Heterogeneous Distribution of Human Papillomavirus (HPV) Integration Sites in Cervical Precancers Compromises the Diagnostic Accuracy of Integrant-Specific PCR

Human papillomavirus (HPV) DNA integration into the host genome is a frequent event in cervical carcinogenesis and may drive clonal expansion of the affected cells. Based on viral cellular junction (vcj) sequences, highly specific vcj-PCRs can be designed to detect viral integrants in DNA from cervical cell scrapes or tissue samples. In a recent study, such patient-specific vcj-PCR assays were employed for the detection of recurrent high-grade squamous intraepithelial lesions (HSIL) during postoperative surveillance. Although the specificity of vcj-PCR was 100%, only 50% of the recurrences were detected using this approach. The focus of the current study was to analyze the cause of this limited sensitivity. Using chemical microdissection and subsequent vcj-PCR analysis, we could demonstrate that the majority of lesions have a heterogeneous integrant pattern. Only 2 of 16 cones showed a homogeneous distribution of the respective integrants throughout the entire lesion. The other lesions displayed clonal outgrowths harboring the integrant in a background HPV16/18 DNA-positive HSIL tissue. In 4 cases, the respective integrant was undetectable in the lesion. These findings indicate that vcj-PCR has limited sensitivity for the detection of recurrent disease owing to intralesional heterogeneity. The observed heterogeneous integrant pattern may thus reflect the multifocal nature of most large HSIL. Alternatively, the possibility that HPV integration may be a late event in the carcinogenic process also needs to be considered.