Imaging, Diagnosis, Prognosis p14ARF ProteinExpressionIsaPredictorofBothRelapseandSurvival in Squamous Cell Carcinoma of the Anterior Tongue Rhonda A. Kwong,1Larry H. Kalish,1Tuan V. Nguyen,2 James G. Kench,1,4 Ronaldo J. Bova,3 Ian E. Cole,3 Elizabeth A. Musgrove,1and Robert L. Sutherland 1 Abstract Purpose: The INK4A-ARF locus at chromosome 9p21is frequently altered in head and neck squamous cell carcinoma (SCC) and encodes two distinct tumor suppressors, p16INK4A and p14ARF.This study addressed the role of p14ARF as a potential prognostic marker in this disease. Experimental Design: p14ARF protein expression was assessed by immunohistochemistry in a cohort of 140 patients with SCC of the anterior tongue. Using univariate and multivariate Cox’s proportional hazards models, the outcomes examined were time to disease recurrence or death, with or without clinicopathologic covariates, including nodal status, disease stage, treatment status, Ki-67 staining, and molecular markers with known functional or genetic relationships with p14ARF (p16INK4A,p53,pRb,p21WAF1/CIP1,E2F-1). Results: On multivariate analysis, p14ARF positivity (nucleolar p14ARF staining and/or nuclear p14ARF staining in z30% of tumor cells) was an independent predictor of improved disease-free survival (DFS; P = 0.002) and overall survival (OS; P = 0.002).This was further enhanced when p14ARF positivity was cosegregated with positive (z 1%) p16INK4A staining (DFS, P < 0.001; OS, P < 0.001). Patients whose cancers were p14ARF negative and p53 positive (>50%) had the poorest outcome (DFS, P < 0.001; OS, P < 0.001) of any patient subgroup analyzed. Conclusions: These data show that in patients with SCC of the tongue, combined nuclear and nucleolar expression of p14ARF protein predicts for improved DFS and OS independent of established prognostic markers. Head and neck cancers account for 3.8% of all newly clinical management and suggest new targets for therapy, as diagnosed cancers in men in Australia (1). Almost all (90%) well as provide insights relevant to a better understanding of head and neck cancers are squamous cell carcinomas (SCC) the biology of HNSCC. and >50% arise in the oral cavity, with the majority represent- Dysregulation of the normal cell cycle regulatory machinery ing SCC of the anterior tongue. Improvements in cancer is integral to the neoplastic process and there is now compelling treatment have not translated into improved disease-free evidence implicating loss of cell cycle control in the develop- survival (DFS) and overall survival (OS) in head and neck ment and progression of most human cancers (3). Conse- SCCs (HNSCC) over the past 20 years (2). The identification of quently, cell cycle regulatory molecules are attractive potential prognostic markers with potential clinical utility may assist in prognostic markers. The INK4A-ARF locusisfrequently disrupted in human cancers and consists of two overlapping genes that encode two unrelated proteins, p16INK4A and p14ARF (4). p16INK4A and p14ARF act through the retinoblastoma 1 2 Authors’Affiliations: Cancer Research Program and Bone and Mineral Research protein (pRb) and p53 pathways, respectively (3), to regulate Program, Garvan Institute of Medical Research; 3Department of ENT Surgery, St. Vincent’s Hospital, Darlinghurst, New South Wales, Australia and 4Institute of cell cycle progression, and these important oncogenic pathways Clinical Pathology and Medical Research, Westmead Hospital, Westmead, New are frequently dysregulated in HNSCC. SouthWales, Australia The pRb pathway comprises INK4A proteins, such as Received 10/5/04; revised 2/18/05; accepted;3/7/05. p16INK4A, the cyclin D/E-dependent kinases, and pRb family Grant support: Garnett Passe and Rodney Williams Memorial Foundation, National Health and Medical Research Council of Australia, Cancer Council New proteins. Cyclin D1 and cyclin-dependent kinases 4 and 6 South Wales, and R.T. Hall Trust. (Cdk4/6) act in concert with cyclin E-Cdk2 to phosphorylate The costs of publication of this article were defrayed in part by the payment of page pRb, leading to release of E2F-1 in late G1 phase. E2F-1 then charges.This article must therefore be hereby marked advertisement in accordance initiates transcription of genes essential for DNA synthesis. The with 18 U.S.C. Section 1734 solely to indicate this fact. p16INK4A protein specifically inhibits the cyclin D–dependent Note: R.A. Kwong and L.H. Kalish contributed equally to this work and are recipi- ents of National Health and Medical Research Council postgraduate research fel- kinases, at least in part by preventing cyclin D1 association lowships. R.A. Kwong is also a recipient of a Garnett Passe and Rodney Williams with Cdk4/6. pRb-E2F-1 complexes actively repress gene research fellowship. E.A. Musgrove is a Cancer Institute NSW Fellow. expression and, consequently, derangement of the pRb Requests for reprints: Robert L. Sutherland, Cancer Research Program, pathway releases this inhibitory control resulting in deregu- Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010, Australia. Phone: 612-9295-8322; Fax: 612-9295-8321; E-mail: lated S-phase entry and uncontrolled cell proliferation (3). [email protected]. Alterations to this pathway through frequent Cdk6 hyper- F 2005 American Association for Cancer Research. activation, cyclin D1 overexpression, and p16INK4A mutations www.aacrjournals.orgClin Cancer Res 2005;11(11) June 1,4107 2005 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Imaging, Diagnosis, Prognosis have been shown in tongue cell lines and carcinomas (5, 6); specimens in Kwong et al.’s study (26) and because one further ARF overall, aberrations in the pRb pathway are found in >80% of patient lacked tissue for the present study. Thus, p14 expression HNSCC (7). was examined on 144 patients. Overall, 140 patients had a complete A second critical oncogenic pathway involves the tumor clinicopathologic and molecular staining data set, and it is these 140 patients whose characteristics are presented in Table 1 and further suppressor p53, its negative regulator Hdm2 (Mdm2 in mice), ARF analyzed in this study. All patients were treated with curative intent in and p14 . Whereas mutated p53 is found in >50% of HNSCC the Departments of Head and Neck Surgery at St. Vincent’s Hospital (8, 9), the roles of other components of this pathway are not and Westmead Hospital, Sydney, New South Wales, Australia. Clinical well understood in this disease. p53 accumulates in response to follow-up was recorded to a minimum of 2 years or when the patient DNA damage, hypoxia, and oncogene activation. Nuclear p53 was diagnosed with recurrent disease. Updated survival data on all is then stabilized and activated, which may result in cell cycle eligible patients increased the median duration of follow-up to arrest (via the Cdk inhibitor p21WAF1/CIP1) or apoptosis 61 months (range: 6-161 months). Forty-one patients (29.3%) had (10, 11). Hdm2/Mdm2 binds to p53 and reduces nuclear p53 recurrence of their disease, corresponding to an annual incidence of levels by transporting p53 to the cytoplasm where it is degraded 6.8% [95% confidence interval (95% CI), 4.6-8.7]. The median time (12, 13). This results in the inhibition of p53-mediated cell to disease recurrence was 41 months, with the 25th and 75th percentiles being 16 and 77 months, respectively. During the follow- cycle arrest and apoptosis. p14ARF abrogates this function of up period, 33 patients (23.6%) died from their disease. The median Hdm2/Mdm2 through direct binding and nucleolar sequestra- ARF OS was 49 months, with the 25th and 75th percentiles being 24 and tion of Hdm2/Mdm2 (14, 15). Excess p14 restrains cell 87 months, respectively. growth by not only inhibiting Hdm2/Mdm2 function but also Tissue microarray. Tissue microarrays were constructed as outlined by independently stabilizing p53, triggering a p53-dependent by Horvath et al. (27). H&E-stained template sections of the original transcriptional response (16). p14ARF activity has mainly been paraffin-embedded tissue block were marked up for areas of invasive attributed to this p53-dependent mechanism. However, p14ARF is also able to suppress growth independent of p53 (17) through delaying S-phase progression (18). Although the Ta b l e 1. Clinicopathologic, treatment, and outcome precise mechanisms remain to be elucidated, it is possible features of 140 patients with SCC of the anterior ARF that these p53-independent effectors of p14 may include tongue either Mdm2 or E2F-1. Moreover, ARF is an E2F-1 target gene (19, 20) and thereby provides a link between the pRb and Clinicopathologic No. patients Percentage of p53 pathways (3). parameter (n =140) patients (%) The majority of previous studies of p16INK4A and p14ARF in HNSCC have focused on genetic aberrations rather than Tumor stage* alterations in protein expression. The overlapping INK4A-ARF I7553.6 genes are altered through homozygous deletion, promoter II 53 37.9 methylation of exon 1a, or mutation of 1h in up to 50% of III 10 7. 1 HNSCC (21, 22). Extensive analysis of INK4A methylation in IV 2 1.4 human HNSCC has also shown a high level of inactivation that Overall stage* correlated with a lack of p16INK4A protein expression by I7251.4 immunohistochemistry (23). We have previously identified II 37 26.4 p16INK4A as an independent prognostic indicator in SCC of the III 16 11.4 anterior tongue (6). IV 15 10.8 The ARF gene has an increased frequency of inactivation in Lymph node stage* recurrent HNSCC lesions compared with the primary lesion N0 119 8 5 . 0 ARF zN1 21 15.0 (22). Expression of p14 protein has not been investigated in c HNSCC despite frequent aberrations in both the INK4A-ARF Tu m o r g r a d e (21, 22) and TP53 gene loci (8, 9) and, thus, the goal of this Well differentiated 37 26.4 study was to determine the relationship between p14ARF protein Moderately differentiated 72 51.4 expression and disease recurrence or death in SCC of the ante- Poorly differentiated 3 1 22.1 rior tongue.