Abstract
Different methodological approaches are available to assess DNA methylation biomarkers. In this study, we evaluated two sodium bisulfite conversion-dependent methods, namely pyrosequencing and methylation-specific qPCR (MS-qPCR), with the aim of measuring the closeness of agreement of methylation values between these two methods and its effect when setting a cut-off. Methylation of tumor suppressor gene p16/INK4A was evaluated in 80 lung cancer patients from which cytological lymph node samples were obtained. Cluster analyses were used to establish methylated and unmethylated groups for each method. Agreement and concordance between pyrosequencing and MS-qPCR was evaluated with Pearson’s correlation, Bland–Altman, Cohen’s kappa index and ROC curve analyses. Based on these analyses, cut-offs were derived for MS-qPCR. An acceptable correlation (Pearson’s R2 = 0.738) was found between pyrosequencing (PYRmean) and MS-qPCR (NMP; normalized methylation percentage), providing similar clinical results when categorizing data as binary using cluster analysis. Compared to pyrosequencing, MS-qPCR tended to underestimate methylation for values between 0 and 15%, while for methylation >30% overestimation was observed. The estimated cut-off for MS-qPCR data based on cluster analysis, kappa-index agreement and ROC curve analysis were much lower than that derived from pyrosequencing. In conclusion, our results indicate that independently of the approach used for estimating the cut-off, the methylation percentage obtained through MS-qPCR is lower than that calculated for pyrosequencing. These differences in data and therefore in the cut-off should be examined when using methylation biomarkers in the clinical practice.
Highlights
Epigenetic mechanisms play an important role in the regulation of gene activity and expression
Methylation data for 18 CpG sites in p16/INK4A were obtained by both pyrosequencing and methylation-specific qPCR (MS-qPCR)
Our study showed that methylation measurements based on MS-qPCR and pyrosequencing have good correlation (Pearson’s R2 = 0.738) when the latter is expressed as pyrosequencing methylation (PYRmean)
Summary
Epigenetic mechanisms play an important role in the regulation of gene activity and expression. Aberrant DNA methylation is the most extensively studied epigenetic alteration and is crucial for cancer initiation and development [1]. Hypermethylation of promoters of well-known cancer-related genes has been reported in many studies [2,3,4]. Aberrant methylation has been detected in liquid biopsy, including blood, sputum, bronchial washings, bronchoalveolar lavage, bronchial aspirates and cytohistological material from endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) [4,5,6]. EBUS-TBNA is a minimally invasive method that allows the sampling of mediastinal and hilar lymph nodes for diagnosis and staging of lung cancer [7]. Samples obtained by EBUS-TBNA are typically analyzed using cytological techniques, though molecular markers, including epigenetics, are considered very promising for the detection of occult lymph node metastasis [6,7,8]
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