Presence of E6 and E7 mRNA from human papillomavirus types 16, 18, 31, 33, and 45 in the majority of cervical carcinomas

Abstract

The oncogenic potential of the human papillomavirus (HPV) early genes E6 and E7 is well established and a source of interest with regard to HPV testing for cervical carcinoma. Here we present a study performed with 204 histologically confirmed invasive cervical squamous cell carcinomas (SCCs) in which we evaluated the HPV E6 and E7 mRNA detection assay PreTect HPV-Proofer for detection of high-risk HPV types 16, 18, 31, 33, and 45. For further evaluation, detection of E6 and E7 mRNA from HPV types 35, 52, and 58 by real-time multiplex nucleic acid sequence-based amplification was also included. For comparison and to assess the overall prevalence of various HPV types, samples were also tested for HPV DNA by both consensus and type-specific PCR, reverse line blotting, sequencing, and in situ hybridization. The overall prevalence of HPV was 97%. HPV E6 and E7 transcripts were detected in 188 of 204 (92%) biopsy specimens, of which 181 contained one of the following HPV types: 16, 18, 31, 33, or 45. Consensus PCR and type-specific PCR detected HPV in 187 of 204 and 188 of 204 (92%) specimens, respectively. In conclusion, this study verifies the presence of HPV E6 and E7 mRNA in SCCs and demonstrates that HPV infections among Norwegian women with SCCs are limited mainly to the five high-risk types, 16, 18, 31, 33, and 45. This, together with the fact that PreTect HPV-Proofer detects the HPV oncogenic transcripts, suggests that the assay is a valuable approach in the field of HPV detection in cervical carcinoma.

FIG. 1.

Flow chart showing the different HPV test methods used with different specimens. Symbols: +, paraffin-embedded tissue; ±, fresh frozen biopsy specimen. Proofer, PreTect HPV-Proofer assay; PCR, type-specific PCR (for HPV types 16, 18, 31, 33, 35, 45, 52, and 58, as indicated); n, number of samples tested.

FIG. 2.

Amplification of HPV RNA by PreTect HPV-Proofer. The PreTect HPV-Proofer assay detects HPV E6 and E7 mRNA from the high-risk HPV types 16, 18, 31, 33, and 45. The assay is based on real-time multiplex NASBA, detecting in complex HPV-16 and the sample performance control U1A (a), HPV-33 and HPV-45 (b), and HPV-31 and HPV-18 (c). A positive reaction result is revealed as a sigmoid curve.

FIG. 3.

Examination of serial sections by ISH. (a) Punctuate and diffuse ISH signal, indicating the presence of both integrated and episomal HPV-16; (b) punctuate ISH signal, indicating integrated HPV-18. Some tumor cells were infected with both HPV-16 and HPV-18 (arrows).