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© 2002 American Society for Clinical Oncology Clinical Significance of p21 Expression in Non–Small-Cell Lung CancerByFrom the Department of Thoracic Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan. Address reprint requests to Hiromi Wada, MD, Department of Thoracic Surgery, Faculty of Medicine, Kyoto University, Shogoin-kawahara-cho 54, Sakyo-ku, Kyoto 606-8397, Japan; email: wadah{at}kuhp.kyoto-u.ac.jp
PURPOSE: The clinical significance of p21 expression remains unclear, whereas many experimental studies have demonstrated that p21, the product of the WAF1/CIP1/SDI1 gene, plays an important role in regulation of the cell cycle as an inhibitor of cyclin-dependent kinases. The purpose of this study was to clarify the clinical significance in resected non–small-cell lung cancer (NSCLC). PATIENTS AND METHODS: A total of 233 consecutive patients with completely resected pathologic stage I to IIIA NSCLC were retrospectively reviewed. Expression of p21 and the status of p53 were examined immunohistochemically. Proliferative activity was also evaluated immunohistochemically. The incidence of apoptotic cell death was evaluated by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick end-labeling staining. RESULTS: Expression of p21 was positive in 120 patients (51.5%). The 5-year survival rate of p21-positive patients was 73.8%, significantly higher than that of p21-negative patients (60.7%; P = .006). Aberrant expression of p53 was positive in 98 patients (42.1%). When combined with p53 status, the prognostic value of p21 status was enhanced: the 5-year survival rate of p21-positive and p53-negative patients was 80.7%, markedly higher than that of p21-negative and p53-positive patients (50.0% for both; P = .001). Multivariate analysis confirmed that positive expression of p21 was a significant factor for predicting a favorable prognosis. There was no significant correlation between p21 expression and p53 status, proliferative activity, or incidence of apoptosis. CONCLUSION: p21 expression was shown to be an independent prognostic factor in NSCLC.
IN MOST INDUSTRIALIZED countries, non–small-cell lung cancer (NSCLC) is the leading cause of cancer death and has a poor prognosis.1 To improve the prognosis, clinical markers that may predict the prognosis and response toward a specific therapy should be established. Although many biologic markers, including p53 abnormality, have been investigated, no biologic marker has been established as a clinical marker in the treatment of NSCLC.2 p21, the product of the WAF1/CIP1/SDI1 gene, is an inhibitor of cyclin-dependent kinases3-5 and is activated through p53-dependent6,7 or p53-independent pathways.8,9 Whereas many experimental studies have clearly indicated that p21 plays an important role in regulation of the cell cycle, especially in G1 arrest,3-7 the clinical significance of p21 expression in NSCLC remains unclear. Among many clinical studies on p21 expression in NSCLC,8,10-16 only a few documented a significant correlation between p21 expression and prognosis.15,17 In this study, therefore, the clinical significance of p21 expression in completely resected NSCLC was assessed to clarify the clinical significance. In addition, the correlations between p21 expression and the incidence of apoptosis, proliferative activity, and p53 status were also assessed.
Clinical Characteristics of Patients We reviewed a total of 237 consecutive patients with pathologic stage I to IIIA NSCLC who underwent complete tumor resection and mediastinal lymph node dissection without any preoperative therapy at the Department of Thoracic Surgery, Kyoto University, between January 1, 1985, and December 31, 1990 (Table 1). This study was approved by the institutional review board of Kyoto University (G27, clinical study of gene expression in lung cancer; G28, clinical study of gene mutation in lung cancer). Complete tumor resection was considered to have been achieved when no microscopic cancer cells were identified in either the margin of resection of the tumor or the highest mediastinal lymph nodes.18 Pathologic stage was re-determined according to the current tumor-node-metastasis classification.19 Histologic type was also re-determined according to the classification by the World Health Organization as revised in 1999.20 One patient was excluded from the study because of operation-related death, and specimens were not available for three patients. Thus, 233 patients were finally evaluated in this study. For all these patients, the inpatient medical records, chest x-ray films, whole-body computed tomography films, bone and gallium scanning data, and records of surgery were reviewed without knowledge of the results of immunohistochemical staining (IHS) or terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) staining. Details of postoperative adjuvant therapy were described previously.21,22 In brief, cisplatin-based chemotherapy, radiation, and oral administration of tegafur (a fluorouracil derivative drug) were prescribed after surgery for 55, 35, and 57 patients, respectively. Intraoperative therapy was not performed on any patient. The day of thoracotomy was considered the starting day for counting postoperative survival days.
Tissue Preparation All tumor specimens were fixed immediately in 10% (vol/vol) formalin and then embedded in paraffin. Serial 4-µm sections were prepared from each sample and used for routine hematoxylin and eosin (HE) staining. IHS was used to detect p21 expression, as well as proliferative cell nuclear antigen (PCNA) expression and p53 aberrant expression, and TUNEL staining was used to detect apoptotic cells. For IHS, dewaxed sections were heated in a microwave oven for 5 minutes three times each to retrieve their antigenicities. For the TUNEL staining, dewaxed sections were digested with proteinase K 20 µg/mL of (Boehringer Mannheim, Mannheim, Germany) for 20 minutes at 25°C for the TUNEL staining.21 Endogenous peroxidase was inactivated by incubating the sections with 0.03% H2O2 in methanol (Nakalai Tesque, Kyoto, Japan) for 30 minutes at 25°C.
Immunohistochemistry A total of 1,000 tumor cells were counted to assess positive staining, and the percentages of positive-staining cells were determined. The slide was judged to exhibit positive expression of p21 or aberrant expression of p53 when the percentage of the cells with positive nuclear staining exceeded 5%. We chose the cutoff value of p21-positive expression on the basis of the results of previous studies8,15,17; a significant correlation between p21 expression determined with IHS and expression of the mRNA determined with Northern blot analysis has been proven in NSCLC, when the cutoff value of 5% was used in the evaluation of p21 expression with IHS. In the p53 analysis, we chose the cutoff value of 5%, referring to our previous studies.22-25 In 27 patients (11.6%), slides were judged to positively express p21 by one author but were judged to be negative by the other author; such slides were re-evaluated until the evaluations coincided. In case of a discordant evaluation after re-evaluation, the slides were evaluated by another author (F.T.). Proliferative index (PI) was defined as the percentage of PCNA positive-staining cancer cells. The details of the evaluation of PI were described previously.21
Detection of Apoptosis
Statistical Methods
Expression of p21 in NSCLC The percentage of p21-positive cells among tumor cells was 7.9% ± 0.7% (mean ± SE) for all patients. When the cutoff value of 5% was used in evaluation of p21 positivity, as described in Patients and Methods, expression of p21 was positive in 120 patients (51.5% of all patients). The mean percentages (± SEs) of p21-positive staining tumor cells were 14.3% ± 1.0% and 1.1% ± 0.2% for p21-positive patients and p21-negative patients, respectively. Expression of p21 according to patients’ characteristics is listed in Table 1. There was no significant correlation between the p21 expression and age, sex, performance status, histologic type, degree of cancer cell differentiation, or pathologic stage.
p21 Expression and Postoperative Survival Postoperative survival according to p21 status is listed in Table 2. For all patients, the 5-year survival rates of the p21-positive and p21-negative patients were 73.8% and 60.7%, respectively, demonstrating a significantly better prognosis for p21-positive patients (P = .006; Fig 1). In addition, the mean disease-free survival of the p21-positive patients was significantly higher than that of the p21-negative patients (2,994 days [95% CI, 2,601 to 3,388 days] and 2,202 days [95% CI, 1,913 to 2,491 days], respectively; P = .012).
Postoperative survival was also analyzed after stratification by pathologic stage. For pathologic stage IA disease, the 5-year survival rates of p21-positive and p21-negative patients were 97.1% and 74.3%, respectively, demonstrating a significantly better prognosis for the p21-positive patients (P = .007; Table 2). For pathologic stage II disease, the 5-year survival rate of p21-positive patients was also significantly higher than that of p21-negative patients (82.5% and 51.6%, respectively; P = .038; Table 2). No significant difference in the prognosis between the p21-positive patients and the p21-negative patients was demonstrated for pathologic stage IB and IIIA (cN0 to cN1 or cN2) disease (Table 2).
p21 and p53 Expression in NSCLC
To clarify the prognostic significance of p21 expression combined with p53 status, postoperative survival was analyzed after all of the patients were divided into the following four groups on the basis of p21 and p53 status: (1) p21-positive and p53-positive [p21(+)/p53(+)] patients; (2) p21-positive and p53-negative [p21(+)/p53(-)] patients; (3) p21-negative and p53-positive [p21(-)/p53(+)] patients; and (4) p21-negative and p53-negative [p21(-)/p53(-)] patients. The p21(+)/p53(-) patients had the most favorable prognosis, with a 5-year survival rate of 80.7%, which was significantly higher than that of the p21(-)/p53(+) patients (50.0%; P < .001) or that of p21(+)/p53(+) patients (62.9%; P = .032; Fig 2).
In an attempt to investigate prognosis in a more homogeneous patient group, only the pathologic stage I patient prognosis was examined, because the majority of the patients studied had pathologic stage I disease. The results were similar to those obtained for all patients. The p21(+)/p53(-) patients had the most favorable prognosis; the 5-year survival rate was 92.4%, significantly higher than that of p21(-)/p53(+) patients and that of p21(+)/p53(+) patients (Fig 3).
p21 Expression, PI, and AI The mean AI values (± SEs) of the p21-positive patients and the p21-negative patients were 18.1 ± 2.0 and 19.4 ± 2.0, respectively, with no significant difference between the two groups (P = .651). The percentage of p21-positive tumor cells in correlation with the AI value in each patient is indicated in Fig 4, with no significant correlation between p21 status and AI.
The mean PI values (± SEs) of the p21-positive patients and the p21-negative patients were 48.8% ± 2.6% and 44.5% ± 2.6%, respectively, with no significant difference between the groups (P = .235). No significant correlation between the percentage of p21-positive tumor cells and PI was demonstrated (Fig 5).
Multivariate Analysis of Prognostic Factors Multivariate analysis confirmed that positive expression of p21 was an independent and significant factor to predict a favorable prognosis. Aberrant expression of p53, pathologic stage, and oral administration of tegafur were also significant prognostic factors (Table 4).
This study clearly demonstrated that positive expression of p21 was a significant factor to predict a favorable prognosis in completely resected NSCLC, whereas it should be also noted that other factors might have made a difference in the prognosis of these patients. These factors are more difficult to evaluate and include other intercurrent illness. Although many studies on p21 status in correlation with the postoperative survival of NSCLC patients have been conducted,8,10-16 the results are not consistent, which may be partly due to the small number and heterogeneity of the patients studied. The reason why the prognostic significance of p21 status was revealed only in this series may be that it used an adequate number (> 200) of patients and had a homogenous population (most patients with pathologic stage I disease). In fact, the percentage of patients with p21-positive tumors was smaller than that documented in a previous study conducted by Marchetti et al,8 even though a similar IHS procedure was used in both studies. The number of patients reviewed in this study was much higher than that in the previous study, which may make a difference not only in the frequency of p21-positive patients but also in the prognostic significance of p21 status. In this study, we examined p21 expression immunohistochemically, and the cutoff value of 5% was used for p21 positivity on the basis of results in previous studies. Although one study documented a significant correlation between p21 immunohistochemical positivity and p21 RNA expression in NSCLC,8 it should be noted that a critical issue remains in the immunohistochemical evaluation of p21 positivity. More quantitative analyses of p21 status, such as real-time reverse transcriptase polymerase chain reaction for the evaluation of mRNA and Western blotting for the evaluation of p21, should be used in the future to clarify the clinical significance of p21 status. This study also demonstrated that the prognostic significance of p21 was enhanced in combination with p53 status. These results may suggest that a combination of p21 status with p53 status may be more useful in deciding the treatment for each NSCLC patient, which should be examined in prospective studies in the future. Whereas many experimental studies demonstrated that p21 was a downstream target of wild-type p53,6,7 this study indicated no significant correlation between p21 status and p53 status, which was consistent with the results of other previous studies.8,10,11,13-16,26 These findings documented in clinical studies may be due to the activation of p21 through p53-independent pathways.9 However, it should be also noted that both p21 expression and p53 status were evaluated immunohistochemically in this study, and the accuracy of IHS for predicting p53 mutation has been reported to be as low as 70%.27,28 In the future, the correlation between p21 expression and p53 status, as determined by mutation analyses, should be analyzed. This study did not find a correlation between p21 status and PI, which was consistent with the results of clinical studies conducted by Groeger et al.12 It was shown that the p53 to p21 pathway inhibits DNA replication by means of p21’s interaction with PCNA29 but does not affect the DNA repair abilities of PCNA.30 These mechanisms will buy time for the machinery involved in DNA repair to effectively correct alterations in genomic DNA. In this study, no correlation was found between p21 expression and PI. Our results may indicate, as suggested by Groeger et al,12 that the control of DNA repair by PCNA is the most important activity of p21 during lung carcinogenesis, compared with its contribution to cell proliferation. In other studies, the mutational status of k-ras has been extensively studied as a prognostic marker in NSCLC, and k-ras mutation has been reported to be a negative prognostic factor.31-36 In this study, we did not evaluate the k-ras status; however, it will be important to examine the relationship between p21 expression and k-ras mutation in NSCLC in future investigations. In conclusion, positive expression of p21 is a significant factor to predict a favorable prognosis, and it may be an im-portant clinical marker in therapy for NSCLC. Patients without p21 expression can be candidates for adjuvant therapy, although further studies examining the relationship between p21 status and the efficacy of adjuvant therapies should be performed.
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test. Continuous data were compared by using Student’s t test if the distribution of the samples was normal or by using the Mann-Whitney U test if the sample distribution was asymmetric. The postoperative survival rate was analyzed by the Kaplan-Meier method, and differences in the survival rates were assessed by the log-rank test. Death from any cause was included in the calculation of postoperative survival. Multivariate analysis of the prognostic factors was performed with Cox’s regression model. Differences were considered significant when the P value was less than .05. All statistical manipulations were performed with SPSS for Windows (SPSS Inc., Chicago, IL). 
