F. Bonvicini

Presence of high-risk mucosal human papillomavirus genotypes in primary melanoma and in acquired dysplastic melanocytic naevi

Summary  Background  Some studies have shown that cutaneous and mucosal melanoma biopsy specimens harbour human papillomavirus (HPV), suggesting that this virus may play a role in development and progression of the tumour.

PNA-based probe for quantitative chemiluminescent in situ hybridisation imaging of cellular parvovirus B19 replication kinetics

To allow the ultrasensitive localization and the quantitative detection of parvovirus B19 nucleic acids in single infected cells at various times post-infection, a peptide nucleic acid (PNA)-based in situ hybridisation (ISH) assay with chemiluminescent detection has been developed. The assay is based on the use of a biotin-labelled PNA probe detected by a streptavidin-linked alkaline phosphatase and a chemiluminescent dioxetane phosphate derivative substrate. The luminescent signal was quantified and imaged with an ultrasensitive nitrogen-cooled CCD camera connected to an epifluorescence microscope. The assay was used to analyze the parvovirus B19 infection process in cell cultures and to quantify the amount of viral nucleic acids at different times after infection. The chemiluminescent ISH-PNA assay is characterized by high resolution providing a sharp localization of B19 nucleic acids within single cells, with higher sensitivity with respect to conventional colorimetric ISH detection. Thanks to the high detectability and wide linear range of chemiluminescence detection, an objective evaluation of the percentage of infected cells, which reached its maximum at 24 h after infection, following a B19 virus infectious cycle could be accurately evaluated. Chemiluminescence detection also allowed the quantitative analysis of viral nucleic acids at the single-cell level, showing a continuous increase of the content of viral nucleic acids in infected cells with time after infection. The developed chemiluminescent ISH-PNA assay could thus represent a potent tool for the assessment of viral infections and for the quantitative evaluation of the virus nucleic acid load of infected cells in virus studies and diagnostics.

Assessment of the presence of mucosal human papillomaviruses in malignant melanomas using combined fluorescent in situ hybridization and chemiluminescent immunohistochemistry

Background  The vast majority of studies aimed at detecting human papillomavirus (HPV) DNA in skin cancer have used sensitive polymerase chain reaction (PCR) methods but the PCR technique, despite its high sensitivity, is not suitable to ascertain whether (i) the presence of HPV can be related only to few cells harbouring the virus, (ii) the presence of HPV is due to a tumour surface contamination and (iii) the presence of HPV is localized in cancer cells, rather than in normal keratinocytes present in the tumour biopsy. In a recent work we have found mucosal high‐risk (HR) HPV genotypes in primary melanoma by PCR.

Efficient treatment of paraffin-embedded cervical tissue for HPV DNA testing by HC-II and PCR assays

BACKGROUND AND OBJECTIVES High-risk human papillomaviruses (HR-HPVs) are the primary cause of cervical cancer. In order to meet with clinical requirements, a direct capture test with signal amplification (HC-II), able to detect the 13 prevalent HR-HPVs, has been developed and validated for cytological specimens. STUDY DESIGN the use of HC-II assay with formaldehyde-fixed paraffin-embedded cervical biopsies, for retrospective studies or to support histological findings, was investigated by analysing three different sample treatments. The efficacy of this test was compared with a reference PCR-ELISA, using MY09/11 and GP5+/6+ consensus primers and the use of a single extraction method for both HC-II and PCR-ELISA assays was validated. RESULTS protease treatment of dewaxed biopsy sections allowed an optimal performance of HC-II and has also been validated for PCR-ELISA. Overall, on the analysis of 50 cervical samples HC-II and PCR-ELISA assays showed a high concordance (K=0.80). Compared with PCR-ELISA, the HC-II had a sensitivity of 86.4% and a specificity of 92.9%. The results showed that short amplimers are necessary for a sensitive PCR-ELISA from formaldehyde-fixed paraffin-embedded biopsies, while HC-II showed a relatively low sensitivity for HPV18 within the HR probe pool. CONCLUSIONS HC-II can be a valid tool for the diagnosis of HPV infection in biopsy material. The possibility to use the same specimen preparation material for both HC-II and PCR-ELISA allows HC-II positive specimens to be further processed by PCR-ELISA if specific genotyping is needed.

Human papillomavirus in melanoma: reply from authors

SIR, We are grateful to Dr Guerrini and colleagues for their interest in our study and we take this opportunity to clarify our opinions. We demonstrated the presence of high-risk mucosal human papillomavirus (HPV) genotypes from a considerable number of dysplastic naevi and primary melanomas using two different polymerase chain reaction (PCR)–enzyme-linked immunosorbent assay methods, concluding that the presence of the virus may be a cofactor in development and progression of these pathologies. We agree with other colleagues that our results need further evaluation. In particular, PCR is a highly sensitive technique, able to detect the presence of total DNA from tissue samples at a level of about one genome per cell. All previous studies aimed to detect HPV DNA in skin cancer using PCR procedures were in most cases unable to conclude that the DNA detected was entirely derived from cancer cells. In fact, PCR procedures are not suitable to ascertain if the HPV DNA detected is derived exclusively from cancer cells, but rather if its presence is due to a tumour surface contamination. On the other hand, different methods, including immunohistochemistry (IHC) and in situ hybridization (ISH), have been demonstrated as more specific, but less sensitive than PCR methods. On the basis of these considerations, we are submitting for publication a second part of our studies, concerning the evaluation of the presence of mucosal high-risk HPV in primary melanoma and its colocalization with a tumoral melanocytic marker in the same section, using a very sensitive method that combines an enzyme-amplified fluorescent ISH with a chemiluminescent IHC method for the detection of the tumoral melanocytic marker HMB-45. The antimelanoma monoclonal antibody HMB-45 used in the chemiluminescent IHC is widely used in diagnostic pathology owing to its great specificity and sensitivity in identifying pigmented tumours such as malignant melanoma, while normal melanocytes are unreactive.