Detection of high risk human papillomavirus in routine cervical smears:strategy for screening

AIM: To develop a methodology for direct detection of high risk human papillomavirus (HPV) infection in routine cervical smears by non-isotopic in situ hybridisation (NISH) which can be compared with cytopathological assessment of the same cells. METHODS: The methodology was established using cultured cells and routine cervical smears hybridised with digoxigenin labelled probes for HPV, 16, 18, 31, and 33. The technique was applied to the analysis of 53 patients from a sexually transmitted disease clinic. RESULTS: The optimal sensitivity achieved for single HPV detection in cultured cells was 1-2 copies of HPV 16 per cell and that for detection of a cocktail of HPV types in routine cervical smears was 2.5-12 copies per cell. Of parallel smears taken from patients with a normal Papinacolau-stained smear 33.3% (24) contained a HPV 16, 18, 31, and 33 signal indicating an occult HPV infection. The prevalence of these HPV types was similar in women in whom a cytopathological diagnosis of wart virus infection was made (64.7%, 17) and in patients with mild dyskaryosis (75%, 12). CONCLUSIONS: The methodology evolved localises HPV sequences directly to epithelial cell nuclei, which can be morphologically assessed by haematoxylin counterstaining. Sample contamination with exogenous viral sequences can be distinguished from true infection. In this study, a HPV signal was not found in morphologically normal epithelial cells. The methods described will permit the detection of HPV sequences in routinely collected cervical smears and the evaluation of the natural history and potential clinical relevance of HPV infection without changes in clinical practice

Episomal and integrated human papillomavirus in cervical neoplasia shown by non-isotopic in situ hybridisation

It was postulated that non-isotopic in situ hybridisation (NISH) signal types 1-3 for human papillomavirus in cervical biopsy specimens represent episomal or integrated virus. The aim of this study was to validate this hypothesis by independent molecular techniques. Fresh cervical intraepithelial neoplasia (CIN) and squamous cell cancer (SCC) tissue were examined for NISH signal pattern by hybridising with digoxigenin labelled HPV 16. DNA was extracted from the same samples and analysed by restriction endonuclease digestion and Southern blotting to determine the physical state of the viral genome. Six CIN biopsy specimens showed a type 1 NISH signal for HPV 16. On Southern analysis these biopsy specimens contained only episomal HPV 16. Three SCC with a type 2 NISH signal contained integrated HPV 16 by Southern analysis. Two specimens, a CIN 3 and an SCC with a type 3 NISH signal for HPV 16, showed the presence of both episomal and integrated HPV 16 with conventional Southern analysis and two dimensional gel electrophoresis. These results show that episomal HPV can be reliably determined by NISH type 1 signal, integrated HPV by type 2, and a combination of both episomal and integrated HPV, by a type 3 signal in archival paraffin wax embedded cervical biopsy specimens. This will add another variable to the epidemiological studies of HPV infection. In particular, it will now allow retrospective studies to be done to define the role of episomal and integrated HPV in the evolution of cervical intraepithelial neoplasia and other cervical disease associated with this virus

Simultaneous in situ genotyping and phenotyping of human papillomavirus cervical lesions:comparative sensitivity and specificity

The sensitivity and specificity of immunocytochemistry were compared with those of non-isotopic in situ hybridisation (NISH) for the direct detection of human papillomaviruses in biopsy specimens. Four monoclonal antibodies raised to the capsid protein of HPV16 were less specific than NISH: all four reacted with lesions containing HPV33, and HPV18. Absolute discrimination of HPV types, therefore, was not possible with the monoclonal antibodies used in this study. The relative sensitivities of these antibodies were also lower than NISH. Sequential immunocytochemistry and NISH on the same section showed that 2.9-13.0 times as many cells were positive by NISH than by immunocytochemistry using the most sensitive monoclonal antibody. These data indicate that NISH has higher diagnostic specificity and sensitivity than immunocytochemistry using monoclonal antibodies to the HPV16 capsid protein

Role of human papillomavirus in determining the HLA associated risk of cervical carcinogenesis

AIMS--To investigate the role of human papillomavirus (HPV) in the association between HLA DQw3 and squamous cell cancer of the cervix (SCCC). METHODS--Tissue from 194 cervical samples, ranging from normal, through cervical intraepithelial neoplasia, to SCCC, were typed for HPV by amplification of the L1 gene using degenerate consensus primers, followed by oligonucleotide probing. HLA DQw3 typing was undertaken in the same samples using a new PCR amplification system using primers common to all DQ loci, followed by restriction digestion with Mlu 1 to differentiate HLA DQw3 types--null, heterozygous, and homozygous. The data were analysed using chi 2 analysis and by calculating relative risks with the 95% confidence interval. RESULTS--Samples (n = 188) were successfully typed for HPV and 177 were typed for HLA DQw3. There was a nonsignificant rise in the prevalence of HLA DQw3 in SCCC (64.3%) compared with the group with normal histology (53.2%). Analysis of the prevalence of HLA DQw3 on the basis of HPV infection rather than histology showed that 63 of 95 (66.3%) of the HPV positive samples contained HLA DQw3 alleles, compared with 39 of 78 (50.0%) of the HPV negative samples (chi 2 4.06; p < 0.05). CONCLUSIONS--There was a significant association between HLA DQw3 and cervical HPV infection. This may be because people with HLA DQw3 are less able to mount an effective immune response to HPV, which predisposes them to the development of SCCC