This retrospective case-control study assessed human papillomavirus 16 (HPV16) viral load and E2/E6 ratio as risk markers for cervical intraepithelial neoplasia (CIN) >or=2 lesions in HPV16-positive women in a routine liquid-based cytology setting. Triplex quantitative PCR for HPV16 E6, E2, and beta-globin was done to determine the HPV16 load and the E2/E6 ratio, as a surrogate marker for integration, for women with a negative histologic endpoint (200 controls: 83 normal histology and 117 CIN1) and women with a >or=CIN2 endpoint (180 cases: 41 CIN2, 122 CIN3, and 17 invasive carcinoma). Our analysis showed a significantly higher HPV16 load in the case group, which was completely attributable to the high viral load of samples with invasive carcinoma as histologic endpoint. There was no significant difference in viral load between the other histologic groups. The E2/E6 ratio proved to be lower for the cases. However, the E2/E6 ratio indicated the presence of HPV integration in a considerable amount of control samples (44.3%), which suggests that HPV integration occurs early in the development of cancer and undermines the clinical value of viral integration. Overall, the intrinsic heterogeneous nature of the cervical cytology samples caused a substantial overlap of the HPV16 load and the E2/E6 ratio between controls and cases, which precludes the determination of cutoff values for risk prediction and hampers the clinical applicability in a cervical screening setting.