| Summary: | <p>Abstract</p> <p>Background</p> <p>The aim of this study is to analyse <it>CDKN2A</it> 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.</p> <p>Methods</p> <p>Primary colorectal cancers and matched non-neoplastic tissues from 432 patients underwent <it>CDKN2A</it> methylation analysis by pyrosequencing (PyroMarkQ96). Methylation was then related to clinical outcome, microsatellite instability (MSI), and <it>BRAF</it> and <it>KRAS</it> mutation. Different amplification conditions (35 to 50 PCR cycles) using a range of 0-100% methylated DNA were tested.</p> <p>Results</p> <p>Background 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. <it>CDKN2A</it> methylation positivity was associated with MSI (p = 0.025), <it>BRAF</it> mutation (p < 0.0001), higher tumor grade (p < 0.0001), mucinous histology (p = 0.0209) but not with <it>KRAS</it> mutation. <it>CDKN2A</it> methylation had an independent adverse effect (p = 0.0058) on prognosis.</p> <p>Conclusion</p> <p>The non-negligible <it>CDKN2A</it> 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. <it>CDKN2A</it> methylation is robustly detected by pyrosequencing, even at low levels, suggesting that this unfavorable prognostic biomarker warrants investigation in prospective studies.</p>
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