Nonradioactive RNA in situ hybridization detection of human papillomavirus 16-E7 transcripts in squamous cell carcinomas of the uterine cervix using confocal laser scan microscopy.

Paraffin-embedded squamous cell carcinomas of the uterine cervix selected for the presence of human papillomavirus (HPV) genotype 16 (n = 19) by polymerase chain reaction, were studied for transcription of the early open reading frame E7 (ORF E7). Nonradioactive RNA in situ hybridization (RISH) was performed using in vitro generated biotinylated probes. Hybrids were visualized by streptavidin gold and silver enhancement staining in combination with confocal laser scan microscopy. Quality of mRNA was verified by detection of beta-actin gene transcripts before E7 expression was studied. In all carcinomas containing HPV 16 DNA and showing beta-actin mRNA signals (n = 13), clear E7 ORF transcription could be found. Additional RNA-PCR on purified cytoplasmic RNA of snapfrozen tissue of identical carcinomas (n = 7) showed E6-E7 specific transcripts in all E7 RISH positive samples. These results indicate continuous expression of E7 ORF in all cervical carcinomas containing HPV 16 DNA and support an active role of the E7 ORF in the pathogenesis of cervical cancer.