HPV DNA integration into the host genome is a characteristic step in cervical carcinogenesis and seems to contribute to clonal expansion of malignant lesions. Indirect techniques for assessing the physical state of the viral genome such as the determination of E2/E6 ratios suggest that virus integration occurs frequently already in low grade lesions. However, this has not been confirmed at the sequence level. The aim of this study is to identify and sequence the integration sites in CIN3 and to use these sequence data to trace the integration sites in serial cervical swabs taken during the course of disease development. By this approach we will determine the time points in the natural history of CIN lesions at which driver-integrates can be detected first.
We included cervical swab samples from 22 patients with histologically confirmed HPV16-positive CIN3 who were treated between 2011 and 2015 at Jena University Hospital. Sample material comprised on average 4 cervical swabs per patient, collected at different time points up to 2 years prior to the diagnosis of CIN3. DNA was isolated using the NucleoSpin® Tissue kit from Macherey Nagel. HPV DNA integration sites were identified and sequenced in swabs taken from CIN3 lesions applying our recently published TEN16 approach (1). Validation of integration sites is done via PCR with primers flanking the transition of HPV and human DNA (viral-cellular junction PCR, vcj-PCR). In case of successful integrate validation, vcj-PCR is used to screen swabs taken prior to the diagnosis of CIN3.
So far, we have analysed the integration state in 18 of 22 cases with CIN3. For 4 patients HPV16 DNA integration sites could be identified and validated by vcj-PCR. The latter technique allows the detection of a single integrate in a background of 1000 integrate-free cells. Up to 3 different patient-specific integration sites were detected. The vcj-PCR is now being used to detect the integration sites in swabs collected prior to the diagnosis of CIN3.
We have demonstrated that the identification of integrated HPV16 DNA from cervical swabs of patients with CIN3 is feasible. The integration rate in this cohort is only 22% up to now. Further analysis and optimization of the TEN16 assay may reveal a higher integration rate since integration rates of up to 50 % have been described in other studies. Should we be able to trace driver integration events to early stages of disease the TEN16 platform may become a powerful prognostic tool.
B. Xu et al., Multiplex Identification of Human Papillomavirus 16 DNA Integration Sites in Cervical Carcinomas. PLoS One 8, e66693 (2013).