Summary
The parameter Tmt has been defined by non-isotopic in situ hybridization and describes the tissue melting temperature (Tmt) of human papillomavirus (HPV) DNA sequences. In this study, multiple in situ hybridization signals for HPV types 16, 31 and 33 in individual archival biopsies hybridized with genomic probes are shown by polymerase chain reactions to be due to cross-hybridization of probe sequences to a single tissue target. Tmt is independent of viral type but depends on the homology between probe and target when using nick-translated whole genomic probes. The difference between Tm and Tmt is not due to the presence of viral capsid protein. Multiple HPV signals in archival material should not therefore be interpreted as indicative of multiple HPV infection unless adequate stringency conditions have been employed or they are present in morphologically distinct areas of the biopsy.
Furthermore, extrapolation of calculated DNA homologies to non-isotopic in situ hybridization analysis may not be appropriate. A hybridization signal does not imply probe and target identity: this has implications for HPV typing in clinical material.
Similar content being viewed by others
References
Anderson, M. L. M. & Young, B. D. (1985) Quantitative filter hybridisation. In Nucleic Acid Hybridisation: a Practical Approach (edited by Higgins, S. & Hames, B. D.), pp. 73–111. Oxford: Oxford University Press.
Bauer, H. M., Ting, Y., Greer, C. E., Chambers, J. C., Tashiro, C. J., Chimera, J., Reingold, A. & Manos, M. M. (1991) Genital human papillomavirus infection in female university students as determined by a PCR-based method. J. Amer. Med. Assoc. 265, 472–7.
Bauer, H. M., Greer, C. & Manos, M. M. (1992) Determination of genital human papillomavirus infection by consensus polymerase chain reaction amplification. In Diagnostic Molecular Pathology: a Practical Approach. Vol 2, pp. 131–52 (edited by Herrington, C. S. & McGee, J.O'D.). Oxford: Oxford University Press.
Coleman, D. V. & Evans, D. M. D. (1988) Biopsy Pathology and Cytology of the Cervix. London: Chapman & Hall.
Cooper, K., Herrington, C. S., Graham, A. K., Evans, M. F. & McGee, J. O'D. (1991a) In situ human papillomavirus (HPV) genotyping of cervical intraepithelial neoplasia in South African and British patients: evidence for putative HPV integration in vivo. J. Clin. Pathol. 44, 400–5.
Cooper, K., Herrington, C. S., Graham, A. K., Evans, M. F. & McGee, J. O'D. (1991b) In situ evidence for HPV 16, 18, 33 integration in cervical squamous cell cancer in Britain and South Africa. J. Clin. Pathol. 44, 406–9.
Cooper, K., Herrington, C. S., Stickland, J. E., Evans, M. F. & McGee, J. O'D. (1991c) Episomal and integrated HPV in cervical neoplasia demonstrated by nonisotopic in situ hybridization. J. Clin. Pathol. 44, 990–6.
Dartmann, K., Schwarz, E., Gissmann, L. & Zur Hausen, H. (1986) The nucleotide sequence and genome organisation of human papilloma virus type 11. Virology 151, 124–30.
Goldsborough, M. D., Di-Silvestre, D., Temple, G. F. & Lorincz, A. T. (1989) Nucleotide sequence of human papillomavirus type 31, a cervical neoplasia-associated virus. Virology 171, 306–11.
Graham, A. K., Herrington, C. S. & McGee, J. O'D. (1991) Simultaneous in situ genotyping and phenotyping of HPV cervical lesions: comparative sensitivity and specificity. J. Clin. Pathol. 44, 96–101.
Greer, C. E., Peterson, H. T., Kiviat, N. B. & Manos, M. M. (1991) PCR amplification from paraffin embedded tissues. Am. J. Clin. Pathol. 95, 116–24.
Gupta, J. W., Saito, K., Saito, A., Fu, Y. S. & Shah, K. (1989) Human papillomaviruses and the pathogenesis of cervical neoplasia. Cancer 64, 2104–10.
Herrington, C. S. & McGee, J. O'D. (1992) Nonisotopic in situ hybridization on cells and tissues: use of biotin and digoxigenin labelled probes. In Methods in Molecular Biology (edited by Manson, M.), Vol. 10, pp. 409–19. Clifton: Humana Press.
Herrington, C. S., Burns, J., Graham, A. K., Evans, M. F. & McGee, J. O'D. (1989a) Interphase cytogenetics using biotin and digoxigenin labelled probes I: relative sensitivity of both reporters for detection of HPV16 in CaSki cells. J. Clin. Pathol. 42, 592–600.
Herrington, C. S., Burns, J., Graham, A. K., Bhatt, B. & McGee, J. O'D. (1989b) Interphase cytogenetics using biotin and digoxigenin labelled probes II: simultaneous detection of two nucleic acid species in individual nuclei. J. Clin. Pathol. 42, 601–6.
Herrington, C. S., Burns, J., Graham, A. K. & McGee, J. O'D. (1990) Discrimination of closely homologous HPV types by in situ hybridization: definition and derivation of Tmts. Histochem. J. 22, 545–54.
Herrington, S. C., Graham, A. K. & McGee, J. O'D. (1991) Interphase cytogenetics using biotin and digoxigenin labelled probes: III. Increased sensitivity and flexibility for detecting HPV in cervical biopsy specimens and cell lines. J. Clin. Pathol. 44, 33–8.
Patel, D., Shepherd, P. S., Naylor, J. A. & McCance, D. J. (1989) Reactivities of polyclonal and monoclonal antibodies raised to the major capsid protein of human papillomavirus type 16. J. Gen. Virol. 70, 69–77.
Schwarz, E., Dürst, M., Demankowski, C., Lattermann, O., Zech, R., Wolfsperger, E., Suhai, S. & Zur Hausen, H. (1983) DNA sequence and genome organisation of genital human papillomavirus type 6b. EMBO J. 2, 2341–8.
Seedorf, K., Krammer, G., Dürst, M., Suhai, S. & Rowekamp, W. G. (1985) Human papillomavirus type 16 DNA sequence. Virology 145, 181–5.
Ting, Y. & Manos, M. M. (1990) Detection and typing of genital human papillomaviruses. In PCR Protocols: a Guide to Methods and Applications (edited by Innis, M. A., Gelfand, D. H., Sninsky, J. J. & White, T. J.), pp. 356–67. San Diego: Academic Press.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Herrington, C.S., Anderson, S.M., Graham, A.K. et al. The discrimination of high-risk HPV types by in situ hybridization and the polymerase chain reaction. Histochem J 25, 191–198 (1993). https://doi.org/10.1007/BF00163814
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00163814