Prognostic Significance of P16 Protein Levels in Oropharyngeal Squamous Cell Cancer

5684 Vol. 10, 5684–5691, September 1, 2004 Clinical Cancer Research

Featured Article Prognostic Significance of p16 Protein Levels in Oropharyngeal Squamous Cell Cancer

Paul M. Weinberger,1 Z. Yu,2 B.G. Haffty,3 swallowing impairment because of treatment-related morbidity. D. Kowalski,4 M. Harigopal,4 C. Sasaki,2 HNSCC are characterized by extensive heterogeneity at the D. L. Rimm,4 and A. Psyrri1 histologic, biological, and clinical level. Therefore, considerable 1 2 3 interest lies in classifying patients in relation to prognosis to Departments of Medical Oncology, Otolaryngology, Therapeutic guide treatment decisions. Radiology, and 4Pathology Yale University School of Medicine, New Haven, Connecticut HNSCC provides an ideal model for understanding carcinogenic progression due to defined risk factors (tobacco and alcohol consumption) and emerging molecular characteristics ABSTRACT (3, 4). According to the molecular progression model for Purpose: Functional inactivation of p16 is an early and HNSCC, loss of 9p21-22 is the most frequent event and is also frequent event in head and neck squamous cell cancers. In present in the earliest definable lesions, including dysplasia and this study, we sought to determine whether p16 expression is carcinoma in situ (5). The p16 (CDKN2) protein resides within of prognostic importance in oropharyngeal squamous cell this region. Thus, it has been hypothesized that p16 is the carcinoma. candidate tumor suppressor gene within this critically deleted Experimental Design: p16 protein expression was eval- area (6). The p16 protein exerts a tumor suppressor function by uated by immunohistochemistry in a tissue microarray com- binding to the cyclin D1 CDK4/CDK6 complex preventing posed of 123 oropharyngeal squamous cell cancers with a phosphorylation of the retinoblastoma (Rb) protein (7). P105Rb mean patient follow-up time of 33 months. and the Rb family members p107 and p130 in turn regulate the Results: p16 overexpression was associated with more activity of E2F transcription factors (8, 9). Complexes consist- advanced Tumor-Node-Metastasis stage and higher histo- ing of E2F and hypophosphorylated p105Rb repress the tran- logic grade. Despite this association with unfavorable fea- scription of genes such as cyclin A required for cell cycle tures, p16 overexpression was associated with decreased progression, and repression is relieved by cyclin dependent 5-year local recurrence rates (11 versus 53%) and increased kinase (CDK)-mediated phosphorylation of p105Rb (10). Thus, 5-year disease-free survival (62 versus 19%) and overall p16 is a CDK inhibitor, which, by inhibition of Rb phosphoryl-

survival (60 versus 21%). In multivariate analysis, p16 ex- ation, preserves the integrity of the G1-S checkpoint and also pression status remained an independent prognostic factor regulates the transcriptional program involved in cell prolifera- for local recurrence, disease-free survival, and overall sur- tion. Although point mutation of p16 gene in HNSCC is rare, vival. alternative mechanisms of abrogation of p16 function such as Conclusions: In patients with oropharyngeal squamous homozygous deletions and methylation of the 5Ј-CpG promoter cell carcinoma, overexpression of p16 as determined by region of p16 have frequently been identified, suggesting that immunohistochemistry is associated with significantly im- functional inactivation of p16 is a common event (11–13). proved prognosis and lower local recurrence rates. Studies have also suggested that loss of p16 function may be associated with tobacco use (14, 15). INTRODUCTION As loss of p16 function is quite common in HNSCC and Over 500,000 patients worldwide are diagnosed with head has important functional implications, we sought to determine and neck squamous cell carcinoma (HNSCC) each year (1). whether p16 expression levels, as determined immunohisto- Despite numerous advances in diagnosis and treatment over the chemically, were associated with clinical outcome in a cohort of last several decades, mortality rates have not changed signifi- 123 oropharyngeal squamous cell cancer patients with a mean cantly (2). Even cured patients suffer significant speech and follow-up of 33 months. Our study demonstrates an independent prognostic value of p16 in determining patient outcome.

MATERIALS AND METHODS Received 3/9/04; revised 5/12/04; accepted 5/21/04. Tissue Microarray Construction. The cohort was as- Grant support: Yale University Institutional Startup Fund (A. Psyrri) sembled from patients with either primary or recurrent oro- and the Doris Duke Charitable Foundation (P. Weinberger). The costs of publication of this article were defrayed in part by the pharyngeal cancer of squamous cell histology treated at Yale- payment of page charges. This article must therefore be hereby marked New Haven hospital between 1980 and 1999. Patients were advertisement in accordance with 18 U.S.C. Section 1734 solely to treated with primary external beam radiotherapy (EBRT) or indicate this fact. gross total surgical resection and postoperative radiotherapy. Requests for reprints: Amanda Psyrri, Yale Cancer Center, P. O. Box 208032, New Haven CT 06520. Phone (203) 737-2476; Fax (203) 785- Exclusion criteria included presentation with metastatic disease 7531; E-mail [email protected]. or failure to receive a full course of radiation therapy. After ©2004 American Association for Cancer Research. Institutional Review Board approval, the tissue microarray was

constructed as previously described (16), including 123 cases oropharyngeal subsite, and drinking and tobacco usage were that met inclusion criteria. Tissue cores were obtained from made using ␹2 analysis. All calculations and analyses were paraffin-embedded, formalin-fixed tissue blocks from the Yale performed with SPSS 11.5 for Windows (SPSS, Inc., Chicago, University Department of Pathology archives. Slides from all IL) and, where appropriate, were two-tailed. blocks were reviewed by a pathologist to select representative areas of invasive tumor to be cored. The cores were placed on RESULTS the recipient microarray block using a Tissue Microarrayer Clinical and Pathological Variable Analysis (Beecher Instrument, Silver Spring, MD). All tumors were rep- There were 123 patients in this cohort that met inclusion resented with 2-fold redundancy. Previous studies have demon- criteria. Tumors were primary 93 (76%) or recurrent 27 (22%). strated that the use of tissue microarrays containing one to two Sixteen (13%) of patients were TNM stage II, 39 (32%) stage histospots provides a sufficiently representative sample for anal- III, and 68 (55%) stage IV. Oropharyngeal subsites included 48 ysis by immunohistochemistry (17–19). Addition of a duplicate (39%) tonsillar fossae, 64 (52%) base of tongue, and 11 (9%) histospot, although not necessary, does provide marginally im- other oropharynx. Seventy-three (59%) patients were managed proved reliability. Cores from human papillomavirus (HPV) with primary EBRT and 44 (36%) with surgical excision fol- type 18-positive HeLa and HPV type 16-positive Caski cell lowed by EBRT. Twenty-two (18%) patients were also treated lines fixed in formalin and embedded in paraffin were selected by chemotherapy. Demographic and clinicopathological vari- for positive controls and included in the array. The tissue micro- ables for the cohort are summarized in Table 1. array was then cut to yield 5-␮m sections and placed on glass slides using an adhesive tape transfer system (Instrumedics, Inc., Immunohistochemistry for p16 Expression Hackensack, NJ) with UV cross-linking. Of the oropharyngeal tumors meeting inclusion criteria, Immunohistochemistry. Tissue microarray slides were 120 of 123 (98%) were interpretable for p16 staining. Seven of deparaffinized and stained using a commercially available kit eight cell-line positive controls were interpretable for p16 stain- (Dako p16INK4a kit; Dako Cytomation, Carpinteria CA). In ing. Tumors displayed one of three distinct immunostaining brief, slides were deparaffinized in xylene followed by absolute phenotypes (Fig. 1): intense diffuse cytoplasmic and nuclear ethanol and subsequent rehydration in graded 95 and 70% ethanol. After a brief rinse in deionized water, heat-induced antigen retrieval was performed using the included epitope retrieval solution at 95°C for 40 minutes. Endogenous peroxidase blocking was performed using the peroxidase blocking Table 1 Demographic, clinical, and pathologic data reagent supplied for 5 minutes at room temperature. The tissue Age (yrs) 41–79 microarray slide was subsequently incubated with the included (median 62) mouse monoclonal antibody against p16 at a 1:25 dilution for 30 Gender Male 92 minutes at room temperature. The negative control slide was Female 31 incubated with supplied negative control reagent (mouse mono- Site clonal antibody to Aspergillus niger glucose oxidase) at a 1:25 Tonsils 48 dilution for 30 minutes at room temperature. Secondary ampli- Base of tongue 64 Other oropharynx 11 fication was accomplished using the included goat antimouse TNM stage dextran polymer-conjugated horseradish peroxidase visualiza- II 16 tion reagent for 30 minutes at room temperature. The antibody III 39 was subsequently visualized using the supplied 3,3Ј-diamino- IV 68 benzidine chromagen for 10 minutes at room temperature fol- Grade Well differentiated 9 lowed by acidified hematoxylin counterstain. Slides were Moderately differentiated 56 mounted with Cytoseal 60 (Richard-Allan Scientific, Kalama- Poorly differentiated 45 zoo, MI). p16 status was independently determined by two Not recorded 13 investigators (M. Harigopal and P. Weinberger) and was re- Tumor type Ͼ Primary 93 garded as overexpression if it was strong and diffuse ( 80% of Recurrent 27 tumor cells) and was regarded as nonoverexpression if absent or Not recorded 3 weak. Management Statistical Analysis. Histospots containing Ͻ10% of a Primary radiotherapy 73 tumor were excluded from additional analysis. Disease-free Post-op radiotherapy 44 Not recorded 6 survival, overall survival, and local recurrence were assessed by Chemotherapy 22 Kaplan-Meier analysis with log-rank score for determining sta- No chemotherapy 101 tistical significance. All survival analysis was performed at Ever drink 5-year cutoffs. Relative risk was assessed by the univariate and No 14 Yes 73 multivariate Cox proportional hazards model. Comparison of Not recorded 36 p16 overexpression with the clinical and pathological variables Ever smoke gender, Tumor-Node-Metastasis (TNM) stage, histologic grade, No 2 tumor type (primary versus recurrent), treatment method (pri- Yes 85 mary EBRT versus primary surgical excision plus radiotherapy), Not recorded 36

Fig. 1 p16 expression in oropharyngeal squamous cell carcinoma and HeLa cell line. p16 expression was determined by using monoclonal mouse anti- p16 antibody (Dako Cytoma- tion). A, squamous cell carcinoma demonstrating strong and diffuse staining. B, HeLa cell line (positive control) demonstrating strong and diffuse staining. C, squamous cell carcinoma demonstrating weak diffuse staining. D, squamous cell carcinoma demonstrating absence of immunohistochemical staining. Only strong and diffuse staining (A and B) was scored as p16 overexpression. Weak or absent staining (C and D) was scored as negative for this study.

staining, weak diffuse cytoplasmic and nuclear staining, and tion with local recurrence using Kaplan-Meier survival analysis absence of immunostaining. In keeping with previous investi- with log-rank statistic for determining significance. This analy- gators’ methods, p16 overexpression was defined as tumors that sis (Fig. 2A) demonstrated that p16 overexpression is associated displayed intense diffuse cytoplasmic and nuclear staining in with decreased 5-year local recurrence rates. Patients with p16 Ͼ80% of tumor cells. There were 24 of 120 (20%) tumors overexpression had a local recurrence rate of 11.1% compared designated as p16 overexpressors, as were 7 of 7 (100%) cell with 53.0% for p16-nonoverexpressing patients (log rank ϭ 8.0, line-positive controls. No staining was observed on any spots for P ϭ 0.0046). Univariate Cox analysis revealed a hazard ratio for the negative control reagent. Comparisons were made between p16 nonoverexpressors of 6.1 (P ϭ 0.013). clinical/pathological variables and p16 status by ␹2 analysis. Disease-Free Survival. The expression status of p16 was There was a trend for p16-overexpressing tumors to be associ- also evaluated for association with disease-free survival. ated with primary versus recurrent, as 22 of 24 (92%) of Kaplan-Meier survival analysis (Fig. 2b) demonstrated that p16 p16-overexpressing tumors were primary tumors, but this did overexpression is associated with improved disease-free sur- not reach statistical significance (P ϭ 0.055). p16 overexpres- vival. Patients with p16 overexpression had a 5-year disease- sion was associated with more advanced TNM stage, with 20 of free survival of 62.2% compared with 18.5% for p16 nonover- 24 (83%) of p16-overexpressing tumors being stage IV (P ϭ expressors (log rank ϭ 8.2, P ϭ 0.0042). Univariate Cox 0.002). p16 overexpression was also associated with higher analysis revealed a hazard ratio for p16 nonoverexpressing histologic grade with 14 of 24 p16-overexpressing tumors being patients of 2.8 (P ϭ 0.006). poorly differentiated (P ϭ 0.001). There was no association between p16 status and the other clinical/pathological variables. Overall Survival These results are summarized in Table 2. The expression status of p16 was also evaluated for association with overall survival. Kaplan-Meier survival analysis Survival Analysis (Fig. 2C) demonstrated that p16 overexpression is associated Local Recurrence. The expression status of p16 as de- with improved overall survival. Patients with p16 overexpres- termined by immunohistochemistry was evaluated for associa- sion had a 5-year overall survival of 59.8% compared with

Table 2 Comparison of p16 status to demographic, clinical and ent prognostic factors (P ϭ 0.038). The results for multivariate pathologic data survival analysis are summarized in Table 4. p16 status* n Ϫϩ P DISCUSSION In this study, we found that p16 expression status in the Patient cohort Interpretable for p16 120 96 24 tissue of patients with oropharyngeal cancer is a strong inde- Noninterpretable 3 pendent prognostic factor for local recurrence as well as disease- Positive controls free and overall survival. Patients with p16-nonoverexpressing Interpretable for p16 7 0 7 tumors had a 6-fold increase in risk of local recurrence and an Noninterpretable 1 Gender almost 3-fold increase in risk of death from any cause. In Male 92 69 21 0.11 multivariate analysis, p16 expression status remained an inde- Female 31 27 3 Site Tonsils 48 39 8 0.42 Base of tongue 64 47 15 Other oropharynx 11 10 1 TNM stage II 16 15 0 0.002† III 39 35 4 IV 68 46 20 Grade‡ Well differentiated 9 9 0 0.001† Moderately differentiated 56 48 5 Poorly differentiated 45 31 14 Tumor type‡ Primary 93 68 22 0.055 Recurrent 27 25 2 Management‡ Primary radiotherapy 73 57 14 0.88 Post-op radiotherapy 44 34 9 Chemotherapy 22 17 5 0.72 No chemotherapy 101 79 19 Ever drink‡ No 14 10 4 0.44 Yes 73 58 14 Ever smoke‡ No 2 1 1 0.31 Yes 85 67 17 * p16 status: (Ϫ) ϭ nonoverexpressor; (ϩ) ϭ overexpressor. † Significant at the 0.01 level. ‡ Does not include patients for whom this information was not available (see Table 1).

21.4% for p16 nonoverexpressors (log rank ϭ 5.7, P ϭ 0.0174). Univariate Cox analysis revealed a hazard ratio for p16-nonoverexpressing patients of 2.4 (P ϭ 0.021). Results for univariate survival analysis of local recurrence, disease-free survival, and overall survival are summarized in Table 3.

Multivariate Analysis Using the Cox proportional hazards model, we performed multivariate analysis to assess the independent predictive value of p16 expression for 5-year disease-free survival, overall survival, and local recurrence. The following prognostic variables were also included: tumor type (primary versus recurrent), management (primary EBRT versus surgery plus EBRT), TNM stage, and histologic grade. For disease-free survival, only p16 expression status remained an independent prognostic factor Fig. 2 Kaplan-Meier estimates of 5-year local recurrence (A), disease-free ϭ survival (B), and overall survival (C) by p16 status. p16 overexpressors had (P 0.01). For local recurrence, both p16 status and manage- significantly higher disease-free and overall survival rates. Additionally, ment were independent prognostic factors (P ϭ 0.029). For p16 overexpressors had lower local recurrence rates than nonoverexpres- overall survival, both p16 status and TNM stage were independ- sors.

Table 3 Univariate Cox regression analysis by p16 expression levels Mean time to Percent recurrence Hazard ratio p16 status recurrence (mos) (95% confidence interval) (95% confidence interval) P Local recurrence Overexpressor 55.0 11.1% (0–26) 0.165 (0.04–0.7) 0.013 Nonoverexpressor 37.0 53.0% (40–66) 6.062 (1.5–25.1) Disease-free survival Overexpressor 42.2 62.2% (41–83) 0.359 (0.2–0.7) 0.006 Nonoverexpressor 24.7 18.5% (10–27) 2.786 (1.3–5.8) Overall survival Overexpressor 30.5 59.8% (38–82) 0.422 (0.2–0.9) 0.021 Nonoverexpressor 43.2 21.4% (12–31) 2.370 (1.1–4.9)

pendent prognostic factor. This result is striking, given that p16 (32, 33). The molecular events in cervical carcinogenesis, which overexpression was also associated with poor prognostic fea- is known to be HPV associated, have been described exten- tures such as advanced TNM stage and high histologic grade. sively. During carcinogenic progression, integration of the viral The relationship between p16 expression levels and prog- DNA into the host cell genome often disrupts the gene that nosis is controversial. Loss of p16 expression has been found to encodes the E2 protein, the main viral transcription/replication be associated with worse prognosis in several cancers, including factor (34–37). The E2 protein functions as a transcriptional non-small cell lung cancer, melanoma, nasopharyngeal, hepato- repressor of the HPV E6 and E7 oncogenes. Loss of the E2 cellular, and colorectal carcinoma (15, 20–24). This is in con- protein results in dysregulated expression of the E6 and E7 trast to reports that overexpression of p16 is associated with genes. These genes encode oncoproteins that neutralize the p53 poor survival in prostate cancer and neuroblastoma (25–27). In and Rb tumor suppressor pathways, respectively (38–40). Rb HNSCC, conflicting data also exists regarding the prognostic acts as a negative regulator of p16 expression at the transcrip- significance of p16. Loss of p16 expression has been found to be tional level (41, 42). It has been demonstrated in a wide variety associated with worse prognosis in carcinoma of the anterior of cancers that p16 and Rb inactivation are almost always tongue (28). In a similar fashion, p16 overexpression has been mutually exclusive (43). Khleif et al. (44) studied cervical correlated with improved outcome in tonsillar carcinoma (29). cancer cell lines and demonstrated that p16 protein is expressed To the contrary, other investigators have found no prognostic in cells that contain a wild-type Rb functionally inactivated by significance of p16 in HNSCC (30, 31). To our knowledge, our E7. Additionally, the authors found that inactivation of Rb was study is the largest of its kind in evaluating the prognostic tightly correlated with subsequent up-regulation of p16, con- significance of p16 expression in oropharyngeal tumors. firming a feedback loop between p16 and Rb. Oropharyngeal cancers are a heterogeneous group in terms Overexpression of p16 has been repeatedly reported in of etiology, pathogenesis and clinical behavior. Besides tobacco HPV-associated cancers. In cervical and genital lesions, levels and alcohol use, evidence has accumulated that some of the of p16 protein expression have been correlated with HPV on- oncogenic HPVs, especially high-risk HPV types 16 and 18, are cogenic potential (45, 46). A recent study of lymph node me- associated with a subset of patients with oropharyngeal tumors tastases in HNSCC reported that p16 overexpression is a surro-

Table 4 Multivariate analysis by Cox regression Variable Hazard ratio 95% confidence interval P Local recurrence Tumor type (primary/recurrent) 1.675 0.8–3.7 0.20 Management (EBRT/surgery ϩ EBRT) 0.407 0.2–0.97 0.043* TNM stage 1.412 0.8–2.4 0.20 Histologic grade 0.965 0.6–1.6 0.90 p16 overexpressor 5.293 1.2–23.6 0.029* Disease-free survival Tumor type (primary/recurrent) 1.616 0.9–3.0 0.13 Management (EBRT/surgery ϩ EBRT) 0.581 0.3–1.1 0.08 TNM stage 1.453 1.0–2.1 0.06 Histologic grade 1.033 0.7–1.5 0.87 p16 overexpressor 3.000 1.3–7.0 0.010* Overall survival Tumor type (primary/recurrent) 1.648 0.9–3.1 0.12 Management (EBRT/surgery ϩ EBRT) 0.648 0.4–1.2 0.14 TNM stage 1.679 1.1–2.5 0.014* Histologic grade 0.984 0.7–1.5 0.94 p16 overexpressor 2.449 1.1–5.7 0.038* * Significant at the 0.05 level.

gate marker for oropharyngeal origin and HPV association (47). overexpression may identify those cancers that would benefit This confirms previous reports associating p16 overexpression from HPV-targeted therapies such as vaccines or antiviral phar- with HPV positivity in oropharyngeal squamous cell carcinomas macotherapy. (29, 48, 49). If indeed p16 overexpression is a surrogate marker of HPV association, then our finding of a 20% incidence of p16 over- REFERENCES expressors would be inconsistent with most studies reporting an 1. Greenlee RT, Hill-Harmon MB, Murray T, Thun M. Cancer statis- incidence of 50% HPV positivity in oropharyngeal squamous tics, 2001. CA - Cancer J Clin 2001;51(1):15–36. cell carcinomas (32, 50, 51). An alternative explanation of our 2. Al-Sarraf M. Treatment of locally advanced head and neck cancer: results would be that p16 overexpression defines a subgroup of historical and critical review. Cancer Control 2002;9(5):387–99. 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Paul M. Weinberger, Z. Yu, B.G. Haffty, et al.

Clin Cancer Res 2004;10:5684-5691.

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