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.To localize mucosal HR-HPV nucleic acids and tumoural melanocytic marker in the same sections of primary melanoma samples in order to understand the relationship between HPVs and melanoma cells.We have developed a very sensitive method that combines an enzyme-amplified fluorescent in situ hybridization (ISH) for the detection of HPV nucleic acids (types 16 and 18) with a chemiluminescent immunohistochemistry (IHC) method for the detection of the tumoural melanocytic marker HMB-45 sequentially in the same section. Digital images of fluorescent ISH and chemiluminescent IHC were separately recorded, assigned different colours and merged using specific software for image analysis.The combined fluorescent ISH and chemiluminescent IHC demonstrated a sharp colocalization (in the range 60-80%) of HPV nucleic acids and melanoma marker inside the same sections of melanoma biopsies, with a strong specificity and sensitivity.The strong colocalization of mucosal HR-HPV nucleic acids and HMB-45 melanocytic marker emphasized that viral nucleic acids were specifically present in melanoma cells and supported a possible active role of HPV in malignant melanoma.
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.To investigate the presence of HPV DNA and the prevalence of different high-risk mucosal HPV genotypes in primary melanoma (PM) and in acquired dysplastic melanocytic naevi (ADMN).Fifty-one PMs from 18 men and 33 women (median age 55.5 years), 33 ADMN from 15 men and 18 women (median age 35.1 years) and 20 control skin samples from nine men and 11 women (median age 43.5 years) were studied. All diagnoses were made after histological analysis. HPV DNA analysis was made using two different polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) methods, namely MY-PCR and GP-PCR.Using GP-PCR, mucosal HPVs were detected in 14 PMs (27%; P = 0.0166) and eight ADMN (24%; P = 0.0367), while with MY-PCR, mucosal HPVs were found in 11 PMs (22%; P = 0.04) and five ADMN (15%; P not significant). All control skin samples were negative for mucosal HPVs with both DNA amplification procedures.Using our PCR-ELISA methods, the detection of mucosal high-risk HPV genotypes in 24% of precursor lesions (ADMN) and in 27% of PMs adds to the body of evidence indicating a colocalization of mucosal HPV and tumoral melanocytic pathologies.
An enzyme linked immunosorbent assay (ELISA) for detecting antibodies against cytomegalovirus induced immediate early antigens and early antigens was developed using purified nuclear antigens and was compared with the indirect immunofluorescence test. The tests were comparable in their ability to detect positive and negative sera, and antibody titres determined by both assays were similar. The use of ELISA for the detection of antibodies against cytomegalovirus induced immediate early and early antigens is advocated in diagnostic and research laboratories.
