Characterization of CDKN2Ap16 methylation and impact in colorectal cancer: systematic analysis using pyrosequencingReportar como inadecuado




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Journal of Translational Medicine

, 10:173

Personalized medicine

Abstract

BackgroundThe aim of this study is to analyse CDKN2A methylation using pyrosequencing on a large cohort of colorectal cancers and corresponding non-neoplastic tissues. In a second step, the effect of methylation on clinical outcome is addressed.

MethodsPrimary colorectal cancers and matched non-neoplastic tissues from 432 patients underwent CDKN2A methylation analysis by pyrosequencing PyroMarkQ96. Methylation was then related to clinical outcome, microsatellite instability MSI, and BRAF and KRAS mutation. Different amplification conditions 35 to 50 PCR cycles using a range of 0-100% methylated DNA were tested.

ResultsBackground methylation was at most 10% with ≥35 PCR cycles. Correlation of observed and expected values was high, even at low methylation levels 0.02%, 0.6%, 2%. Accuracy of detection was optimal with 45 PCR cycles. Methylation in normal mucosa ranged from 0 to >90% in some cases. Based on the maximum value of 10% background, positivity was defined as a ≥20% difference in methylation between tumor and normal tissue, which occurred in 87 cases. CDKN2A methylation positivity was associated with MSI p = 0.025, BRAF mutation p < 0.0001, higher tumor grade p < 0.0001, mucinous histology p = 0.0209 but not with KRAS mutation. CDKN2A methylation had an independent adverse effect p = 0.0058 on prognosis.

ConclusionThe non-negligible CDKN2A methylation of normal colorectal mucosa may confound the assessment of tumor-specific hypermethylation, suggesting that corresponding non-neoplastic tissue should be used as a control. CDKN2A methylation is robustly detected by pyrosequencing, even at low levels, suggesting that this unfavorable prognostic biomarker warrants investigation in prospective studies.

KeywordsColorectal cancer CDKN2A p16 Methylation Pyrosequencing AbbreviationsMSIMicrosatellite instability.

Electronic supplementary materialThe online version of this article doi:10.1186-1479-5876-10-173 contains supplementary material, which is available to authorized users.

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Autor: Michel P Bihl - Anja Foerster - Alessandro Lugli - Inti Zlobec

Fuente: https://link.springer.com/







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