title: Assessment of the presence of mucosal human papillomaviruses in malignant melanomas using combined fluorescent in situ hybridization and chemiluminescent immunohistochemistry.
abstract: 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.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Section of Microbiology, Department of Clinical and Experimental Medicine, University of Bologna, Bologna, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Ambretti
authors.forename: S
authors.initials: S
authors.lastname: Venturoli
authors.forename: M
authors.initials: M
authors.lastname: Mirasoli
authors.forename: M
authors.initials: M
authors.lastname: La Placa
authors.forename: F
authors.initials: F
authors.lastname: Bonvicini
authors.forename: M
authors.initials: M
authors.lastname: Cricca
authors.forename: M
authors.initials: M
authors.lastname: Zerbini
authors.forename: A
authors.initials: A
authors.lastname: Roda
authors.forename: M
authors.initials: M
authors.lastname: Musiani
mesh_terms: D000328:Adult; D000368:Aged; D001706:Biopsy / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods; D017404:In Situ Hybridization, Fluorescence; D008163:Luminescent Measurements; D008297:Male; D008545:Melanoma / Q000821:virology*; D008875:Middle Aged; D009062:Mouth Neoplasms / Q000821:virology*; D009092:Mucous Membrane / Q000821:virology; D027383:Papillomaviridae / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000175:diagnosis*