Methylation status of the E2 binding sites of HPV16 in cervical lesions determined with the Luminex® xMAP™ system

Cervical carcinogenesis is driven by deregulated E6/E7 expression in dividing cells. A potential deregulating mechanism is methylation of E2 binding sites in the viral long control region, thereby prohibiting HPVE2-mediated transcription regulation. Here the frequency of HPV16E2BS methylation in cervical lesions (SCC, n = 29; CIN3, n = 17) and scrapes (controls, n = 17; CIN3, n = 21) was investigated. Three E2BSs were amplified using methylation independent PCR followed by specific detection of methylated CpGs using the Luminex® xMAP™ system. The frequency of E2BS1, E2BS3 and E2BS4 methylation was significantly higher in SCC compared to CIN3, i.e. 93% vs. 21% (p < 0.01), 90% vs. 47% (p < 0.01) and 69% vs. 5% (p < 0.01), respectively and ranged from 6 to 15% in controls. In scrapings of women with CIN3 methylation ranged from 24 to 33%.In conclusion, we showed that the MIP-Luminex system is a highly sensitive method for methylation analysis. HPV16 E2BSs methylation appeared highly frequent in SCC, with particularly E2BS3 methylation occurring proportional to severity of cervical disease.

► MIP-Luminex assay is a sensitive and reliable method for DNA methylation detection. ► Methylation of HPV16 E2BSs is highly frequent in cervical SCCs. ► E2BS methylation is a rather late event in cervical carcinogenesis. ► E2BS3 methylation occurs proportional to severity of cervical disease.