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Figure 1.
Expression of p21WAF1/CIP1 in relation to survivors and nonsurvivors in 80 patients with tongue squamous cell carcinoma. The horizontal line indicates the 23% cutoff level (P = .03).

Expression of p21WAF1/CIP1 in relation to survivors and nonsurvivors in 80 patients with tongue squamous cell carcinoma. The horizontal line indicates the 23% cutoff level (P = .03).

Figure 2.
Expression of p21WAF1/CIP1 in relation to survivors and nonsurvivors in 33 cases of p53-negative tongue squamous cell carcinoma. The horizontal line indicates the 23% cutoff level (P = .002).

Expression of p21WAF1/CIP1 in relation to survivors and nonsurvivors in 33 cases of p53-negative tongue squamous cell carcinoma. The horizontal line indicates the 23% cutoff level (P = .002).

Figure 3.
Disease-specific survival curves (Kaplan-Meier) for p21WAF1/CIP1 expression in 80 patients with tongue squamous cell carcinomas (P = .04).

Disease-specific survival curves (Kaplan-Meier) for p21WAF1/CIP1 expression in 80 patients with tongue squamous cell carcinomas (P = .04).

Figure 4.
Disease-specific survival curves (Kaplan-Meier) for the p53 − p21WAF1/CIP1 combination score in 80 patients with tongue squamous cell carcinomas (P<.001).

Disease-specific survival curves (Kaplan-Meier) for the p53 − p21WAF1/CIP1 combination score in 80 patients with tongue squamous cell carcinomas (P<.001).

Figure 5.
Disease-specific survival curves (Kaplan-Meier) for the Bax + p21WAF1/CIP1 combination score in 80 patients with tongue squamous cell carcinomas (P<.001).

Disease-specific survival curves (Kaplan-Meier) for the Bax + p21WAF1/CIP1 combination score in 80 patients with tongue squamous cell carcinomas (P<.001).

Table 1 
Log-Rank and Proportional Hazard Regression Analysis (Cox Method) for Clinical Histologic Variables and p21WAF1/CIP1 Expression in Relation to Disease-Specific Survival for 80 Patients With Tongue Squamous Cell Carcinoma*
Log-Rank and Proportional Hazard Regression Analysis (Cox Method) for Clinical Histologic Variables and p21WAF1/CIP1 Expression in Relation to Disease-Specific Survival for 80 Patients With Tongue Squamous Cell Carcinoma*
Table 2 
Selected Cutoff Levels for the Classification of p21WAF1/CIP1 Expression, p53 Accumulation, and Bax Expression for 80 Patients With Squamous Cell Carcinoma of the Tongue*
Selected Cutoff Levels for the Classification of p21WAF1/CIP1 Expression, p53 Accumulation, and Bax Expression for 80 Patients With Squamous Cell Carcinoma of the Tongue*
Table 3 
Log-rank Analysis for Individual and Combined Parameters Including p21WAF1/CIP1 Expression, p53 Accumulation, and Bax Expression in Relation to the Disease-Specific Survival for 80 Patients With Tongue Squamous Cell Carcinoma*
Log-rank Analysis for Individual and Combined Parameters Including p21WAF1/CIP1 Expression, p53 Accumulation, and Bax Expression in Relation to the Disease-Specific Survival for 80 Patients With Tongue Squamous Cell Carcinoma*
1.
Sherr  CJ Cancer cell cycles. Science.1996;274:1672-1677.
2.
el-Deiry  WSTokino  TVelculescu  VE  et al WAF1, a potential mediator of p53 tumor suppression. Cell.1993;75:817-825.
3.
Harper  JWAdami  GRWei  NKeyomarsi  KElledge  SJ The p21 cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell.1993;75:805-816.
4.
Noda  ANing  YVenable  SFPereira-Smith  OMSmith  JR Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res.1994;211:90-98.
5.
Xiong  YHannon  GJZhang  HCasso  DKobayashi  RBeach  D p21 is a universal inhibitor of cyclin kinases. Nature.1993;366:701-704.
6.
Waga  SHannon  GJBeach  DStillman  B The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature.1994;369:574-578.
7.
Chen  JJackson  PKKirschner  MWDutta  A Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature.1995;374:386-388.
8.
Parker  SBEichele  GZhang  P  et al p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells. Science.1995;267:1024-1027.
9.
Jiang  HLin  JSu  ZZCollart  FRHuberman  EFisher  PB Induction of differentiation in human promyelocytic-HL-60-leukemia cells activates p21WAF1/CIP1 expression in the absence of p53. Oncogene.1994;9:3397-3406.
10.
Jiang  MShao  ZMWu  J  et al p21/waf1/cip1 and mdm-2 expression in breast carcinoma patients as related to prognosis. Int J Cancer.1997;74:529-534.
11.
Komiya  THosono  YHirashima  T  et al p21 expression as a predictor for favorable prognosis in squamous cell carcinoma of the lung. Clin Cancer Res.1997;3:1831-1835.
12.
Anttila  MAKosma  VMHongxiu  J  et al p21/WAF1 expression as related to p53, cell proliferation and prognosis in epithelial ovarian cancer. Br J Cancer.1999;79:1870-1878.
13.
Caffo  ODoglioni  CVeronese  S  et al Prognostic value of p21(WAF1) and p53 expression in breast carcinoma: an immunohistochemical study in 261 patients with long-term follow-up. Clin Cancer Res.1996;2:1591-1599.
14.
Zlotta  ARNoel  JCFayt  I  et al Correlation and prognostic significance of p53, p21WAF1/CIP1 and Ki-67 expression in patients with superficial bladder tumors treated with bacillus Calmette-Guerin intravesical therapy. J Urol.1999;161:792-798.
15.
Jeannon  JPSoames  JLunec  JAwwad  SAshton  VWilson  JA Expression of cyclin-dependent kinase inhibitor p21(WAF1) and p53 tumour suppressor gene in laryngeal cancer. Clin Otolaryngol.2000;25:23-27.
16.
Erber  RKlein  WAndl  T  et al Aberrant p21(CIP1/WAF1) protein accumulation in head-and-neck cancer. Int J Cancer.1997;74:383-389.
17.
Jin  YTKayser  SKemp  BL  et al The prognostic significance of the biomarkers p21WAF1/CIP1, p53, and bcl-2 in laryngeal squamous cell carcinoma. Cancer.1998;82:2159-2165.
18.
Pruneri  GPignataro  LCarboni  N  et al Clinical relevance of expression of the CIP/KIP cell-cycle inhibitors p21 and p27 in laryngeal cancer. J Clin Oncol.1999;17:3150-3159.
19.
Xie  XClausen  OPFDe Angelis  PBoysen  M Prognostic value of spontaneous apoptosis, Bax, Bcl-2, and p53 in tongue squamous cell carcinoma. Cancer.1999;86:913-920.
20.
Bukholm  IKNesland  JMKaresen  RJacobsen  UBorresen  AL Relationship between abnormal p53 protein and failure to express p21 protein in human breast carcinomas. J Pathol.1997;181:140-145.
21.
Schmider  AGee  CFriedmann  W  et al p21 WAF1/CIP1 protein expression is associated with prolonged survival but not with p53 expression in epithelial ovarian carcinoma. Gynecol Oncol.2000;77:237-242.
22.
Agarwal  SMathur  MShukla  NKRalhan  R Expression of cyclin-dependent kinase inhibitor p21waf1/cip1 in premalignant and malignant oral lesions: relationship with p53 status. Oral Oncol.1998;34:353-360.
23.
Nio  YDong  MUegaki  K  et al Comparative significance of p53 and WAF/1-p21 expression on the efficacy of adjuvant chemotherapy for resectable invasive ductal carcinoma of the pancreas. Pancreas.1999;18:117-126.
24.
Xie  XClausen  OPFDe Angelis  PBoysen  M Bax expression has prognostic significance that is enhanced when combined with AgNOR counts in glottic carcinomas. Br J Cancer.1998;78:100-105.
25.
Xie  XDe Angelis  PClausen  OPFBoysen  M Prognostic significance of proliferative and apoptotic markers in oral tongue squamous cell carcinomas. Oral Oncol.1999;35:502-509.
Original Article
August 2002

Prognostic Significance of p21WAF1/CIP1 Expression in Tongue Squamous Cell Carcinomas

Author Affiliations

From the Department of Otolaryngology (Drs Xie and Boysen) and the Institute for Pathology (Drs Xie and Clausen), The National Hospital, University of Oslo, Oslo, Norway.

Arch Otolaryngol Head Neck Surg. 2002;128(8):897-902. doi:10.1001/archotol.128.8.897
Abstract

Objective  To investigate the prognostic significance of p21WAF1/CIP1 expression and its relationship with p53 accumulation and other apoptotic markers such as Bax, Bcl-2, and apoptotic index in relation to disease-specific survival in oral tongue squamous cell carcinoma (SCC).

Design  A 10-year retrospective clinical study. Information about clinical findings, treatment, and follow-up has been recorded prospectively.

Patients and Methods  Diagnostic, formalin-fixed, paraffin-embedded sections taken from 80 randomly selected patients before treatment for T1 to T4 oral tongue SCC were stained immunohistochemically with p21WAF1/CIP1. The percentages of positive nuclei that stained positive for tumor were determined.

Main Outcome Measures  The significance of prognostic parameters was tested by log-rank and Kaplan-Meier methods for the univariate analysis. The Cox proportional hazards regression model was used for multivariate analysis.

Results  Expression of p21WAF1/CIP1 correlated inversely with T classification and clinical stage (P = .02 and P = .006, respectively) but not with N classification, tumor differentiation, or apoptosis-related variables such as p53 accumulation, apoptotic index, and Bax and Bcl-2 expression. Patients with tumors expressing high p21WAF1/CIP1 values had increased disease-specific survival time (P = .03). The correlation between p21WAF1/CIP1 expression and disease-specific survival was even more significant when restricted to p53-negative tumors (P = .002). A 2-parameter combination between p21WAF1/CIP1 expression and Bax expression or p21WAF1/CIP1 expression and p53 accumulation, respectively, revealed an enhanced prognostic potential (P<.001) when compared with single parameters.

Conclusion  The expression of p21WAF1/CIP1, particularly in combination with p53 accumulation or Bax expression, has prognostic value in oral tongue SCC.

TUMORIGENESIS and progression of malignant tumors are characterized by uncontrolled cell proliferation. In mammalians, the cell cycle progression is regulated by the cyclin-dependent kinases, which are activated by cyclin and inactivated by inhibitors of cyclin-dependent kinases including p21WAF1/CIP1. The p21 protein is the gene product of the WAF1, CIP1, or SD11 gene, which play an important role in regulating the G1-S transition of the cell cycle.14 The biological significance of p21WAF1/CIP1 in controlling cell proliferation is not fully understood. However, in response to DNA damage, wild-type p53 accumulates and binds to the promoter region of the p21WAF1/CIP1 gene.2,5 This induces the expression of the p21WAF1/CIP1 that inhibits the activity of the cyclin/cyclin-dependent kinase complex to block cell cycle progression.3,5 In addition, p21WAF1/CIP1 may block DNA synthesis by direct binding to the proliferating cell nuclear antigen that is important in DNA replication.6,7 The levels of p21WAF1 may also be elevated by non–p53-dependent mechanisms.6,8 Furthermore, p21WAF1/CIP1 is associated with terminal differentiation and cell senescence, and thus may play a role in cell maturation and cell death.4,9

Results presented by previous studies regarding the correlation between expression of p21WAF1/CIP1 and prognosis are controversial. In several tumor types, expression or overexpression of p21WAF1/CIP1 seems to be associated with favorable clinical outcome.1012 Such correlation has, however, not been confirmed by others.13,14 These conflicting findings have also been reported in head and neck cancers.1518 So far, the prognostic significance of p21WAF1/CIP1 expression has scarcely been reported in tongue squamous cell carcinoma (SCC).

In the present study, we investigate the expression of p21WAF1/CIP1 in a series of oral tongue SCCs and the possible correlation between this parameter and clinical, histopathologic, p53, and apoptosis-related markers such as apoptotic index (AI) and Bax and Bcl-2 expression.19 Furthermore we have also tested whether a combination between parameters might have predictive value.

PATIENTS, MATERIALS, AND METHODS

We analyzed formalin-fixed, paraffin-embedded pretreatment specimens from 80 randomly selected patients with oral tongue SCCs admitted to the Department of Otolaryngology, National Hospital, University of Oslo, Oslo, Norway, between 1983 and 1992. There were 34 women and 46 men ranging in age from 31 to 88 years (mean age, 63 years). All the tumors were classified according to the International Union Against Cancer (UICC) TNM classification of 1987. There were 51 T1 to T2 tumors and 29 T3 to T4 tumors. Thirty-seven patients had metastasis (N1, 22; N2, 14; and N3, 1). Clinical findings, treatment, and follow-up have been recorded prospectively. None of the patients were lost to follow-up. The range of follow-up is 0.1 to 11.6 years. Of 14 patients who died from diseases other than tongue SCC, only 3 patients had an observation time shorter than 3 years (1.7, 2.3, and 2.8 years, respectively). Thirty-seven patients died of the disease (15 T1-T2 and 22 T3-T4).

Five patients with small primary tumors (T1) were treated with surgery alone. Forty-three patients received combined treatment, 24 with radiotherapy (60-70 Gy) followed by surgery, and 19 with surgery followed by radiotherapy (50-60 Gy). Thirty-two patients with nonresectable tumors or who were unsuited for or refused surgery received radiotherapy alone (70-80 Gy). The dose used for elective radiotherapy of the neck was 50 Gy, whereas patients with neck metastases received 60 Gy followed by surgery whenever possible. Of 41 treatment failures, 4 patients with local recurrence underwent successful salvage procedures (all had T1-T2 tumors).

HISTOPATHOLOGIC PROCEDURES

Serial 5-µm-thick sections were cut from the tissue blocks and mounted on gelatin-coated slides. One section was stained with hematoxylin-eosin for verification of the original histopathologic diagnosis and histologic differentiation. The histologic differentiation was graded by one of us (O.P.F.C.) as 1, good; 2, moderate; 3, poor; and 4, undifferentiated.

IMMUNOHISTOCHEMICAL ANALYSIS
Staining

In brief, after being dewaxed in xylene, the sections were dehydrated in ethanol and rinsed in distilled water. For antigen retrieval, the slides were then incubated 5 times in 10 mM citric acid buffer (pH, 6.3) in a microwave oven (750 W) for 5 minutes. After cooling for 30 minutes at room temperature, the sections were washed in Tris buffer for 5 minutes and incubated overnight at 4°C with 1:100 dilution monoclonal antibody against p21 (WAF, Ab-1; Oncogene Science, Cambridge, Mass). The sections were incubated with biotinylated secondary antibody (1:500), washed with phosphate-buffered saline, incubated for 40 minutes with avidin-biotinylated peroxidase complex solution, then placed in a development solution containing 0.06% diaminobenzidine and 0.1% volume per volume hydrogen peroxide. The sections were finally counterstained with hematoxylin and mounted. For each batch of immunostained sections, 1 positive and 1 negative control (without the primary antibody) were included.

Evaluation

Ten systematically chosen fields were evaluated by means of an ×100 magnification oil-immersion lens. The percentages of tumor cells with, respectively, nuclear staining and cytoplasmic staining were determined. Specimens with fewer than 5% positive cells were considered negative. All percentages were determined by one of us (X.X.), and the results were based on the evaluation of at least 700 tumor cells in each section. Areas with pronounced inflammation, necrosis, or artificial damage were avoided. The assessments were performed without knowledge of the clinical outcome. Six months after the completion of the evaluation, 16 previously examined sections were randomly selected and percentages of tumor cells redetermined to test the reproducibility of the method.

STATISTICAL PROCEDURES

The data were stored and analyzed by means of SAS 6.12 software (SAS Institute, Cary, NC). The χ2 test was used for comparison of group means. Correlation between parameters was performed by the Pearson test. Kaplan-Meier plots and log-rank tests were used to visualize and evaluate the significance of clinical variables and p21WAF1/CIP1 expression in relation to disease-specific survival. The DISCRIM procedure that computes various discriminant functions for classifying observations into groups on the basis of quantitative variables was used to screen possible combinations between 2 variables in relation to disease-specific survival. A case was censored when death resulted from causes other than the original tumor and when there was no evidence of residual tumors or recurrences at the last follow-up consultation. Prognostically significant parameters by the univariate analysis were further analyzed by means of the Cox proportional hazards regression model. Statistical significance was set at P<.05. Reproducibility was tested by means of the least squares regression analysis.

RESULTS

In normal epithelium adjacent to the tumors, p21WAF1/CIP1 was regularly expressed in the suprabasal region with nuclear staining, whereas nuclei in the superficial and/or basal regions were only occasionally stained. Cytoplasmic staining was also seen in some cases. In tumor cells, the p21WAF1/CIP1 expression was mainly nuclear, although cytoplasmic staining was also found in many cases. Some of the tumor cells showed both nuclear and cytoplasmic staining. Seventy-four tumors (92%) showed variable nuclear expression of p21WAF1/CIP1, with a mean value of 32% (range, 0%-88%), among them 25 cases (33%) with additional cytoplasmic staining. Three tumors showed cytoplasmic staining only.

Of 47 cases of p53-positive tumors, 45 showed p21WAF1/CIP1 nuclear expression. Eighteen of them showed a sum of more than 100% tumor nuclei stained for p21WAF1/CIP1 and p53 proteins, which indicates that parts of the tumor cells were positive for both p21WAF1/CIP1 and p53.

Results from p21WAF1/CIP1 nuclear expression were compared with histopathologic and clinical parameters and with our previously investigated apoptosis-related markers, including Bax and Bcl-2 expression, AI, and p53 accumulation.19 Expression of p21WAF1/CIP1 correlated inversely with T classification and clinical stage (P = .02 and P = .006, respectively). We found no correlation between p21WAF1/CIP1 and tumor differentiation, N classification, or any of the apoptosis-related parameters.

No correlation was found between p21WAF1/CIP1 nuclear expression and disease-specific survival when the overall mean value was used as the cutoff level. However, when using a plot diagram, we found that 23% of p21WAF1/CIP1 expression gave the best cutoff level regarding patient clinical outcome. Tumors with p21WAF1/CIP1 nuclear expression exceeding this 23% level had a significantly longer disease-specific survival time than those with values below 23% (P = .03; Figure 1). In all 33 cases of p53-negative tumors, p21WAF1/CIP1 nuclear expression correlated strongly with disease-specific survival using 23% as the cutoff level (P = .002; Figure 2). We found no correlation between p21WAF1/CIP1 cytoplasmic staining and patient clinical outcome.

Table 1 summarizes the findings of the log-rank analyses for clinical parameters, tumor differentiation, and p21WAF1/CIP1 expression. T and N classifications, clinical stage, treatment, and p21WAF1/CIP1 expression were significantly associated with disease-specific survival. Figure 3 shows the Kaplan-Meier plots for p21WAF1/CIP1 expression in relation to disease-specific survival (P = .04). Multiple regression analysis (Cox method) revealed clinical stage as the only significant independent variable.

In an attempt to elucidate a possible synergistic prognostic effect, we tested whether combination between p21WAF1/CIP1 expression and apoptosis-related parameters such as p53 accumulation, AI, and Bax and Bcl-2 expression might improve the prognostic ability with respect to disease-specific survival. The DISCRIM analysis indicated that there was an association between p21WAF1/CIP1 expression and p53 accumulation or Bax expression in discriminating between patients with favorable and unfavorable clinical outcome. We therefore selected 3 to 4 cutoff levels to obtain scores for each of the parameters evaluated according to their distribution in relation to disease-specific survival (Table 2). Since tumors with high values for p21WAF1/CIP1 and Bax expression correlated positively with favorable outcome, whereas high levels of p53 tended to correlate with poor prognosis, the scores of p21WAF1/CIP1 expression were added to the scores of Bax expression, but subtracted from the scores of p53 accumulation to test the prognostic significance of the new combined parameters.

Table 3 summarizes the findings of the log-rank analysis for individual parameters and the new combined parameters in relation to disease-specific survival and indicates that combined parameters seemed to be stronger prognostic indicators than any of the parameters alone. Figure 4 and Figure 5 show the cooperative effect between p21WAF1/CIP1 expression and, respectively, p53 accumulation and Bax expression in discriminating between survivors and nonsurvivors. Least squares regression analysis resulted in the R2 value of 0.8 for p21WAF1/CIP1 expression.

COMMENT

No correlation was found between the expression of p21WAF1/CIP1 and p53 accumulation. This is in agreement with the findings presented by 2 other studies in head and neck SCC.16,18 Previous studies have suggested that p21WAF1/CIP1 expression is induced by wild-type p53.2,5 This was supported by 2 studies in breast and ovarian cancers where p21WAF1/CIP1 expression was inversely correlated with p53 accumulation,12,20 but not confirmed in 2 other studies of breast carcinoma and epithelial ovarian carcinomas.13,21 Consequently, it is possible that p53-dependent and p53-independent mechanisms may be involved in p21WAF1/CIP1 expression.22,23 This is supported by the findings of similar frequencies of p21WAF1/CIP1 expression in p53 wild-type tumors and p53 mutant tumors in head and neck SCC.16,18,22 A study on oral premalignant and malignant lesions also suggested that expression of p21WAF1/CIP1 is induced by p53-dependent as well as p53-independent mechanisms during oral tumorigenesis.22 In the present study, nearly all the p53-positive tumors expressed p21WAF1/CIP1. Eighteen tumors showed a sum of more than 100% tumor nuclei stained for p21WAF1/CIP1 and p53 proteins, which indicates that parts of the tumor cells were positive for both p21WAF1/CIP1 and p53. These findings support the finding that p53-dependent and p53-independent mechanisms are involved in p21WAF1/CIP1 expression in tongue SCC.

In the present study, we show that patients with tumors that have high p21WAF1/CIP1 expression values have a more favorable clinical outcome than those with low values. This is in agreement with the findings from studies of several other types of tumor such as breast carcinoma, lung SCC, epithelial ovarian cancer, and laryngeal carcinoma.1012,15 Contradictory results have also been reported in breast carcinoma, superficial bladder tumors, and head and neck cancer,13,14,16 whereas no correlation was found between p21WAF1/CIP1 expression and prognosis in laryngeal SCC.17 These contradictory results for head and neck cancer as well as for tumors at other sites indicate that the association between prognosis and expression of p21WAF1/CIP1 is complex and may vary from tumor type to tumor type. In addition, we clearly show that the impact of p21WAF1/CIP1 expression on prognosis is stronger among p53-negative tumors than among those with p53 accumulation. This suggests that the p53-dependent regulation of p21 is more important for disease-specific survival than the p53-independent pathway.

Our findings indicate that the evaluation of the combined variable p21WAF1/CIP1 expression and p53 accumulation may have more prognostic value discriminating patients with favorable and unfavorable clinical outcome in tongue SCC than evaluation of single variables. This observation is supported by studies on breast carcinoma and invasive ductal carcinoma of the pancreas.13,23 Unlike the method used by others who compared different groups of patients using 1 cutoff level (positive/negative) for p21WAF1/CIP1 and p53, we selected several cutoff levels to obtain scores for p21WAF1/CIP1 and p53 accumulation according to their distribution with regard to the clinical outcome. With this method we demonstrated a more complex and synergistic relationship between these 2 variables in discriminating patients with good and poor prognosis. Interestingly, p53 in itself was not a statistically significant prognostic variable in this series of patients. However, when p53 was combined with p21WAF1/CIP1, this new variable seemed to be a strong prognostic parameter.

Concerning the relationship between p21WAF1/CIP1 expression and apoptosis-related markers including AI and Bax and Bcl-2 expression, we found no correlation. However, our results showed that evaluation of p21WAF1/CIP1 and Bax expression combined improves prognostic ability over single parameters alone. This finding is in agreement with our previous observation in head and neck carcinomas where evaluation of the combination of proliferative and apoptotic markers was of greater prognostic value than evaluation of individual variables.24,25 This observation may suggest that such a combination of p21WAF1/CIP1 and Bax expression reflects different aspects of cell proliferation and apoptosis.

Our investigation suggests that p21WAF1/CIP1 expression has prognostic value in SCC of the tongue. Evaluation of p21WAF1/CIP1 expression in combination with, respectively, p53 accumulation and Bax expression provides considerably better prognostic information than evaluation of any individual variables alone.

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Article Information

Accepted for publication January 17, 2002.

This study was supported by funding from the Norwegian Cancer Society, Oslo.

We thank the Norwegian Cancer Society for generously supporting this study and Aasa Schjölberg for excellent technical assistance.

Corresponding author and reprints: Xin Xie, MD, PhD, Department of Otolaryngology, The National Hospital, 0027 Oslo, Norway (e-mail: xin.xie@labmed.uio.no).

References
1.
Sherr  CJ Cancer cell cycles. Science.1996;274:1672-1677.
2.
el-Deiry  WSTokino  TVelculescu  VE  et al WAF1, a potential mediator of p53 tumor suppression. Cell.1993;75:817-825.
3.
Harper  JWAdami  GRWei  NKeyomarsi  KElledge  SJ The p21 cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell.1993;75:805-816.
4.
Noda  ANing  YVenable  SFPereira-Smith  OMSmith  JR Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res.1994;211:90-98.
5.
Xiong  YHannon  GJZhang  HCasso  DKobayashi  RBeach  D p21 is a universal inhibitor of cyclin kinases. Nature.1993;366:701-704.
6.
Waga  SHannon  GJBeach  DStillman  B The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature.1994;369:574-578.
7.
Chen  JJackson  PKKirschner  MWDutta  A Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature.1995;374:386-388.
8.
Parker  SBEichele  GZhang  P  et al p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells. Science.1995;267:1024-1027.
9.
Jiang  HLin  JSu  ZZCollart  FRHuberman  EFisher  PB Induction of differentiation in human promyelocytic-HL-60-leukemia cells activates p21WAF1/CIP1 expression in the absence of p53. Oncogene.1994;9:3397-3406.
10.
Jiang  MShao  ZMWu  J  et al p21/waf1/cip1 and mdm-2 expression in breast carcinoma patients as related to prognosis. Int J Cancer.1997;74:529-534.
11.
Komiya  THosono  YHirashima  T  et al p21 expression as a predictor for favorable prognosis in squamous cell carcinoma of the lung. Clin Cancer Res.1997;3:1831-1835.
12.
Anttila  MAKosma  VMHongxiu  J  et al p21/WAF1 expression as related to p53, cell proliferation and prognosis in epithelial ovarian cancer. Br J Cancer.1999;79:1870-1878.
13.
Caffo  ODoglioni  CVeronese  S  et al Prognostic value of p21(WAF1) and p53 expression in breast carcinoma: an immunohistochemical study in 261 patients with long-term follow-up. Clin Cancer Res.1996;2:1591-1599.
14.
Zlotta  ARNoel  JCFayt  I  et al Correlation and prognostic significance of p53, p21WAF1/CIP1 and Ki-67 expression in patients with superficial bladder tumors treated with bacillus Calmette-Guerin intravesical therapy. J Urol.1999;161:792-798.
15.
Jeannon  JPSoames  JLunec  JAwwad  SAshton  VWilson  JA Expression of cyclin-dependent kinase inhibitor p21(WAF1) and p53 tumour suppressor gene in laryngeal cancer. Clin Otolaryngol.2000;25:23-27.
16.
Erber  RKlein  WAndl  T  et al Aberrant p21(CIP1/WAF1) protein accumulation in head-and-neck cancer. Int J Cancer.1997;74:383-389.
17.
Jin  YTKayser  SKemp  BL  et al The prognostic significance of the biomarkers p21WAF1/CIP1, p53, and bcl-2 in laryngeal squamous cell carcinoma. Cancer.1998;82:2159-2165.
18.
Pruneri  GPignataro  LCarboni  N  et al Clinical relevance of expression of the CIP/KIP cell-cycle inhibitors p21 and p27 in laryngeal cancer. J Clin Oncol.1999;17:3150-3159.
19.
Xie  XClausen  OPFDe Angelis  PBoysen  M Prognostic value of spontaneous apoptosis, Bax, Bcl-2, and p53 in tongue squamous cell carcinoma. Cancer.1999;86:913-920.
20.
Bukholm  IKNesland  JMKaresen  RJacobsen  UBorresen  AL Relationship between abnormal p53 protein and failure to express p21 protein in human breast carcinomas. J Pathol.1997;181:140-145.
21.
Schmider  AGee  CFriedmann  W  et al p21 WAF1/CIP1 protein expression is associated with prolonged survival but not with p53 expression in epithelial ovarian carcinoma. Gynecol Oncol.2000;77:237-242.
22.
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