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

Abstract

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. 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 (, and ) that showed a significant increase in methylation with severity of disease in both tissue specimens and cervical scrapes ( < 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 ( < 0.0005), characterized by duration of preceding hrHPV infection, and were significantly higher in the presence of a 3q gain ( < 0.05) in the corresponding tissue biopsy. By unbiased genome-wide DNA methylation profiling and comprehensive stepwise verification and validation studies using and patient-derived samples, we identified 3 promising methylation markers (, and associated with a 3q gain for the detection of cervical (pre)cancer..

Host-cell DNA methylation patterns during high-risk HPV-induced carcinogenesis reveal a heterogeneous nature of cervical pre-cancer

“…These results correspond to the finding that only a subset of CIN2/3 will progress to cancer over a long time period [18,19]. Previous studies on copy number changes and DNA methylation levels of only a few genes also showed a cancer-like profile in only a subset of CIN2/3 lesions [11,14,20,21]. These lesions were characterized by a preceding hrHPV infection of >5 years and considered as more advanced lesions, which have a high short-term progression risk to cancer.…”

“…Other unidentified underlying abnormalities may have resulted in increased DNA methylation levels. Considering a potential effect of age, i.e., 55 year for this control, DNA methylation levels of the markers tested were not or minimally affected by age in our previous studies using large cohorts [8–11,42,43]. Therefore, solely age is very unlikely to explain the cancer-like methylation-high pattern in this hrHPV-positive control.…”

“…DNA extraction, quality assessment, sodium bisulfite-treatment, (multiplex) qMSP conditions, and DNA methylation ratio calculations were described previously for ANKRD18CP, C13orf18, EPB41L3 , and JAM3 [8,9]; SOX1 and ZSCAN1 [9,10]; GHSR, SST and ZIC1 [11]; and FAM19A4, PHACTR3 , and PRDM14 [12]. Quality control was routinely performed using a methylation-independent assay for the housekeeping gene ACTB [13].…”

“…Using both targeted and genome-wide approaches, we previously identified 12 genes, including ANKRD18CP, C13orf18, EPB41L3 , and JAM3 [8,9]; SOX1 and ZSCAN1 [9,10]; GHSR, SST , and ZIC1 [11]; and FAM19A4, PHACTR3 , and PRDM14 [12]. These genes were considered promising biomarkers to identify hrHPV-positive women and/or women with abnormal cytology at risk for cervical cancer [1,7–14].…”

Epigenomic profiling of DNA methylation for hepatocellular carcinoma diagnosis and prognosis prediction

“…[9][10][11][12] Most of the earlier studies used a candidate gene approach focused on selected genes; other important genes may have been missed. In recent years, a number of powerful technologies have emerged that allow high-throughput detection of genome-wide epigenetic changes in diseases, [13][14][15] and especially, the bead array technology developed by Illumina could now provide genome-wide methylation status at single-nucleotide resolution. [16][17][18][19] However, these studies mainly were conducted in European or American populations; only a few samples from Asian populations were included.…”