Loss of p16 Expression Has Prognostic Significance in Human Nasopharyngeal Carcinoma 1 Antti A. Ma ¨kitie, Christina MacMillan, James Ho, Wei Shi, Andrew Lee, Brian O’Sullivan, David Payne, Melania Pintilie, Bernard Cummings, John Waldron, Padraig Warde, Jonathan Irish, Dale Brown, Ralph Gilbert, Patrick Gullane, Fei-Fei Liu, 2 and Suzanne Kamel-Reid Departments of Otolaryngology and Surgical Oncology [A. A. M., J. I., D. B., R. G., P. G.], Radiation Oncology [B. O., D. P., B. C., J. W., P. W., F-F. L.], Molecular and Cellular Biology [A. A. M., S. K-R.], Experimental Therapeutics [W. S., A. L., F-F. L.], Pathology [C. M., J. H., S. K-R.], Biostatistics [M. P.], Princess Margaret Hospital/Ontario Cancer Institute, Toronto M5G 2M9, and University Health Network, Departments of Otolaryngology and Surgical Oncology [J. I., D. B., R. G., P. G., S. K-R.], Medical Biophysics [W. S., A. L., F-F. L., S. K-R.], Pathobiology and Laboratory Medicine [C. M., S. K-R.], and Radiation Oncology [B. O., D. P., B. C., J. W., P. W., F-F. L.], University of Toronto, Toronto, Ontario, Canada ABSTRACT Purpose: p16 is an important inhibitor of cell cycle progression; absence of p16 can thus result in increased cellular proliferation. In nasopharyngeal carcinoma (NPC), absence of p16 has been reported in association with pres- ence of the EBV and pRb. We therefore examined p16, pRb, and EBV-encoded RNA (EBER) expression in biopsy spec- imens from 84 patients with newly diagnosed NPC, who were treated primarily with curative radiation therapy. Our objective was to determine whether there was a correlation between these parameters and clinical outcome in NPC. Experimental Design: Sections were cut from archival formalin-fixed, paraffin-embedded tumor blocks from NPC patients. p16 and pRb expression were determined using polyclonal and monoclonal antibodies, respectively. The presence of EBV was determined by in situ hybridization for EBER. The percentage of positively staining tumor nuclei was scored for p16 or pRb immunoreactivity. Relative in- tensity and proportion of cells with EBER signals were also documented. Results: p16 expression was reduced (<5% positive immunoexpression) in 59 of 84 (70%) NPC samples; in contrast, pRb was observed in all (100%) tumors. EBER signals were detected in 67 of 83 (81%) NPC specimens. There was a weak correlation between EBER presence and loss of p16 (P  0.1). Using a Cox regression model control- ling for known prognostic parameters, such as age, weight loss, and tumor stage, complete absence of p16 expression (0%, i.e., no immunostaining identified throughout the spec- imen) was associated with an inferior overall survival rate (P  0.022). In addition, EBER-positive NPC was strongly associated with improved overall survival (P  0.005) as reported previously (Shi et al., Cancer, 94: 1997, 2002). Conclusion: These results provide the first evidence suggesting that inactivation of p16 appears to be a signifi- cant predictor for poor overall survival in NPC. Given that reduced p16 expression is observed in the majority of pa- tients with NPC, this indicates that therapeutic strategies targeting the p16 pathway may be a biologically rational approach for NPC. The favorable prognostic value of EBER suggests that future clinical trials with NPC should consider stratifying for EBER status. INTRODUCTION NPC 3 is distinct from other epithelial malignancies in the head and neck region in that it affects a relatively young pop- ulation and has a predilection for developing distant metastases (1). It has unique epidemiological characteristics, such as geo- graphic distribution in Southeast Asia or the Mediterranean basin. In addition, its intimate association with the EBV is well established. Its anatomical location with close proximity to the skull base renders it a significant challenge for effective delivery of modalities, such as RT. With conventional RT, the overall 5-year survival rate hovers 70% (2, 3), potentially related to inadequate coverage of the tumor volume (4). With the advent of intensity-modulated RT, the 4-year local control rate is su- perior, but the metastasis rate remains high at 40% (2, 5). The role of Cis-platinum-based chemotherapy combined with RT remains controversial (3, 6 –9), underscoring an urgent need to develop novel therapeutic strategies to improve outcome. To design novel therapies rationally, a thorough under- standing of the behavior of NPC is essential. Information de- Received 12/9/02; accepted 2/11/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by funds from the Canadian Institutes of Health Research and the National Cancer Institute of Canada, Pathology As- sociates Research Fund (Department of Pathology, the University Health Network), Finska La ¨karesa ¨llskapet and Meritza and Reino Sa- lonen Foundation (Finland), and Elia Chair in Head and Neck Cancer Research. 2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, Princess Margaret Hospital/Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario, M5G 2M9. Phone: (416) 946-2123; Fax: (416) 946-4586; E-mail: Fei-Fei.Liu@rmp. uhn.on.ca. 3 The abbreviations used are: NPC, nasopharyngeal carcinoma; RT, radiation therapy; ISH, in situ hybridization; LMP, latent membrane protein; DFS, disease-free survival; OS, overall survival; EBER, EBV- encoded RNA. 2177 Vol. 9, 2177–2184, June 2003 Clinical Cancer Research