title: Combining routine morphology, p16(INK4a) immunohistochemistry, and in situ hybridization for the detection of human papillomavirus infection in penile carcinomas: a tissue microarray study using classifier performance analyses.
abstract: OBJECTIVE
  Infection by high-risk human papillomavirus (HR-HPV) plays an important role in the pathogenesis of penile cancer in approximately 50% of the patients. The gold standard for human papillomavirus (HPV) detection is the polymerase chain reaction (PCR) assay. However, technical requirements and associated costs preclude the worldwide use of PCR assays on a routine basis. Herein, we evaluated the predictive abilities of tumor morphology, immunohistochemistry for p16(INK4a) expression, and in situ hybridization (ISH) for HR-HPV detection in defining HPV status, as established by PCR.
  
  
  METHODS
  Tissue samples from 48 patients with HPV-positive penile squamous cell carcinoma (SCC) were included in 4 tissue microarrays (TMA).
  
  
  RESULTS
  Sensitivities and specificities were as follows: tumor morphology, 70% and 68%; p16(INK4a) immunohistochemistry, 65% and 90%; HR-HPV ISH, 47% and 100%. Regarding combinations of the predictors, the best performance was seen when HR-HPV ISH and p16(INK4a) immunohistochemistry were combined, regardless of the tumor morphology: sensitivity, 88%; specificity, 64%; area under the receiver-operating characteristic (AUC) curve, 0.83. Combinations of tumor morphology with p16(INK4a) immunohistochemistry or with HR-HPV ISH performed similarly well.
  
  
  CONCLUSIONS
  In penile SCC, both p16(INK4a) immunohistochemistry and ISH for HR-HPV increase the predictive ability of routine morphology in defining HPV status. These tests can be interpreted differentially, depending on the necessity of a higher sensitivity or a higher specificity. For research/screening studies, we recommend combining tumor morphology, p16(INK4a) immunohistochemistry, and HR-HPV ISH. To increase sensitivity, positivity in any of these predictors should be considered as indicative of HPV infection. For routine diagnosis of clinical cases, criteria should be more stringent, and, to achieve the highest specificity in classifying a case as HPV-related, all predictors should be consistently positive. The data generated in the present study could be used in algorithms for defining HPV status in penile carcinomas.
authors.affiliation: Department of Pathology, the Johns Hopkins University School of Medicine, Baltimore, MD; Norte University School of Medicine, Asuncion, Paraguay. Electronic address: alcideschaux@uninorte.edu.py.
authors.forename: Alcides
authors.initials: A
authors.lastname: Chaux
authors.affiliation: Instituto de Patología e Investigación, Asuncion, Paraguay.
authors.forename: Antonio L
authors.initials: AL
authors.lastname: Cubilla
authors.affiliation: Department Oncology, the Johns Hopkins University School of Medicine, Baltimore, MD.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Haffner
authors.affiliation: Department of Pathology, the Johns Hopkins University School of Medicine, Baltimore, MD.
authors.forename: Kristen L
authors.initials: KL
authors.lastname: Lecksell
authors.affiliation: Department of Pathology, the Johns Hopkins University School of Medicine, Baltimore, MD.
authors.forename: Rajni
authors.initials: R
authors.lastname: Sharma
authors.affiliation: Department Urology, the Johns Hopkins University School of Medicine, Baltimore, MD.
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Burnett
authors.affiliation: Department of Pathology, the Johns Hopkins University School of Medicine, Baltimore, MD; Department Urology, the Johns Hopkins University School of Medicine, Baltimore, MD; Department Oncology, the Johns Hopkins University School of Medicine, Baltimore, MD.
authors.forename: George J
authors.initials: GJ
authors.lastname: Netto
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000175:diagnosis* / Q000378:metabolism / Q000821:virology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis*; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008297:Male; D027383:Papillomaviridae / Q000235:genetics*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000378:metabolism / Q000821:virology; D010412:Penile Neoplasms / Q000175:diagnosis* / Q000378:metabolism / Q000821:virology; D011379:Prognosis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D046888:Tissue Array Analysis / Q000379:methods
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16
keywords: Human papillomavirus; In situ hybridization; Penile cancer; Sensitivity and specificity; Squamous cell carcinoma; p16(INK4a)