HPV DNA, E6  I-mRNA expression and p16 INK4A immunohistochemistry in head and neck cancer – How valid is p16 INK4A as surrogate marker? Markus Hoffmann a,⇑ , Silke Tribius b , Elgar Susanne Quabius c,d , Hannes Henry a , Saskia Pfannenschmidt a , Claudia Burkhardt a , Tibor Görögh a , Gordana Halec e , Anna Sophie Hoffmann f , Tomas Kahn g , Christoph Röcken h , Jochen Haag h , Tim Waterboer e , Markus Schmitt e a Department of Otorhinolaryngology, Head and Neck Surgery, Christian-Albrechts-University Kiel, Arnold-Heller-Straße 3, House 27, 24105 Kiel, Germany b Department of Radiation Oncology, University Medical Center Hamburg–Eppendorf, D-Martinistr. 52, 20246 Hamburg, Germany c Department of Prosthodontics, Propaedeutics and Dental Materials, Christian-Albrechts-University Kiel, Arnold-Heller-Straße 3, 24105 Kiel, Germany d Institute of Immunology, Christian-Albrechts-University Kiel, Arnold-Heller-Straße 3, 24105 Kiel, Germany e German Cancer Research Center (DKFZ), Infection and Cancer Program (F020), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany f Department of Otorhinolaryngology, University of Schleswig-Holstein, Campus Luebeck, Ratzeburger Allee 160, D-23538 Luebeck, Germany g Expert Team Life Sciences, Deutsche Bank AG, Große Gallusstr. 10-14, D-60311 Frankfurt, Germany h Institute for Pathology, Christian-Albrechts-University Kiel, Arnold-Heller-Straße 3, 24105 Kiel, Germany article info Article history: Received 23 January 2012 Received in revised form 5 March 2012 Accepted 29 March 2012 Keywords: BSGP5+/6+-PCR–MPG Multiplex HPV genotyping Human papillomavirus HPV E6mRNA Head and neck cancer HNSCC p16 Viral load abstract It has been proposed that p16 INK4A qualifies as a surrogate marker for viral oncogene activity in head and neck cancer (HNSCC). By analyzing 78 HNSCC we sought to validate the accuracy of p16 INK4A as a reliable marker of active HPV infections in HNSCC. To this end we determined HPV DNA (HPVD) and E6  I mRNA (HPVR) expression status and correlated these results with p16 INK4A staining. In tonsillar SCC 12/20 were HPVD+ and 12/12 of these showed active HPV infections whereas in non-tonsillar SCC 10/58 were HPVD+ and 5/10 showed active HPV infections. Thus, we prove about 8% of non-tonsillar SCC to be also correlated with HPV-associated carcinogenesis. Strikingly, 3/14 (21.4%) of tonsillar and non-tonsillar HPVD+/HPVR+ cases did not show p16 INK4A overexpression and these cases would have been missed when applying ini- tial p16 INK4A staining only. However, in 13 cases negative for HPV, DNA p16 INK4A was overexpressed. In conclusion, our data confirm tonsillar SCC to be predominantly but not only associated with active HPV infections. Furthermore, our data show that p16 INK4A overexpression is not evident in a subgroup of HNSCC with active HPV infection. Definitive HPV data should therefore be utilized in diagnostics and treatment modalities of HPV positive and HPV negative HNSCC patients, resulting in a paradigm shift regarding these obviously different tumor entities. Ó 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Head and neck squamous cell carcinoma (HNSCC) arises in the oral cavity, oropharynx, larynx or hypopharynx and is the sixth leading cancer incidence worldwide representing an estimated 5% of all cancers [1]. It is likely that approximately 570,000 cases will arise each year worldwide and that only 40–50% of patients will survive for 5 years [2,3]. Besides tobacco and alcohol consumption [4], the etiological involvement of human papillom- aviruses (HPVs), specifically HPV genotype 16 [5,6], in the carcino- genesis of HNSCC has proven to be of significant importance in both initiation and progression of these tumors [7]. Starting from first reports showing that HPV plays a role not only in the anogen- ital but also in the upper aerodigestive tract [8–10] there has been a continuous increase in knowledge on HPV prevalence, socioepi- demiological characteristics of HPV positive vs. HPV negative pa- tients, and impact on recurrence free and overall survival of HPV positive vs. HPV negative cases in head and neck cancer [11–15]. Epidemiologic data show HPV DNA prevalences of about 25–35% concerning all anatomical tumor sites of the head and neck region [5,6,16], with the SCC of the Waldeyer’s tonsillar (TSCC) ring showing a HPV DNA positive rate of up to 60% as particularly pre- destined for an infection with HPV [11,17]. However, there still is an ongoing debate on the true prevalence rate of HPV infections that can be expected in different anatomical sites of HNSCC, a de- bate that is stimulated by diverging reports on the issue until now: While data from Sweden describe HPV DNA infections in tonsillar carcinomas in even up to 85% of the investigated patients and while there is accumulating data supporting a general increase of HPV associated HNSCC, specifically of the oropharynx [18–20], Ribeiro and coworkers report low HPV prevalences in head and 0304-3835/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2012.03.033 ⇑ Corresponding author. Tel.: +49 (0)431 597 2307; fax: +49 (0)431 597 2272. E-mail address: mhoffmann@hno.uni-kiel.de (M. Hoffmann). Cancer Letters 323 (2012) 88–96 Contents lists available at SciVerse ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet