Primary high-risk Human Papillomavirus (hrHPV) screening has recently become an accepted standalone or co-test with conventional cytology. Unfortunately, hrHPV singularly lacks specificity for cytopathological grade. However, mechanisms and markers of evolving virus-host interactions at the epigenome level may be harnessed as a better predictor of carcinogenesis. This study aimed to validate and expand the clinical performance of a multiparametric biomarker panel, referred to as the "Molecular Pap smear" based, on HPV genotype and ADCY8, CDH8 and ZNF582 CpG-methylation as a predictive classifier of cervical cytology. This prospective, cross-sectional study used an independent cohort of residual liquid-based cytology for HPV genotyping and epigenetic analysis. Extracted DNA underwent parallel PCR using 3 primer sets for HPV DNA amplification. HPV-infected samples were genotyped by Sanger sequencing. Promoter methylation levels of 3 tumor suppressor genes were quantified by bisulfite-pyrosequencing of genomic DNA on the newest high-resolution PyroMark Q48 platform. Logistic model performance was compared, and model parameters were used to predict and classify binary cytological outcomes. A total of 883 samples were analyzed. HPV DNA positivity correlated with worsening grade: 125/237 (53%) NILM; 136/235 (58%) ASCUS; 222/229 (97%) LSIL; and 157/182 (86%) HSIL samples. The proportion of carcinogenic HPV-types in PCR-positive sequenceable samples correlated with worsening grade: NILM 34/98 (35%); ASCUS 50/113 (44%); LSIL 92/214 (43%); HSIL 129/152 (85%). Additionally, ADCY8, CDH8, and ZNF582 methylation levels increased in direct correlation with worsening grade. Overall, the multi-marker modeling parameters predicted binarized cytological outcomes better than HPV-type alone with significantly higher area under the receiver operator curve (AUC)s, respectively: NILM vs. > NILM (AUC 0.728 vs. 0.709); NILM/ASCUS vs. LSIL/HSIL (AUC 0.805 vs. 0.776); and

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