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Background Anal cancer rates have increased, particularly in human immunodeficiency virus (HIV)–infected (HIV+) women. We assessed factors associated with anal precancer in HIV+ and at-risk HIV-negative women from the Atlanta Women's Interagency HIV Study cohort. Methods All participants underwent high-resolution anoscopy and anal cytology and had anal and cervical samples collected. Specimens were tested for 37 human papillomavirus (HPV) types and for FAM19A4 and microRNA124-2 promoter methylation. Binary logistic regression and multivariate analysis were conducted with histologic anal high-grade squamous intraepithelial lesion (A-HSIL) as the dependent variable. Results Seventy-five women were enrolled: 52 (69%) were HIV+ with three-fourths having undetectable viral load; 64 (86%) were black; mean age was 49 ± 8 years. Forty-nine (65%) anal cytology samples were abnormal, and 38 (51%) of anal samples were positive for at least 1 of 13 high-risk HPV (hrHPV) types. Thirteen (18%) anal biopsies identified A-HSIL. Hypermethylation of FAM19A4 and/or microRNA124-2 was found in 69 (95%) anal samples and 19 (26%) cervical samples. In multivariate analyses, the odds of having A-HSIL were >6 times higher in women with anal hrHPV (adjusted odds ratio [aOR], 6.08 [95% confidence interval {CI}, 1.27–29.18], P =.02) and with positive cervical methylation (aOR, 6.49 [95% CI, 1.66–25.35], P =.007), but not significantly higher in women with positive anal methylation. Conclusions Anal hrHPV and promoter hypermethylation in the cervix show promise as biomarkers for anal cancer screening in HIV+ and at-risk HIV-negative women. Greater understanding of gene silencing by promoter hypermethylation in anal carcinogenesis is needed. [ABSTRACT FROM AUTHOR]