title: Molecular Pap smear: HPV genotype and DNA methylation of ADCY8, CDH8, and ZNF582 as an integrated biomarker for high-grade cervical cytology.
abstract: 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. 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.
  
  
  
  This 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).
  
  
  
  Two 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.
  
  
  
  HPV 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.
authors.affiliation: Gynecologic Oncology & Clinical Investigation, Department of Clinical Investigation, Brooke Army Medical Center, 3698 Chambers Pass, Fort Sam Houston, TX 78234-6315 USA.
authors.forename: Jane
authors.identifier: 0000-0002-0876-9338
authors.initials: J
authors.lastname: Shen-Gunther
authors.affiliation: Department of Molecular Medicine, Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229 USA.
authors.forename: Chiou-Miin
authors.initials: CM
authors.lastname: Wang
authors.affiliation: Department of Clinical Investigation, Brooke Army Medical Center, Fort Sam Houston, TX 78234 USA.
authors.forename: Graham M
authors.initials: GM
authors.lastname: Poage
authors.affiliation: Department of Molecular Medicine, Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229 USA.
authors.forename: Chun-Lin
authors.initials: CL
authors.lastname: Lin
authors.affiliation: Department of Clinical Investigation, Brooke Army Medical Center, Fort Sam Houston, TX 78234 USA.
authors.forename: Luis
authors.initials: L
authors.lastname: Perez
authors.affiliation: Department of Pathology and Area Laboratories, Brooke Army Medical Center, Fort Sam Houston, TX 78234 USA.
authors.forename: Nancy A
authors.initials: NA
authors.lastname: Banks
authors.affiliation: Department of Molecular Medicine, Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229 USA.
authors.forename: Tim Hui-Ming
authors.initials: TH
authors.lastname: Huang
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics*; D000328:Adult; D015820:Cadherins / Q000235:genetics*; D045744:Cell Line, Tumor; D003430:Cross-Sectional Studies; D019175:DNA Methylation; D004279:DNA, Viral / Q000032:analysis; D055088:Early Detection of Cancer; D005260:Female; D005838:Genotype; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics*; D008875:Middle Aged; D060787:Neoplasm Grading; D065006:Papanicolaou Test / Q000379:methods; D027383:Papillomaviridae / Q000235:genetics*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000453:epidemiology; D011401:Promoter Regions, Genetic; D011446:Prospective Studies; D017422:Sequence Analysis, DNA / Q000379:methods; D065310:Squamous Intraepithelial Lesions of the Cervix / Q000453:epidemiology / Q000235:genetics / Q000473:pathology / Q000821:virology; D002583:Uterine Cervical Neoplasms / Q000453:epidemiology* / Q000235:genetics / Q000473:pathology* / Q000821:virology; D014626:Vaginal Smears / Q000379:methods; D055815:Young Adult; D002578:Uterine Cervical Dysplasia / Q000453:epidemiology / Q000235:genetics / Q000473:pathology / Q000821:virology
chemical_list: C112316:CDH8 protein, human; D015820:Cadherins; D004279:DNA, Viral; D051741:Kruppel-Like Transcription Factors; C577358:ZNF582 protein, human; D000262:Adenylyl Cyclases; C118292:adenylyl cyclase 8
keywords: DNA methylation; HPV; HPV genotyping; Molecular biomarkers; Molecular diagnostics; Pap smear; Pyrosequencing