tumors, including non-small cell lung cancers (NSCLCs), through promoter hypermethylation. Presence of p16(INK4A) 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(INK4A) hypermethylation detection in plasma limits its consideration in a diagnostic grid.


METHODS
A fluorescent methylation-specific PCR assay (F-MSP) was established to evaluate p16(INK4A) 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 mutant-enriched PCR), and quantification of circulating DNA. Assay results were analyzed by two-sided chi(2) or Fisher's exact tests.


RESULTS
p16(INK4A) 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(INK4A) 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(INK4A) promoter hypermethylation detection in plasmas from NSCLC patients. Microsatellite alterations, circulating DNA quantification, and p16(INK4A) hypermethylation might contribute to a diagnostic grid for NSCLC.
Bearzatto
Alessandra
A
Department of Experimental Oncology, Unit 10, Istituto Nazionale Tumori, 20133 Milan, Italy.
Conte
Davide
D
Frattini
Milo
M
Zaffaroni
Nadia
N
Andriani
Francesca
F
Balestra
Debora
D
Tavecchio
Luca
L
Daidone
Maria Grazia
MG
Sozzi
Gabriella
G
D014408:Biomarkers, Tumor / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D002893:Chromosomes, Human, Pair 3 / Q000235:genetics; D015331:Cohort Studies; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000097:blood*; D005260:Female; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D018895:Microsatellite Repeats; D008875:Middle Aged; D009367:Neoplasm Staging; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011446:Prospective Studies; D015996:Survival Rate; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm