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Originally published as JCO Early Release 10.1200/JCO.2004.05.174 on February 23 2004 © 2004 American Society of Clinical Oncology. Combined Effects of p53, p21, and pRb Expression in the Progression of Bladder Transitional Cell CarcinomaFrom the Departments of Pathology, Urology, and Preventive Medicine, University of Southern California Keck School of Medicine; and University of Southern California/Kenneth Norris Comprehensive Cancer Center, Los Angeles, CA Address reprint requests to Richard J. Cote, MD, FRCPath, Department of Pathology, University of Southern California/Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, 1441 Eastlake Ave, Los Angeles, CA 90033; e-mail: cote_r{at}norsur.hsc.usc.edu
PURPOSE: To determine the combined effects of p53, p21, and pRb alterations in predicting the progression of bladder transitional cell carcinoma. PATIENTS AND METHODS: p53, p21, and pRb expression was examined immunohistochemically on archival radical cystectomy samples from 164 patients with invasive or high-grade recurrent superficial transitional cell carcinoma (TCC; lymph node–negative, 117 patients; lymph node–positive, 47 patients). Median follow-up was 8.6 years. Based on percentage of nuclear reactivity, p53 was considered as wild-type (0% to 10%) or altered (> 10%); p21 was scored as wild-type (>10%) or altered (< 10%); and pRb status was considered wild-type (1% to 50%) or altered (0% or > 50%). RESULTS: As individual determinants, the p53, p21, and pRb status were independent predictors of time to recurrence (P < .001, P < .001, and P < .001, respectively), and overall survival (P < .001, P = .002, and P = .001, respectively). By examining these determinants in combination, patients were categorized as group I (no alteration in any determinant, 47 patients), group II (any one determinant altered, 51 patients), group III (any two determinants altered, 42 patients), and group IV (all three determinants altered, 24 patients). The 5-year recurrence rates in these groups were 23%, 32%, 57%, and 93%, respectively (log-rank P < .001), and the 5-year survival rates were 70%, 58%, 33%, and 8%, respectively (log-rank P < .001). After stratifying by stage, the number of altered proteins remained significantly associated with time to recurrence and overall survival. CONCLUSION: This study suggests that alterations in p53, p21, and pRb act in cooperative or synergistic ways to promote bladder cancer progression. Examining these determinants in combination provides additional information above the use of a single determinant alone.
Tumorigenesis and tumor progression are thought to result from the accumulation of multiple genetic alterations, including the activation of oncogenes, loss of distinct chromosomal regions, and inactivation of tumor suppressor genes [1-3]. Over- or underexpression and alterations in the proteins encoded by these genes leads to a perturbation in the intricate pathways involving signal transduction and cell cycle control. Furthermore, comprehensive analyses of human tumors and cell lines have demonstrated that tumor suppressor gene alterations can be grouped according to their functional role within independent cascades of the cell cycle control machinery [4,5]. Thus knowledge of the biologic roles and the effects of these genetic changes may potentially aid clinicians in predicting the clinical outcome (ie, recurrence and survival), as well as the response of individual patients to treatment. In human bladder transitional cell carcinoma (TCC), alterations in tumor suppressor genes such as p53 and Rb are known to be common events and have been reported to be associated with tumor progression [6-14]. (The terms TCC and urothelial carcinoma are interchangeable and both are used to denote primary neoplasms of the bladder. We use the term TCC here, to maintain consistency, because it is the designation we have used in our previous studies.) We previously demonstrated that abnormal nuclear accumulation of p53 along with either altered p21 or altered pRb (absent pRb or highly expressed pRb) may have cooperative effects in promoting tumor progression [7,8]. Furthermore, cell line studies using mouse embryo fibroblasts have indicated that loss of pRb and p21 have additive effects in cell cycle dysregulation and subsequent tumor development [15,16]. However, the effect of alterations in p21 and pRb, independent of p53 alterations, and the effect of alterations in p53, p21, and pRb has remained incompletely described. In the present study, we used immunohistochemistry to analyze samples of surgically resected bladder TCC to determine whether there is a cooperative or independent relationship between p53, p21, and pRb alterations in predicting tumor progression. We further extended our analysis to include classic prognostic indicators to determine the importance that each variable plays in predicting overall patient survival and tumor recurrence.
Patient Population This study includes 164 patients who underwent en bloc radical cystectomy, pelvic lymphadenectomy, and urinary reconstruction for muscle-invasive TCC of the bladder (n = 131) or recurrent high-grade superficial TCC of the bladder that had become refractory to intravesicle therapy (n = 33) at the University of Southern California/Norris Comprehensive Cancer Center from April 1983 to December 1988. We have previously reported on the association of p53, p21, and Rb—each examined individually—with outcome [6-8]; this series includes all patients for whom immunohistochemical results are available on all three proteins. Patients with pure adenocarcinoma, squamous cell carcinoma, or small-cell carcinoma were not included in the analysis. Five patients received systemic chemotherapy before cystectomy and 27 patients received adjuvant chemotherapy after cystectomy. At the time of cystectomy, no patients received adjuvant or neoadjuvant radiation. Patient follow-up consisted of evaluations every 3 months for the first year, every 4 months during the second year postoperatively, and annually thereafter. Follow-up in all cases included a serum biochemical profile (serum electrolytes and CBC), chest radiography, and a physical examination. Further radiographic studies, with computed tomography or bone scan, were performed in suspected cases of recurrent disease. At time of recurrence, the majority of patients (62%) received chemotherapy; 14% received radiation therapy alone. Tumor samples from the cystectomy specimens were preserved as archival paraffin-embedded tissue blocks and were available in all cases. This study was approved by the University of Southern California/Norris Comprehensive Cancer Center institutional review board. Histologic grading (grades 1 to 4) was performed on the basis of the Bergkvist grading system [17], and the pathologic stage was classified according to the tumor-node-metastasis system [18].
Antibodies and Immunohistochemistry
Statistical Analysis
Association of p53, p21, and pRb Status With Tumor Stage and Grade Associations of p53, p21, and pRb expression with standard TCC clinicopathologic features are listed in Table 1. p53 expression was significantly associated with pathologic tumor stage and lymph node status (P = .008); higher stage disease was associated with a higher proportion of cases demonstrating altered p53 expression. However, there was no significant association between p53 expression and tumor grade (P = .32). p21 expression was associated with stage of disease (P = .005) but not with tumor grade (P = .40). On the other hand, altered pRb protein expression (ie, tumors with either no detectable expression or very high pRb expression) was significantly associated with both stage (P = .002) and grade (P = .011).
Relationship Between p53, p21 and pRb Status Analysis of bladder TCCs for the expression of p53, p21, and pRb revealed a strong association between the expression of p53-wt and p21-wt proteins (P < .001). As previously reported [8], seventy-seven percent of those tumors with no evidence of p53 alterations demonstrated p21-wt expression. Also, there was a significant association between the p21 expression and pRb expression (P = .007); the majority (78%) of tumors displaying pRb-wt (moderate pRb) expression also had p21-wt protein expression. Of 54 tumors with no p21 expression, the majority (70%) also showed pRb alteration.
Association of p53, p21, and pRb as Individual Determinants With Clinical Outcome
Combined p53, p21, and pRb Status: Association With Clinical Outcome On the basis of the combined p53, p21, and pRb status, we further classified the patients into four groups: group I (no alteration in any determinant, 47 patients), group II (alteration in any one determinant, 51 patients), group III (alteration in any two determinants, 42 patients), and group IV (alteration in all three determinants, 24 patients). The 5-year recurrence rates in these groups were 23%, 32%, 57%, and 93%, respectively (P < .001; Fig 1), and the 5-year survival rates were 70%, 58%, 33%, and 8%, respectively (P < .001, Fig 2). The median times to recurrence were greater than 11, greater than 11, 3.3, and 0.75 years, and the median survival times were 11, 6.8, 1.9, and 1.6 years, respectively. Thus alteration in two or more determinants showed the greatest effect on recurrence-free survival, whereas an alteration in only one determinant had a less drastic effect on clinical outcome. Indeed, the most dramatic impact of these alterations was in patients whose tumors were altered in all three determinants (group IV), where more than 90% of patients experienced a tumor recurrence, and only 8% of patients survived at 5 years.
Analyzing each protein, taken one at a time, using a stratified (by stage) Cox proportional hazards model, p53 status, p21 status, and pRb status were all predictors of time to recurrence; p53 and pRb were strongly associated with overall survival, whereas the association between p21 status and survival was not significant at the .05 level, with P = .10 (Table 3). Similarly, when the number of altered genes was included in a Cox regression analysis stratified by stage, it was strongly statistically significantly associated with time to recurrence and overall survival (Table 3).
As we and others have previously reported, altered expression of p53, p21, or pRb are independent predictors of bladder cancer progression when examined as individual determinants [7-12,14,22]. This study focused on the combined analysis of these three important determinants of bladder cancer progression. When none of the three determinants are altered, patients demonstrate extremely low rates of recurrence and increased survival. However, a substantial proportion of patients with alteration in p53 alone, p21 alone, or pRb alone will experience recurrence. We and others have shown that the combination of p53 and p21 status, as well as p53 and pRb status, provides additional clinical outcome information, above that obtained from examining a single determinant [7,8]. Here we show that alterations in two or three determinants cause a significant reduction in survival and increase in recurrence. In fact, the majority of patients with tumors showing alteration in all three determinants experienced recurrence and died within 5 years. This study demonstrates that analysis of p53, p21, and pRb in combination provide additional prognostic information beyond that obtained from single-marker analysis. The relationship between p53, p21, and pRb is now becoming clear; these proteins form an integral foundation of an intricate pathway involved in the control of cell growth and proliferation. Functional inactivation of p53 is the most common event in human malignancies, occurring in at least half of all tumors [23-26]. One of the primary functions of the p53 protein as a cell cycle regulatory protein is to upregulate the expression of p21, a universal cyclin/cyclin dependent kinase inhibitor with an important role in G1 arrest [27,28]. In response to DNA damage, p53 induces the expression of p21, which inhibits cyclin/CDK complexes, and thereby prevents phosphorylation of pRb proteins [29]. In its unphosphorylated form, pRb can bind to and sequester the transcription factor E2F/DP [30], which is known to activate the expression of genes required for transit from G1 to S phase. Thus p53, p21, and pRb are critical components in a series of highly interrelated pathways. Our results indicate that expressions of all three proteins are associated; a majority of tumors with p53-wt expression also expressed p21-wt and pRb-wt. However, we now also know that p21 can be induced during cell growth and differentiation under both p53-dependent and p53-independent conditions by various other stimuli such as cytokines and cell adhesion events [8,31,32]. Our results support this; 50% of tumors maintained p21 expression despite p53 alteration [8]. Similarly, the pRb protein has been shown to be influenced by multiple cell cycle regulatory proteins in addition to p21, such as p16 (INK4a) [33,34]. In our series, 22% and 27% of tumors showed pRb protein expression despite alterations in p21 and p53, respectively. Although the majority of pRb-wt tumors expressed p21-wt, p21 expression was maintained in a substantial proportion of pRb altered tumors as well. Thus although tumor suppressor pathways involving p53, p21, and pRb overlap, and one determinant influences the others significantly, there are additional influences that alter the function of these proteins. Thus it is not surprising that examination of these determinants in combination provides additional information regarding clinical outcome, beyond what is provided by a single determinant alone. Several interesting aspects emerge from this study. First, when no alteration in p53, p21, or pRb is identified, the recurrence rate is low and survival rate is high. However, even in this group, a substantial minority of patients (23%) will experience recurrence in 5 years. Second, alteration in only one determinant shows recurrence and survival rates that are only moderately worse than those with no alteration, indicating that the effect of a single alteration can be at least partially abrogated by maintenance of the remainder of the pathway. Third, alteration in two or more determinants shows greater effects on clinical outcome, indicating that alterations at multiple points in a pathway may show cooperative or synergistic effects on clinical outcome. Finally, patients with alteration in all three determinants show extremely poor survival. These results indicate that although we can predict the outcome for patients with tumors showing multiple alterations rather accurately, it is still difficult to predict outcome for patients with tumors that show relatively few alterations; progression of these tumors might be occurring via alternative proteins or pathways. Although this study has demonstrated the power of assessing multiple alterations in predicting clinical outcome, it is clear that we need to better understand the molecular pathways of progression and to extend the analysis to new pathways involved in tumor progression. In conclusion, our study has demonstrated that the accumulation of molecular abnormalities in bladder TCC are important in predicting cancer behavior. Patients with bladder tumors that have altered p53, p21, and pRb protein products are at a high risk of recurrence and death. Therefore, adjuvant therapeutic strategies may prove to be most beneficial in this subset of patients following cystectomy. Most importantly, this study urges the continuing search for a more complete and precise model of tumor progression, which will ultimately guide us to the earlier detection and more suitable management of bladder cancer.
The authors indicated no potential conflicts of interest.
Supported by the Molecular and Cellular Pathways of Bladder Cancer Progression Grant (grant No. NCI CA 70903), the Cancer Center Core Grant (grant No. NCI CA 14089), and the Bladder Cancer Program Project Grant (grant No. PO1 CA 86871). Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test were used to evaluate the association of p53, p21, and pRb status with tumor grade, pathologic stage, and lymph node status. Kaplan-Meier plots [
