title: Laboratory-developed L1 sequencing and type-specific, real-time polymerase chain reaction for the detection and typing of human papillomaviruses in formalin-fixed, paraffin-embedded tissues.
abstract: BACKGROUND
  The detection and typing of high-risk and low-risk human papillomavirus (HPV) in archival formalin-fixed, paraffin-embedded tissues by nucleic acid amplification testing is an important adjunct to immunohistochemical staining in evaluation of squamous cell proliferations of the oropharynx, larynx, and anal canal.
  
  
  OBJECTIVE
  To evaluate semiautomated, xylene-free extraction from formalin-fixed, paraffin-embedded tissues combined with laboratory-developed HPV L1 sequencing and type-specific HPV 6, 11, 16, and 18 real-time polymerase chain reaction for identification and typing of HPV in the clinical laboratory.
  
  
  METHODS
  We evaluated the adequacy of extraction using β-globin amplification and compared L1 sequencing and real-time polymerase chain reaction methods for typing accuracy using 68 formalin-fixed, paraffin-embedded tissues, including 56 anorectal biopsy or surgical resection specimens and 12 laryngeal papilloma specimens from patients with recurrent respiratory papillomatosis.
  
  
  RESULTS
  Adequate DNA was obtained from 68 of 68 specimens analyzed and all were HPV positive. In 47 cases where L1 sequencing demonstrated that the predominant HPV type was 6, 11, 16, or 18, type-specific, real-time polymerase chain reaction provided concordant results. Sequencing revealed additional low-risk (HPV 40) and high-risk HPV types (HPV 31, 33, 56, and 58) in anorectal specimens, whereas HPV 6 or 11 were the types found in laryngeal papillomas.
  
  
  CONCLUSIONS
  Both L1 sequencing and type-specific, real-time polymerase chain reaction are suitable methods for routine HPV testing of formalin-fixed, paraffin-embedded tissues in a clinical laboratory setting.
authors.affiliation: Department of Pathology, Division of Infectious Diseases, Stanford University School of Medicine, Palo Alto, California 94304, USA.
authors.forename: Anne
authors.initials: A
authors.lastname: Mills
authors.forename: Rajeev
authors.initials: R
authors.lastname: Balasubramaniam
authors.forename: Teri A
authors.initials: TA
authors.lastname: Longacre
authors.forename: Christina S
authors.initials: CS
authors.lastname: Kong
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Pinsky
mesh_terms: D052685:Alphapapillomavirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D001004:Anus Diseases / Q000175:diagnosis / Q000821:virology; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics*; D003218:Condylomata Acuminata / Q000175:diagnosis / Q000821:virology; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D005557:Formaldehyde; D052140:Human papillomavirus 11 / Q000235:genetics / Q000302:isolation & purification; D052162:Human papillomavirus 16 / Q000235:genetics / Q000302:isolation & purification; D052161:Human papillomavirus 18 / Q000235:genetics / Q000302:isolation & purification; D052163:Human papillomavirus 6 / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007822:Laryngeal Neoplasms / Q000175:diagnosis / Q000821:virology; D008297:Male; D008828:Microbiological Techniques / Q000379:methods; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D010212:Papilloma / Q000175:diagnosis / Q000821:virology; D030361:Papillomavirus Infections / Q000175:diagnosis / Q000821:virology; D016612:Paraffin Embedding; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012002:Rectal Diseases / Q000175:diagnosis / Q000821:virology; D016707:Tissue Fixation
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; C114580:HPV L1 protein, Human papillomavirus; D009856:Oncogene Proteins, Viral; D005557:Formaldehyde