Genome-wide DNA Methylation Profiling Reveals Methylation Markers Associated with 3q Gain for Detection of Cervical Precancer and Cancer.

Abstract

<b>Purpose:</b> Epigenetic host cell changes involved in cervical cancer development following a persistent high-risk human papillomavirus (hrHPV) infection, provide promising markers for the management of hrHPV-positive women. In particular, markers based on DNA methylation of tumor suppressor gene promoters are valuable. These markers ideally identify hrHPV-positive women with precancer (CIN2/3) in need of treatment. Here, we set out to identify biologically relevant methylation markers by genome-wide methylation analysis of both hrHPV-transformed cell lines and cervical tissue specimens.<b>Experimental Design and Results:</b> Genome-wide discovery by next-generation sequencing (NGS) of methyl-binding domain-enriched DNA (MBD-Seq) yielded 20 candidate methylation target genes. Further verification and validation by multiplex-targeted bisulfite NGS and (quantitative) methylation-specific PCR (MSP) resulted in 3 genes (<i>GHSR, SST</i>, and <i>ZIC1</i>) that showed a significant increase in methylation with severity of disease in both tissue specimens and cervical scrapes (<i>P</i> < 0.005). The area under the ROC curve for CIN3 or worse varied between 0.86 and 0.89. Within the group of CIN2/3, methylation levels of all 3 genes increased with duration of lesion existence (<i>P</i> < 0.0005), characterized by duration of preceding hrHPV infection, and were significantly higher in the presence of a 3q gain (<i>P</i> < 0.05) in the corresponding tissue biopsy.<b>Conclusions:</b> By unbiased genome-wide DNA methylation profiling and comprehensive stepwise verification and validation studies using <i>in vitro</i> and patient-derived samples, we identified 3 promising methylation markers (<i>GHSR, SST</i>, and <i>ZIC1)</i> associated with a 3q gain for the detection of cervical (pre)cancer. <i>Clin Cancer Res; 23(14); 3813-22. ©2017 AACR</i>.