Marked differences in survival rate between smokers and nonsmokers with HPV 16-associated tonsillar carcinomas Harri€ et C. Hafkamp 1 , J.J. Manni 1 , A. Haesevoets 2 , A.C. Voogd 3 , M. Schepers 2 , F.J. Bot 4 , A.H.N. Hopman 2 , F.C.S. Ramaekers 2 and Ernst-Jan M. Speel 2,4 * 1 Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Maastricht, Maastricht, The Netherlands 2 Department of Molecular Cell Biology, GROW—School for Oncology and Developmental Biology, University of Maastricht, Maastricht, The Netherlands 3 Department of Epidemiology, University of Maastricht, Maastricht, The Netherlands 4 Department of Pathology, University Hospital Maastricht, Maastricht, The Netherlands Oncogenic human papillomavirus (HPV) is a causative agent in a subgroup of head and neck carcinomas, particularly tonsillar squamous cell carcinomas (TSCC). This study was undertaken because controversial data exist on the physical status of HPV- DNA and the use of p16 INK4A overexpression as surrogate HPV marker, and to examine the impact of HPV and tobacco consump- tion on the clinical course of TSCC. Tissue sections of 81 TSCC were analyzed by HPV 16-specific fluorescence in situ hybridiza- tion (FISH) and p16 INK4A -specific immunohistochemistry. Results were correlated with clinical and demographic data. HPV 16 inte- gration was detected by FISH as punctate signals in 33 out of 81 (41%) TSCC, 32 of which showed p16 INK4A accumulation. Only 5 out of 48 HPV-negative tumors showed p16 INK4A immunostaining (p < 0.0001). The presence of HPV furthermore correlates signifi- cantly with low tobacco (p 5 0.002) and alcohol intake (p 5 0.011), poor differentiation grade (p 5 0.019), small tumor size (p 5 0.024), presence of a local metastasis (p 5 0.001) and a decreased (loco)regional recurrence rate (p 5 0.039). Statistical analysis revealed that smoking significantly increases the risk of cancer death from TSCC and that non-smoking patients with HPV-containing TSCC show a remarkably better disease-specific survival rate. HPV 16 is integrated in 41% of TSCC and strongly correlates with p16 INK4A overexpression, implicating the latter to be a reliable HPV biomarker. Patients with HPV-positive tumors show a favorable prognosis as compared to those with HPV-nega- tive tumors, but tobacco use is the strongest prognostic indicator. These findings indicate that oncogenic processes in the tonsils of non-smokers differ from those occurring in smokers, the former being related to HPV 16 infection. ' 2008 Wiley-Liss, Inc. Key words: human papillomavirus; p16 INK4A ; tonsillar carcinomas; FISH; viral integration; survival analysis Head and neck squamous cell carcinomas (HNSCC) account for 4% of all malignancies in the Western world, for up to 50% of all malignancies in Southeast Asian countries and for 6.5% of all annual cancer cases worldwide. 1 HNSCC is associated with severe disease- and treatment-related morbidity and because treatment has not improved greatly in recent years, the 5-year survival rate remains 50%. HNSCC develop in various anatomical defined regions, including the oral cavity, larynx and pharynx. These organ-specific tumors each show specific clinical presentations and outcome, and are treated by different strategies. 2,3 The median age at presentation is 60 years and approximately two-third of patients are male. 1 Well-known risk factors in the etiology of HNSCC are cigarette smoking combined with alcohol consumption in Western coun- tries, or with betel quid chewing in Asia. A history of tobacco use is present in 90% of patients who develop oral cavity cancers. 2,3 Despite these evident associations, the exact mechanisms by which these factors cause tumor initiation and progression are not fully understood. Furthermore, the fact that most tobacco and alcohol users do not develop HNSCC and that in recent years more often individuals without a history of these traditional risk factors have been witnessed, 4 underlines the complexity of HNSCC pathogenesis and a role for additional factors in the dis- ease process. Increasing evidence suggests that human oncogenic papilloma- viruses (HPVs), known to cause uterine cervical and other anogen- ital cancers, may also be of importance in the pathogenesis of HNSCC. 5 The strongest association has been found for oropharyn- geal carcinomas, especially tonsillar carcinomas. 6–11 Sero-positive patients for HPV 16 or with a history of HPV-related anogenital cancer also show increased risk rates of developing oropharyngeal cancer. 12,13 The prevalence of HPV-exhibiting HNSCC, however, varies broadly amongst several studies (2–76%) due to differences in the population, combination of histological subsites, type and number of specimens analyzed, and detection methods used. 7,14 Thus, besides determining the presence of HPV DNA it has been suggested to better define the biological association of oncogenic HPV with these tumors, e.g., by means of assessing the viral copy number per cell, the viral oncoprotein E6/E7 expression levels, perturbation of pRb-dependent cell cycle control, or the physical status of the virus (episomal or integrated). 15 In this way, several reports have shown that HPV 16 is predominantly identified in oropharyngeal carcinomas, with a frequency of 50–70%. 9,16–18 Integration of high-risk HPV DNA, such as HPV 16 DNA, into the human cellular genome is considered an important step in ma- lignant transformation. 19 From studies on lesions of the uterine cervix it has become clear that viral integration marks the transi- tion from a dysplastic lesion to (micro)invasive cancer. 20,21 After integration and disruption of (part of) the viral early gene E2, an upregulation of the oncoproteins E6 and E7 is detected. 7,19 On the one hand the E6 protein interacts specifically with the host-cell tumor suppressor protein p53 and induces its degradation. The subsequent inability to inhibit cell growth and induce apoptosis results in genetic instability. P53 mutations are therefore not a pre- requisite in HPV-related tumorigenesis, and are therefore sel- domly identified. 8,9,18 On the other hand, the E7 protein inacti- vates the retinoblastoma tumor suppressor protein pRb, resulting in release of the transcription factor E2F and upregulation of p14 ARF and p16 INK4A . 22,23 In oropharyngeal and especially tonsil- lar carcinomas, however, the literature is controversial with respect to viral integration. Results range from virus being present only in an episomal form to 100% viral integration, with or with- out concurrent episomal HPV. 18,24,25 Also immunohistochemical detection of p16 INK4A overexpression, which has been postulated as a fast, easy and less expensive alternative for the detection of Grant sponsors: the Medical Research Foundation ‘‘Profileringsfonds’’ and the Research Foundation of the ENT Department, University Hospital Maastricht, Maastricht, The Netherlands; Grant number: PF126. Harri€ et C. Hafkamp’s current address: Department of Otorhinolaryngo- logy (HCH), Reinier de Graaf hospital, Delft, The Netherlands. *Correspondence to: Department of Molecular Cell Biology, UNS50- 17, University of Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Fax: 131-43-388-4151. E-mail: ernstjan.speel@molcelb.unimaas.nl Received 29 July 2007; Accepted after revision 12 December 2007 DOI 10.1002/ijc.23458 Published online 21 March 2008 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 122, 2656–2664 (2008) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer