Study of viral integration of HPV-16 in young patients with LSIL

Abstract

Aims: To investigate the physical status of human papillomavirus 16 (HPV-16) in low grade squamous intraepithelial lesions (LSILs) as a means of determining the percentage of viral integration.

Methods: Ninety two LSIL/HPV positive Thin Prep(TM) samples were initially tested for the E6 gene by the polymerase chain reaction (PCR) to identify the HPV-16 virus. To avoid false positive results, the specificity of the bands obtained from PCR was confirmed by Southern blot hybridisation with internal oligonucleotide probes. Next, a PCR screen for the E2 gene was performed to identify those samples in which the virus was integrated. Viral integration was detected in just over half of them.

Results: Twenty of the 92 samples were HPV-16 positive, as shown by PCR for the E6 gene. Southern blot analysis confirmed that 13 of these samples were positive for the viral E6 gene. Thus, viral integration was detected in just over a half of the samples positive for HPV-16.

Conclusions: These data show that HPV-16 integration occurs in a subset of LSILs. The measurement of HPV-16 integration would be a helpful complementary tool for cytological evaluation in primary cervical screening to identify those patients at risk of developing high grade squamous intraepithelial lesions and cervical cancer.

Figure 1

Identification of human papillomavirus 16 (HPV-16) in low grade squamous intraepithelial lesions. Ninety two samples were tested for the presence of HPV-16 by polymerase chain reaction (PCR) amplification of the E6 gene. (A,C) Some of the samples positive for E6 are shown (lanes 1–15). C+ and C− correspond to the positive and negative controls, respectively. Arrows on the right show the expected molecular weight of the E6 gene fragment (209 bp). (B,D) The E6 gene PCR product (20 μl) was electrophoresed on a 2% agarose gel and hybridised using a single strand oligonucleotide corresponding to the region of the HPV genome spanning nucleotides 136 to 161.

Figure 2

Study of viral integration. For the detection of integration, the E2 open reading frame of HPV-16 was amplified. Examples of viral genomic integration (lanes 1, 5, 6, 9, 11, and 13) and of viral episomal forms (lanes 10, 12, 14, 16, 17, 20, and 21) are shown. Arrows on the right show the expected molecular weight of the E2 gene fragment (351 bp).

Figure 3

Diagnostic method of identification of human papillomavirus 16 (HPV-16) and the detection of viral integration. The figure shows, in a temporal sequence, the steps followed in HPV-16 identification and in the detection of viral integration, ending with the follow up. Results of each phase of the study (numbered from 1 to 4) are shown below. In phase 1, HPV-16 was detected by polymerase chain reaction (PCR) for the E6 gene and 20 of the 92 cases were positive. In phase 2, Southern blot hybridisation was performed on the E6 gene PCR product as a control. Thirteen of the 92 cases were confirmed to be HPV-16 positive. Phase 3 was the study of viral integration among the HPV-16 positive cases. The absence of E2 gene PCR product implies viral integration and this was found in seven of the 13. In the last phase, the clinical outcome is summarised (after two years of follow up) for those patients in whom the virus had been integrated. In five of these seven patients, HPV infection or moderate dysplasia persisted during the two following years.