Abstract
BackgroundThe Pap smear has remained the foundation for cervical cancer screening for over 70 years. With advancements in molecular diagnostics, primary high-risk human papillomavirus (hrHPV) screening has recently become an accepted stand-alone or co-test with conventional cytology. However, both diagnostic tests have distinct limitations. The aim of this study was to determine the association between HPV genotypes and cellular epigenetic modifications in three grades of cervical cytology for screening biomarker discovery.MethodsThis prospective, cross-sectional study used residual liquid-based cytology samples for HPV genotyping and epigenetic analysis. Extracted DNA was subjected to parallel polymerase chain reactions using three primer sets (MY09/11, FAP59/64, E6-E7 F/B) for HPV DNA amplification. HPV+ samples were genotyped by DNA sequencing. Promoter methylation of four candidate tumor suppressor genes (adenylate cyclase 8 (ADCY8), cadherin 8, type 2 (CDH8), MGMT, and zinc finger protein 582 (ZNF582)) out of 48 genes screened was quantified by bisulfite-pyrosequencing of genomic DNA. Independent validation of methylation profiles was performed by analyzing data from cervical cancer cell lines and clinical samples from The Cancer Genome Atlas (TCGA).ResultsTwo hundred seventy-seven quality cytology samples were analyzed. HPV was detected in 31/100 (31 %) negative for intraepithelial lesion or malignancy (NILM), 95/100 (95 %) low-grade squamous intraepithelial lesion (LSIL), and 71/77 (92 %) high-grade squamous intraepithelial lesion (HSIL) samples. The proportion of IARC-defined carcinogenic HPV types in sequenced samples correlated with worsening grade: NILM 7/29 (24 %), LSIL 53/92 (58 %), and HSIL 65/70 (93 %). Promoter methylation of ADCY8, CDH8, and ZNF582 was measured in 170 samples: NILM (N = 33), LSIL (N = 70), and HSIL (N = 67) also correlated with worsening grade. Similar hypermethylation patterns were found in cancer cell lines and TCGA samples. The combination of four biomarkers, i.e., HPV genotype and three-gene promoter methylation, predicted HSIL (AUC 0.89) better than HPV alone (AUC 0.74) by logistic regression and probabilistic modeling.ConclusionsHPV genotype and DNA methylation of ADCY8, CDH8, and ZNF582 are correlated with cytological grade. Collectively, these biomarkers may serve as a molecular classifier of Pap smears.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0263-9) contains supplementary material, which is available to authorized users.
Highlights
The Pap smear has remained the foundation for cervical cancer screening for over 70 years
The results indicate a positive correlation between Pap grade and promoter methylation of adenylate cyclase 8 (ADCY8), CDH8, and zinc finger protein 582 (ZNF582) (Spearman’s rank, p < 0.05) but not methylguanineDNA methyltransferase (MGMT)
The four panels display the chromosomal positions of ADCY8, CDH8, ZNF582, and MGMT with an expanded area showing the CpG probes on the Illumina HumanMethylation 450 K microarray
Summary
The Pap smear has remained the foundation for cervical cancer screening for over 70 years. With advancements in molecular diagnostics, primary high-risk human papillomavirus (hrHPV) screening has recently become an accepted stand-alone or co-test with conventional cytology. With advancements in molecular diagnostics and automation, primary high-risk HPV (hrHPV) cervical screening and alternative strategies that supplant the resource-demanding cytology-based model, such as visual inspection with acetic acid (VIA), have risen to the forefront. Both screening strategies are incorporated into the 2014 World Health Organization (WHO) published guidance on cervical cancer [3]. Full-spectrum HPV genotyping reveals the genotype and phenotype (carcinogenic potential), which are valuable guides for selecting conservative or ablative therapy in the clinical setting
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