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. Because p14ARF seems to play pivotal roles in both Adjuvant radiotherapy the pRb and p53 pathways, independently as well as through its Ye s 5 3 3 7. 8 interaction with E2F-1 (24, 25), we did subgroup analyses to No 87 62. 1 further assess the interaction of p14ARF with either p16INK4A Recurrence or p53 protein expression in SCC of the anterior tongue. Ye s 4 1 2 9 . 3 No 99 70.7 Patients and Methods Died of disease Ye s 3 3 2 3 . 6 Patient and tissue samples. Following Institutional Review Board No 107 76.4 approval (St. Vincent’s Hospital Ethics Committee H01/28), we identified patients with primary operable SCC of the anterior tongue, *Tumor and lymph node stages were determined by pathologic analysis with complete survival data and tissue previously used by Bova et al. according to the tumor node metastases system. Data from 140 patients who INK4A (6) for cyclin D1 (n = 147), p16 (n = 143), p53 (n = 143), and had complete clinicopathologic and molecular staining data sets are pre- Ki-67 (n = 148) immunohistochemistry analyses. Three of these 148 sented. patients (6) were excluded from pRb (n = 145) and E2F-1 (n = 145) cTumor grade was determined by pathologic analysis. immunohistochemistry analysis because of lack of invasive cancer

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Fig. 1. Expression of p14ARF in SCC of the tongue. Original magnification, 400, unless otherwise specified. p14ARF positive control: HBL100 (A) and T-47D (B) breast cancer cell lines (200); negative control: MCF-7 (C)andMDA-MB-231(D) breast cancer cell lines. E, positive p14ARF nuclear staining; F, negative p14ARF nuclear staining. G, positive nucleolar and negative nuclear staining of tongue SCC tissue (1,000, oil immersion). H, negative nucleolar and positive nuclear staining of tongue SCC tissue (1,000, oil immersion). NCL, nucleolus. NUC, nuclear.

SCC of the tongue by a histopathologist (J.G. Kench). Verification of the used for p16INK4A, cyclin D1, E2F-1, pRb, p53, and Ki-67 in the pathology of each tissue core was also similarly done. Each patient case previous studies (6, 26). For each tissue microarray core, a score was was represented by a mean of three (range, two to six) cylindrical accepted if it was within V10% for the two observers; however, if scores 1.5 mm tissue biopsy cores distributed randomly over at least 2 of the were significantly dissimilar (>10%), then the core was reviewed by 14 tissue microarray blocks. both scorers simultaneously on a double-headed microscope until a Immunohistochemistry. Four-micrometer tissue sections were cut consensus was reached. These finalized core scores were then collated from formalin-fixed, paraffin-embedded tissue microarrays, dewaxed in for each tumor sample (represented by two to six tissue cores; i.e., 4-12 xylene, and rehydrated through graded alcohol concentrations. For scores) and averaged to yield the final patient tumor score that was used p14ARF, unmasking was achieved using EDTA citrate buffer (pH 8.0) for statistical analysis. solution at high temperature and pressure for 30 minutes, followed by p14ARF was scored according to the percentage of positively stained rapid cooling within 2 minutes. Endogenous peroxidase activity was tumor cell nuclei and whether positively staining nucleoli were present. ARF then quenched by 0.3% H2O2 treatment and the sections were treated A positive p14 score indicated positively staining nucleoli and/or an with protein blocking solution (DAKO, Carpinteria, CA) to reduce average nuclear expression of z30% in the tumor cores (Fig. 1E). background staining. The samples were processed using a DAKO Conversely, a negative score indicated tumor samples that had <30% autostainer with p14ARF antibody (FL-132, Santa Cruz Biotechnologies, positively stained nuclei and lacked nucleolar staining (Fig. 1F). An Santa Cruz, CA) at a dilution of 1:75. The primary antibody was example of a cell demonstrating nucleolar positivity with intensely visualized with a secondary detection system, using Envision Plus stained nucleoli is presented in Fig. 1G, whereas a nuclear positive cell antirabbit antibody, for 10 minutes. Color development was achieved with no nucleoli staining is shown in Fig. 1H. A summary of using 3,3-diaminobenzidine (DAB kit, Vector Laboratories, Burlingame, immunohistochemistry scoring for the molecular markers studied is CA) and hematoxylin was used as a counterstain. Further immunohis- shown in Table 2. tochemistry staining was done in the control cell lines, with an Statistical analysis. Both univariate and multivariate analyses were additional p14ARF antibody (p14ARF C-18, Santa Cruz Biotechnologies) done to assess the association between positive p14ARF expression and that confirmed the specificity of the p14ARF FL-132 antibody (data not DFS and OS in relation to covariates. The clinicopathologic covariates shown). HBL100 and T-47D breast carcinoma cell lines were used as considered were tumor stage, nodal stage, grade, pathologic stage, positive controls. INK/ARF-deleted breast cancer cell lines MCF-7 and and treatment. The biochemical markers considered were p16INK4A MDA-MB-231 (28) were used as negative controls. Positive and negative (6), cyclin D1 (6), E2F-1 (26), pRb (26), p53 (6), and Ki-67 (6) controls for p14ARF are shown in Fig. 1A to D. p21WAF1/CIP1 was protein expression. Preliminary analyses indicated that the distribu- processed using techniques outlined by Bova et al. (6) with p21WAF1/CIP1 tions of a number of covariates were highly skewed and logarithmic antibody (1:200; Transduction Laboratories, Lexington, KY). Immuno- transformation failed to normalize the distribution; therefore, for histochemistry staining for p16INK4A, cyclin D1, E2F-1, pRb, p53, and Ki- some covariates, the data were reduced into mutually exclusive 67 on specimens in this database was published previously (6, 26). categories. As a result, tumor stage, nodal stage, pathologic stage, and Immunohistochemical scoring. Each immunohistochemistry slide grade were each reduced into categories according to the clinical stained for p14ARF (FL-132) and p21WAF1/CIP1 was scored by two severity of cancer in addition to being assessed as continuous independent blinded observers (R.A. Kwong and L.H. Kalish for p14ARF variables. Expression was analyzed as outlined on Table 2. Briefly, in and R.A. Kwong and R.J. Bova for p21WAF1/CIP1) in the same fashion as line with previous publications, p16INK4A was assessed for complete

www.aacrjournals.orgClin Cancer Res 2005;11(11) June 1,4109 2005 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Imaging, Diagnosis, Prognosis absence of staining (0%) or positive nuclear expression (z1%), 140 tongue SCCs, we assessed nucleolar as well as nuclear whereas >10% and V10% positive tumor nuclei was used for cyclin p14ARF staining because p14ARF sequestration of Hdm/Mdm2 D1 (6). Similarly, E2F-1 and pRb were examined using V35% and in the nucleolus has been implicated in p53 activation and, z >35%, and 50% and <50% positive nuclear expression, respectively therefore, may be indicative of the growth inhibitory (26). For p21WAF1/CIP1, Ki-67, and p53, dichotomizations at each potential of p14ARF overexpression. Human p14ARF localizes 10th percentile had no impact on outcome variables or multivariate predominantly in the nucleolus, which was difficult to models. To maximize the data analysis, trichotomizations (at the 33rd ARF and 66th percentiles) were done for p53, p21WAF1/CIP1, and Ki-67 at accurately assess in patients with high nuclear p14 levels. ARF 0% to V10%, >10% to V70%, and >70%; 0% to V25%, >25% to When p14 is highly expressed, it is possible that enhanced V60%, and >60%; 0% to V20%, >20% to V60%, and >60%, nuclear accumulation could mask the presence of a minor respectively. The outcome variables were assessed as time-to-event, nucleoplasmic pool that is nonetheless capable of executing which was defined as the difference between the time of diagnosis the physiologic functions of p14ARF (30). Therefore, in this and the time of disease recurrence or death. In the univariate analysis, study, positive p14ARF immunostaining included all patients survival curves were constructed using the Kaplan-Meier method. with nucleolar p14ARF staining and/or p14ARF nuclear The differences in survival times between the categories were staining in z30% of HNSCC cells (Fig. 1E and G). compared using the two-tailed log-rank statistic. Subsequent analysis Association of p14ARF expression with clinicopathologic param- involved the use of the Cox’s proportional hazards model, with the eters. The relationships between clinicopathologic parameters, SAS procedure of PHREG (29), to estimate the hazards ratio (HR) ARF and 95% CI associated with each risk factor and covariate. In this previously assessed molecular markers, and p14 are shown analysis, variables meeting an entry criterion of P < 0.15 in the on Table 3. Twenty percent (n = 29) of patients were negative ARF univariate analyses and for which data were complete (n = 140) were for p14 staining. There were no significant associations analyzed in a multivariate model. A backward stepwise selection between p14ARF negativity and tumor grade, overall stage, or procedure was used to confirm the ‘‘final’’ result, with variables being lymph node stage, nor with the other molecular markers removed from the model according to a partial likelihood ratio test. examined. However, p14ARF positivity was significantly associ- All models met the proportional hazards assumption for Cox ated with low tumor stages (I and II; P = 0.0388). proportional test. No significant interactions were detected. All In a univariate Cox survival analysis (Table 4), p14ARF statistical tests were two sided. negativity was highly significantly associated with decreased DFS (HR, 2.865; 95% CI, 1.488-5.525; P = 0.0017) and Results decreased OS (HR, 3.115; 95% CI, 1.517-6.410; P = 0.0020). Kaplan-Meier survival analysis showed that patients with p14ARF immunohistochemistry staining and immunoscoring. p14ARF-negative and p14ARF-positive cancers have a 41.4% In our examination of p14ARF expression in the cohort of and 78.3% 5-year DFS, respectively (Fig. 2A). Similar benefits

Ta b l e 2 . Immunohistochemical scoring system for molecular markers

Covariate Scoring*(percentile) Range (median) p14ARF (negative vs positive) Positive: 0-90% (33; for p14ARF nuclear staining) 1: Any nucleolar positive staining 2: Any nucleolar positive and <30% nuclear staining 3: Any nuclear staining z30% Negative: 1: Only nucleolar negative and <30% nuclear staining (f50th percentile for p14ARF nuclear staining) Cyclin D1 (positive vs negative) >10 % vs V10 % (f25th percentile) 0-90% (50) p16INK4A (negative vs positive) 0% vs z1% (50th percentile) 0-95% (1) pRb (positive vs negative) z50% vs <50% (f50th percentile) 0-95% (45) E2F-1 (negative vs positive) V35% vs >35% (75th percentile) 0-78% (15) c p53 (+1 )0%toV10 % vs >10 % to V70% vs >70% (33rd and 66th percentiles) 0-100% (50) c p21WAF1/CIP1 (+1 )0%toV25% vs >25% to V60% vs >60% (33rd and 66th percentiles) 0-90% (40) c Ki67 (+1 )0%toV20% vs >20% to V60% vs >60% (33rd and 66th percentiles) 5-90% (30) c p14ARF/p53 (+1 )p14ARF-negative and >50% p53 nuclear expression vs p14ARF-positive and >50% p53 nuclear expression vs p14ARF-negative and V50% p53 nuclear expression vs p14ARF-positive and V50% p53 nuclear expression c p14ARF/p16INK4A (+1 )p14ARF positive and p16INK4A positive vs p14ARF positive and p16INK4A negative or p14ARF negative and p16INK4A positive vs p14ARF negative and p16INK4A negative

*Scoring was based on the percentage of positively staining nuclei unless otherwise specified. cScoring was assessed for each unit increase.

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Ta b l e 3 . Association of clinicopathologic, treatment, and outcome features with p14ARF

p14ARF nucleolar and/or z30% p14ARF no nucleolar and <30% Clinicopathologic parameter nuclear expression (n =111) nuclear expression (n =29) C2 P Tumor stage I60150.0388 II 42 11 III 9 1 IV 0 2 Overall stage I 58 14 0.9296 II 29 8 III 13 3 IV 11 4 Lymph node stage

N0 96 23 0.3352

zN1 15 6 Tu m o r g r a d e WD 33 4 0.0910 MD 52 20 PD 26 5 Radiotherapy Yes 67 20 0.3949 No 44 9 Recurrence Ye s 2 7 14 0.0116 No 84 15 Died of disease Ye s 2 1 12 0.0112 No 90 17 pRb <50% 73 15 0.1635 z50% 38 14 E2F-1 0.1635 >35% 82 25 V35% 29 4 p16INK4A 0.3758 z1% 60 13 0% 51 16 Cyclin D1 0.6787 >10 % 72 20 V10 % 39 9 p53 0.5122 0% to V10 % 41 11 >10 % to V70% 36 12 >70% 34 6 Ki67 0.7048 0% to V20% 44 11 >20% to V60% 51 12 >60% 16 6 p21WAF1/CIP1 0.1144 0% to V25% 38 16 >25% to V60% 53 10 >60% 20 3

NOTE: Statistically significant results are shown in bold. Abbreviations: WD, well differentiated; MD, moderately differentiated; PD, poorly differentiated.

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Ta b l e 4 . Univariate Cox’s proportional hazards analysis for clinicopathologic parameters and molecular markers

DFS OS Covariate HR* 95% CI P HR* 95% CI P p14ARF (negative vs positive) 2.865 1.488-5.525 0.0017 3.115 1.517-6.410 0.0020 Cyclin D1 (>10% vs V10 %) 2.985 1.321-6.711 0.0085 4.329 1.520-12.346 0.0061 c p16INK4A (0% vs z1%) 3.077 1.613-5.952 0.0008 3.559 1.667-7.634 0.0012 pRb (z50% vs <50%) 1.798 0.968-3.338 0.0633 1.782 0.897-3.536 0.0991 c E2F-1 (V35% vs >35%) 1.377 0.637-2.985 0.4166 3.333 1.020-10.869 0.0476 b p53 (0% to V10 % vs >10 % to V70% vs >70%) 1.063 0.727 - 1.559 0.7529 1.221 0.800-1.862 0.3555 b p21WAF1/CIP1 (0% to V25% vs >25% to V60% vs >60%) 1.044 0.674 - 1.611 0.8468 1.262 0.787-2.021 0.3347 b Ki-67 (0% to V20% vs >20% to V60% vs >60%) 1.244 0.804- 1.925 0.3264 1.339 0.836-2.140 0.2250 Treatment (surgery alone vs surgery and radiotherapy) 2.058 1.114 -3.800 0.0211 2.938 1.460-5.913 0.0025 N stagex (+1k) 4.204 2.160-8.182 <0.0001 6.876 3.408-13.875 <0.0001 Overall stagex (+1k) 1.461 1.100-1.939 0.0088 1.874 1.388-2.530 <0.0001 Tstagex (+1k) 1.256 0.816-1.933 0.3007 1.687 1.091-2.609 0.0188 Gradex (+1k) 1.716 1.100-2.678 0.0174 1.978 1.195-3.274 0.0079

NOTE: Statistically significant results are shown in bold. *Hazard ratios for p53 and cyclin D1differ from previous publications (6, 26) due to changes in cutoffs; see text for details. cHazards ratios were determined for both p16INK4A and E2F-1as negative (i.e., 0% and V35%, respectively) versus positive (z1% and >35%, respectively) rather than positive versus negative in the previous publication (25). bp53, p21WAF1/CIP1, and Ki-67 were not statistically significant predictors of outcome when dichotomized at each 10th percentile, in line with data for p53 and Ki-67 presented in prior publications (6, 25). Data were thus trichotomized at the 33rd and 66th percentiles. xN stage, overall stage, tumor stage, and grade were assessed as continuous variables rather than dichotomized as in the previous publications (6, 25). kScoring was assessed for each unit increase.

were found for 5-year OS rates: Patients with p14ARF-negative Association of combined p14ARF/p53 expression with clinico- cancers had a 5-year OS of 42.9% compared with an OS of 81% pathologic parameters. p14ARF expression is regulated by pRb for patients with positive p14ARF staining (Fig. 2B). On through the E2F transcription factor family (19, 31), and thus univariate analysis, p16INK4A, cyclin D1, treatment, grade, p14ARF has the capacity to integrate both pRb- and p53- lymph node status, and overall pathologic stage were signifi- dependent mechanisms. Moreover, p53 represses transcription cant on both DFS and OS (Table 4). With updated survival from the human ARF promoter and p14ARF and p53 status are data, E2F-1 (26) was a significant predictor of increased OS inversely correlated in human tumor cell lines (32). To (P = 0.0476), whereas pRb displayed a trend toward investigate the relationship between p14ARF and p53 in SCC significance for DFS (P = 0.0633). of the tongue, we examined the correlations between the Multivariable models were constructed to determine the expression of these molecules in our cohort. Although there independence of p14ARF as a predictor of prognosis in SCC of was no significant association between p14ARF and p53 staining the anterior tongue. Overall, p14ARF negativity was not only an by immunohistochemistry (P = 0.5122), on Kaplan-Meier independent predictor after adjusting for all variables but also analysis, there was a significant survival disadvantage apparent maintained its HR of f2.5 in all three multivariate models in patients who were negative for p14ARF and had high p53 tested. The strongest multivariate model for both DFS and OS expression (>50% positive nuclear staining representing the included p14ARF, p16INK4A, cyclin D1, histopathologic grade, 50th percentile; n = 13) compared with p14ARF-negative and lymph node status and is shown on Table 5. Decreased patients who had low p53 expression (V50% positive nuclear DFS was significantly associated with p14ARF negativity (HR, staining; n = 17) and p14ARF-positive patients, irrespective of 2.506; 95% CI, 1.274-4.926), p16INK4A negativity (HR, 2.809; p53 expression (n = 110; DFS: P = 0.0030; OS: P < 0.0001; 95% CI, 1.439-5.495), and positive lymph node status (HR, Fig. 3A and B). This p14ARF-negative, high p53 expression 3.325; 95% CI, 1.655-6.681). Similarly, decreased OS was subgroup of patients had a 12% 5-year survival rate for both significantly associated with p14ARF negativity (HR, 2.832; 95% DFS and OS compared with 77% to 80% for the remaining CI, 1.340-5.988), p16INK4A negativity (HR, 2.994; 95% CI, patients. When further investigated on multivariate analyses 1.368-6.579), positive lymph node status (HR, 5.459; 95% CI, (Table 6), this combination of molecular markers was the 2.570-11.592), and each unit increase in the histopathologic strongest independent predictor of DFS and OS in multivariate grade (HR, 1.973; 95% CI, 1.110-3.508). When p14ARF was models, including lymph node status, treatment, or overall assessed against cyclin D1, p16INK4A,gradewitheither stage. The p14ARF-negative/high p53 (>50% positive nuclear treatment (p14ARF DFS: HR, 2.571; 95% CI, 1.302-5.076; OS: staining) subgroup with lymph node status (DFS: HR, 6.002; HR, 2.778; 95% CI, 1.330-5.814), or overall stage (p14ARF DFS: 95% CI, 2.275-15.832; OS: HR, 7.319; 95% CI, 2.418-22.156), HR, 2.437; 95% CI, 1.252-4.808; OS: HR, 2.660; 95% CI, with treatment (DFS: HR, 5.747; 95% CI, 2.126-15.353; OS: 1.267-5.556), p14ARF remained an independent prognostic HR, 5.961; 95% CI, 2.621-13.557), or with overall stage (DFS: predictor of outcome for all models examined. HR, 4.979; 95% CI, 1.925-12.879; OS: HR, 6.268; 95% CI,

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correlation between p14ARF and p16INK4A immunoreactivity (P = 0.3758), with p16INK4A negativity equally likely in both p14ARF-positive and p14ARF-negative groups. However, because loss of each protein is associated with deregulation of distinct pathways, we further analyzed the data to assess the effect of loss of one or both proteins. Sixteen patients (11.4%) were negative for both p14ARF and p16INK4A immunostaining. Although this analysis must be interpreted with caution because of the small size of this subgroup, loss of both p14ARF and p16INK4A resulted in significantly decreased DFS (P < 0.0001) and OS (P < 0.0001) on univariate analysis compared with patients who retained either or both p14ARF or p16INK4A. Of the 16 patients with combined p14ARF and p16INK4A loss, 11 patients (69%) had disease recurrence within the first 15 months following surgery and 9 (56%) had died of disease by 25 months. There was no correlation between combined p14ARF/p16INK4A-negative scoring and overall stage (P = 0.373), lymph node status (P = 0.180), treatment (P = 0.856), or histopathologic grade (P = 0.130). To fully assess the independence of this combination of molecular markers, multivariate analysis was done (Table 6). Combined p14ARF/p16INK4A negativity was a strong indepen- dent predictor of both poor DFS and OS in all three models assessed; when assessed with cyclin D1, tumor grade and either lymph node status (DFS: HR, 4.386; 95% CI, 2.105- 9.174; OS: HR, 4.048; 95% CI, 1.808-4.049), or treatment (DFS: HR, 4.695; 95% CI, 2.247-9.804; OS: HR, 4.329; 95% CI, 1.961-9.524), or overall stage (DFS: HR, 4.444; 95% CI, 2.137-9.259; OS: HR, 3.891; 95% CI, 1.748-8.621). Kaplan- Meier analysis was done comparing combined p14ARF/ p16INK4A negativity with combined p14ARF/p16INK4A positivity ARF INK4A Fig. 2. Kaplan-Meier survival curves according to p14ARF status. Cumulative DFS or positive staining for either p14 or p16 alone (A)andcumulativeOS(B) for p14ARF-positive (n; n =29)andp14ARF-negative (Fig. 3C and D). This revealed a significant association (. with broken line; n = 111) p a t i e n t s . between worsening DFS (P < 0.0001) and OS (P < 0.0001) and loss of expression of one or both proteins. Assessment of 2.692-14.594) had a higher HR than most established combined loss, loss of one protein alone, or expression of clinicopathologic parameters in this cohort. These data support both proteins showed a 67%, 26%, and 15% 5-year incidence a potential biological relationship between p14ARF and p53 in of disease recurrence and a 53%, 24%, and 12% 5-year SCC of the tongue, but interpretation is limited by the small incidence of disease-specific death, respectively. patient numbers in these subgroup analyses. ARF INK4A Association of combined p14 /p16 expression with Discussion clinicopathologic parameters. We further investigated whether the influence of p14ARF could be a consequence of influences We have examined the relationship between p14ARF in the pRb pathway via the intimate relationship of the protein expression and outcome in SCC of the anterior overlapping INK4A and ARF loci. Surprisingly, there was no tongue, demonstrating p14ARF as an independent predictor of

Ta b l e 5 . Multivariate Cox’s proportional hazards analysis for clinicopathologic parameters and molecular markers

DFS OS Covariate (n =140) HR 95% CI P HR 95% CI P p14ARF (negative vs positive) 2.506 1.274-4.926 0.0078 2.832 1.340-5.988 0.0064 Cyclin D1 (>10 % vs V10%) 2.101 0.898-4.926 0.0872 2.525 0.847-7.519 0.0965 p16INK4A (0% vs z1%) 2.809 1.439-5.495 0.0025 2.994 1.368-6.579 0.0061 Grade (+1*) 1.604 0.977-2.631 0.0616 1.973 1.110-3.508 0.0206 N stage (+1*) 3.325 1.655-6.681 0.0007 5.459 2.570-11.592 <0.0001

NOTE: Statistically significant results are shown in bold. *Scoring was assessed for each unit increase.

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DFS and OS in the presence of concurrent clinicopathologic predictors, including regional lymph node status. Thus, a predictor of outcome, p14ARF has potential advantages over the existing tumor node metastases staging system (33) where limited predictive ability is illustrated by varied recurrence rates within each tumor node metastases substage, and additionally confounded by the fact that the majority of patients present with no nodal metastasis at the time of diagnosis (34, 35). We found significant advantages in 5-year outcome for both DFS and OS for patients with positive p14ARF immunoscoring (i.e., displaying nucleolar p14ARF and/or >30% nuclear p14ARF staining). This strong relationship was not observed when p14ARF nuclear positivity alone was considered, whereas for patients with positive p14ARF expression there was no difference in survival in patients with only nuclear staining compared with those with only nucleolar staining (data not shown). The nucleolus is best understood as the site for synthesis of rRNA, ribosome biogenesis, and shuttling of some mRNA species, and some recent studies have provided evidence that p14ARF may regulate ribosome biogenesis (36, 37). The predominant nucleolar localization of p14ARF and the murine homologue, p19ARF (14, 32), in contrast with the localization of its targets Hdm2/Mdm2 and p53 in the nucleoplasm, has raised questions over the mechanism by which p14ARF activates p53. In SCC of the cervix (HeLa) cells, levels of endogenous p14ARF and its distribution between the nucleolus and nucleoplasm are sensitive to changes in cell morphology, cell cycle, and nucleolar function (38). However, the role of p14ARF localization in its function remains unclear, with studies showing that nucleolar locali- zation is not sufficient for growth inhibition (17) and is dispensable for relocalization and stabilization of Hdm2/ Mdm2 and p53 (16, 39). This contrasts with the earlier view that sequestration of Hdm2/Mdm2 in the nucleolus is an essential component of p14ARF function. Our observation that incorporating the localization of p14ARF into immunohisto- chemistry scoring is important in identifying the effect of p14ARF on outcome suggests that the subcellular localization of p14ARF may be of functional significance in some aspects of HNSCC biology in vivo. Furthermore, the demonstration that patients with high p14ARF expression had a significantly improved DFS and OS independent of other molecules in the pRb and p53 pathways that impact on prognosis, including cyclin D1, E2F-1, or p53, implicates p14ARF as having a distinct role as a tumor suppressor in SCC of the anterior tongue. The biological and clinical consequences of genetic defects in the INK4A/ARF locus in HNSCC are unclear and p14ARF protein expression has not been analyzed previously in HNSCC, although increased alterations to ARF through promoter methylation and/or point mutations have been

Fig. 3. Kaplan-Meier survival curves according to p14ARF/p53 status and p14ARF/p16INK4A. Cumulative DFS (A)andcumulativeOS(B) for p14ARF/p53 status: p14ARF positive and p53 >50% (! with broken line; n =56); p14ARF negative and p53 V50% (E with dashed line; n =17);p14ARF positive and p53 V50% (n; n =54);andp14ARF negative and p53 >50% (.; n = 13) patients. Cumulative DFS (C)andcumulativeOS(D) for p14ARF/p16INK4A status: for both p14ARF and p16INK4A positive (! with broken line; n =54);eitherp14ARF or p16INK4A (E with dashed line; n = 70); and both p14ARF and p16INK4A negative (.; n = 16) patients.

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Ta b l e 6 . Multivariate Cox’s proportional hazards analysis for clinicopathologic parameters and molecular markers for either p14ARF/p53 or p14ARF/p16INK4A

DFS OS Covariate (n =140) HR 95% CI P HR 95% CI P p14ARF/p53 [p14ARF ()andp53(>50%)vsother*] 6.002 2.275-15.832 0.0003 7.319 2.418-22.156 <0.0001 c p14ARF/p16INK4A (negative vs other ) 4.3 86 2.105-9.174 <0.0001 4.048 1.808-4.049 0.0009 Cyclin D1 (>10 % vs V10%) 1.821 0.776-4.274 0.1687 2.646 0.904-7.752 0.0756 b Grade (+1 ) 1.524 0.931-2.494 0.0937 1.868 1.080-3.231 0.0253 b N stage (+1 ) 3.768 1.888-7.519 0.002 7.367 3.465-15.663 <0.0001

NOTE: Statistically significant results are shown in bold. *Patients were considered in four categories: p14ARF-negative and p53 nuclear expression >50% vs p14ARF-positive and p53 nuclear expression >50% vs p14ARF-nega- tive and p53 nuclear expression V50% vs p14ARF-positive and p53 nuclear expression V50%. cPatients were considered in two categories: patients with both p14ARF and p16INK4A negative vs patients with both p14ARF and p16INK4A positive and patients with either p14ARF or p16INK4A negative but not both. bScoring was assessed for each unit increase.

shown in tumor recurrences of HNSCC (22). One unanswered in the p14ARF-positive and p14ARF-negative subgroups. When question is whether the two gene products of INK4A/ARF are the cohort was dichotomized at the median into low and high simultaneously affected or whether they can be selectively p53 expressors, it was clear that there was an interaction inactivated in HNSCC. We found that few tumors were between this parameter and p14ARF that had implications for negative for p14ARF alone (n = 13) compared with p16INK4A outcome. Patients with both reduced p14ARF expression and alone (n = 51) or both (n = 16). Patients displaying loss of high expression of p53 had the worst prognosis and reduced 5- p14ARF in these cancers (DFS: 46%; OS: 45%) had a trend year DFS and OS (12%) compared with patients who retained toward a shorter 5-year OS and DFS than those lacking only p14ARF expression and low p53 expression (77-80%). Previous p16INK4A (DFS: 73%; OS: 76%). Mice lacking p16INK4A, either studies have indicated that p53 mutation is usually associated through specific inactivating mutations or methylation of its with increased expression of immunoreactive protein (45); promoter, develop spontaneous tumors but at a lower therefore, increased p53 expression in our cohort may reflect frequency and latency than those that lack p19ARF or both p53 mutation. Although this analysis is based on a small p19ARF and p16INK4A (40). Mice hemizygous for p19ARF do number of patients and must therefore be interpreted with not develop tumors unless p16INK4A is also lost, suggesting caution, these data not only support the critical involvement of that p19ARF and p16INK4A cooperate in tumorigenesis (41) and p14ARF in the p53 pathway but also suggest that p14ARF may that p53 pathway control, which utilizes p19ARF, is critical in have significant antineoplastic effects independent of p53 in murine cells. Data from studies of human cells provide a SCC of the anterior tongue. different perspective, suggesting that p16INK4A is the major Activation of p53 by p14ARF is associated with up-regulation tumor suppressor of the INK4A/ARF locus. For example, of the p53-responsive gene, p21WAF1/CIP1 (32, 46). As a potent p16INK4A is more commonly altered by point mutation or Cdk inhibitor, p21WAF1/CIP1 is a major effector of p53-mediated deletion than p14ARF in melanoma (40) and recent in vitro cell cycle arrest. Because p14ARF does not directly interact with studies indicate that a reduction in p14ARF expression cyclins or Cdks (4), a prevailing concept is that p21WAF1/CIP1 enhances growth but is not tumorigenic, whereas loss of mediates p14ARF-induced growth arrest. Thus, in addition to the p16INK4A expression does not enhance growth but can cause induction of p14ARF by E2F-1, connections between p14ARF and transformation when p53 is also inactivated (42). Whatever the pRb pathway are indicated by the observations that p14ARF the implications of loss of either p14ARF or p16INK4A alone, inhibits growth through pRb, based on the up-regulation of loss of both confers very poor outcome. The predictive value p21WAF1/CIP1, inhibition of Cdks, and the presence of hypo- of combined p14ARF/p16INK4A negativity was stronger for both phosphorylated pRb in wild-type p14ARF-arrested cells DFS and OS than that of either p14ARF or p16INK4A alone (4, 32, 47). However, other studies have shown that p14ARF (Tables 5 and 6). can function independent of pRb (48) and p21WAF1/CIP1 is not The TP53 and INK4A/ARF loci encompass the most frequent- required for p14ARF- and p53-mediated growth arrest, indicat- ly inactivated genes in human cancer (43, 44) and numerous ing that multiple downstream effectors exist that mediate the studies support a model where p14ARF and p53 have interde- growth-suppressive functions of p14ARF (49). The observed pendent roles. Because p14ARF stabilizes p53, it has been molecular associations are consistent with proposed biological proposed that the loss of p14ARF may be functionally equivalent molecular interactions that support a role for p14ARF as a tumor to a p53 mutation. Consistent with this idea, many human suppressor, having effects in both pRb and p53 pathways. tumors that retain wild-type p53 suffer loss of p14ARF and are, Outcome in this cohort of SCC of the anterior tongue was therefore, unable to activate p53 in response to abnormal affected by interactions between p14ARF and the pRb pathway proliferative signals. Conversely, cells lacking p53 are refractory (via p16INK4A) as well as between p14ARF and p53. to p14ARF-induced arrest (32, 41). However, in our cohort of Insummary,wehaveshown,forthefirsttime,the HNSCC, high expression of p53 occurred at similar frequency importance of p14ARF as an independent prognostic indicator

www.aacrjournals.orgClin Cancer Res 2005;11(11) June 1,4115 2005 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Imaging, Diagnosis, Prognosis in SCC of the anterior tongue. Until now, the role of p14ARF in tumor cells suggest that reintroduction of p14ARF may have HNSCC has been largely overshadowed by its counterpart, therapeutic utility. The importance of p14ARF in the pathogen- p16INK4A. However, our data indicate that loss of p14ARF esis of SCC of the anterior tongue, given its possible therapeutic expression is associated with poor outcome independent of and prognostic value, warrants further investigation. The other clinicopathologic markers in multivariate analysis. suggestion from our data that interactions between p14ARF Furthermore, the biochemical activity of p14ARF as an activator and p16INK4A or p53 status can have substantial effects on of p53 and its biological role as an inducer of apoptosis in outcome particularly merits further attention.

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