p16INK4A hypermethylation detected by fluorescent methylation-specific PCR in plasmas from non-small cell lung cancer

Alessandra Bearzatto, Davide Conte, Milo Frattini, Nadia Zaffaroni, Francesca Andriani, Debora Balestra, Luca Tavecchio, Maria Grazia Daidone, Gabriella Sozzi

Research output: Contribution to journalArticle

Abstract

Purpose: The p16INK4A tumor suppressor gene is inactivated in many solid tumors, including non-small cell lung cancers (NSCLCs), through promoter hypermethylation. Presence of p16INK4A 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 p16INK4A hypermethylation detection in plasma limits its consideration in a diagnostic grid. Experimental Design: A fluorescent methylation-specific PCR assay (F-MSP) was established to evaluate p16INK4A 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 X2 or Fisher's exact tests. Results: p16INK4A 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. p16INK4A 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 p16INK4A promoter hypermethylation detection in plasmas from NSCLC patients. Microsatellite alterations, circulating DNA quantification, and p16INK4A hypermethylation might contribute to a diagnostic grid for NSCLC.

Original languageEnglish
Pages (from-to)3782-3787
Number of pages6
JournalClinical Cancer Research
Volume8
Issue number12
Publication statusPublished - Dec 1 2002

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Non-Small Cell Lung Carcinoma
Methylation
Polymerase Chain Reaction
Microsatellite Repeats
DNA
Neoplasms
Mutation
Body Fluids
Tumor Suppressor Genes
Early Diagnosis
Research Design
Tissue Donors

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

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p16INK4A hypermethylation detected by fluorescent methylation-specific PCR in plasmas from non-small cell lung cancer. / Bearzatto, Alessandra; Conte, Davide; Frattini, Milo; Zaffaroni, Nadia; Andriani, Francesca; Balestra, Debora; Tavecchio, Luca; Daidone, Maria Grazia; Sozzi, Gabriella.

In: Clinical Cancer Research, Vol. 8, No. 12, 01.12.2002, p. 3782-3787.

Research output: Contribution to journalArticle

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abstract = "Purpose: The p16INK4A tumor suppressor gene is inactivated in many solid tumors, including non-small cell lung cancers (NSCLCs), through promoter hypermethylation. Presence of p16INK4A 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 p16INK4A hypermethylation detection in plasma limits its consideration in a diagnostic grid. Experimental Design: A fluorescent methylation-specific PCR assay (F-MSP) was established to evaluate p16INK4A 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 X2 or Fisher's exact tests. Results: p16INK4A 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. p16INK4A 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 p16INK4A promoter hypermethylation detection in plasmas from NSCLC patients. Microsatellite alterations, circulating DNA quantification, and p16INK4A hypermethylation might contribute to a diagnostic grid for NSCLC.",
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AU - Andriani, Francesca

AU - Balestra, Debora

AU - Tavecchio, Luca

AU - Daidone, Maria Grazia

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