van der Zee RP, Meijer CJLM, Cuming T, et al. - Despite a high prevalence of human papillomavirus (HPV)-induced anal intraepithelial neoplasia (AIN, graded 1-3) in HIV-positive (HIV+) men-who-have-sex-with-men (MSM), progression to cancer appear only in a minority of lesions. In this study, anal tissue samples from a cross-sectional series (n = 104) of HIV + MSM and longitudinal series (n = 40) of AIN2/3 progressing to cancer were described comprehensively using different biomarkers. With increasing severity of anal lesions, there was a decrease in E4 positivity, whereas an increase was observed in p16 and Ki-67 scores and methylation marker positivity. A heterogeneous biomarker pattern was observed concerning E4, p16, and methylation status within AIN2, suggesting these lesions as biologically heterogeneous. In the longitudinal series, high p16 and Ki-67 expression, strong methylation positivity, and occasional E4 positivity were observed in AIN2/3 and carcinomas. High methylation levels are earlier linked with progression to cancer. E4 expression is observed in some AIN2/3 during the course of progression to cancer and there was absence of E4 in a considerable number of AIN1 lesions; this finding created difficulty in interpreting the potential clinical significance of E4 expression. Overall data indicated a possible value of IHC biomarkers to characterize AIN, however their prognostic value for cancer risk stratification, next to objective methylation analysis, seems to be limited.