Differential mechanisms of CDKN2A (p16) alteration in oral tongue squamous cell carcinomas and correlation with patient outcome Annette M. Lim 1,2,3 , Hongdo Do 3,4,5 , Richard J. Young 3 , Stephen Q. Wong 3,4 , Christopher Angel 4,6 , Marnie Collins 7 , Elena A. Takano 3,4 , June Corry 2,8 , David Wiesenfeld 2,9 , Stephen Kleid 10 , Elizabeth Sigston 11 , Bernard Lyons 12 , Stephen B. Fox 2,4 , Danny Rischin 1,2 , Alexander Dobrovic 2,3,4,5 and Benjamin Solomon 1,2,3 1 Department of Medical Oncology, Peter MacCallum Cancer Centre, East Melbourne, Australia 2 University of Melbourne, Australia 3 Research Division, Peter MacCallum Cancer Centre, East Melbourne, Australia 4 Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, Australia 5 Ludwig Institute for Cancer Research, Heidelberg, Australia 6 Department of Pathology, Royal Melbourne Hospital, Australia 7 Department of Biostatistics and Clinical Trials, Peter MacCallum Cancer Centre, East Melbourne, Australia 8 Department of Radiation Oncology, Peter MacCallum Cancer Centre, East Melbourne, Australia 9 Department of Surgery, Royal Melbourne Hospital, Australia 10 Department of Surgical Oncology, Peter MacCallum Cancer Centre, East Melbourne, Australia 11 Department of Surgery, Monash Medical Centre, Australia 12 Department of Surgery, St. Vincent’s Hospital, Australia CDKN2A (p16) disruption is reported as a frequent event in head and neck squamous cell carcinomas that confers poor prog- nosis. We investigated the frequency of different potential mechanisms of CDKN2A inactivation in oral tongue squamous cell carcinomas (OTSCC) and their impact on patient outcome. From a cohort of 153 OTSCC patients, 131 formalin fixed paraffin embedded blocks of pre-treatment primary tumours were suitable for further molecular analysis. We assessed CDKN2A (p16) levels by immunohistochemistry (IHC), promoter methylation status by methylation-sensitive high resolution melting, mutation status by Sanger sequencing, gene copy number variation by fluorescence in situ hybridisation, and correlated these with patient outcome. We found that the majority of OTSCC did not overexpress p16 (110/116, 95%), assessed by IHC. The fre- quency of CDKN2A mutations was 20% (21/103), homozygous loss was 7% (7/97), hemizygous loss 31% (30/97), and pro- moter methylation was 18% (20/113). We found no evidence of these mechanisms in 24/106 (23%) p16 IHC negative tumours. No significant correlation was identified between any potential mechanism of CDKN2A inactivation and clinical fea- tures, including smoking status and age. There was a non-significant trend for worse overall survival for p16 IHC negative patients versus positive patients (HR 5 1.81, 95% CI 5 0.44–7.47, p 5 0.40). No relationship was found between mecha- nisms of CDKN2A disruption and patient outcome. In conclusion, we demonstrate that CDKN2A alteration is a frequent event in OTSCC tumourigenesis. However, no correlation was identified between different potential mechanisms of CDKN2A disrup- tion and clinical characteristics or patient outcome. Oral tongue squamous cell carcinomas (OTSCC) have been reported to have the worst prognosis for early stage disease compared with any other head and neck subsite. 1 Despite treatment with combinations of surgery, radiotherapy and chemotherapy, which result in significant morbidity, failure rates remain unacceptably high. 2,3 A comprehensive Key words: p16, CDKN2A, methylation, FISH, mutation, “tongue cancer,” “oral cancer” Additional Supporting Information may be found in the online version of this article. This article was published online on 30 January 2014. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 12 February 2014. Grant sponsor: Cancer Australia grant, Cancer Council of Victoria grant, Cancer Council of Victoria Post-doctoral Fellowship, and Cancer Council of Victoria Postgraduate Scholarship DOI: 10.1002/ijc.28727 History: Received 18 Oct 2013; Accepted 30 Dec 2013; Online 16 Jan 2014 Correspondence to: Benjamin Solomon, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, Victoria, Australia 3002, Tel.: 161-3-9656-1118, Fax.: 161-3-9656-1408, E-mail: ben.solomon@petermac.org Early Detection and Diagnosis Int. J. Cancer: 135, 887–895 (2014) V C 2014 UICC International Journal of Cancer IJC