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p53 Gene and Protein Status: The Role of p53 Alterations in Predicting Outcome in Patients With Bladder Cancer

Ben George, Ram H. Datar, Lin Wu, Jie Cai, Nancy Patten, Stephen J. Beil, Susan Groshen, John Stein, Donald Skinner, Peter A. Jones, Richard J. Cote

From the Departments of Pathology, Urology, Preventive Medicine, and Biochemistry, University of Southern California, Keck School of Medicine, Los Angeles; and Roche Molecular Systems, Pleasanton, CA

Address reprint requests to Richard J. Cote, MD, FRCPath, Department of Pathology and Urology, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, NOR 2424, Los Angeles, CA 90033; e-mail cote_r{at}ccnt.usc.edu

	ABSTRACT

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Purpose The p53 gene status (mutation) and protein alterations (nuclear accumulation detectable by immunohistochemistry; p53 protein status) are associated with bladder cancer progression. Substantial discordance is documented between the p53 protein and gene status, yet no studies have examined the relationship between the gene-protein status and clinical outcome. This study evaluated the clinical relationship of the p53 gene and protein statuses.

Materials and Methods The complete coding region of the p53 gene was queried using DNA from paraffin-embedded tissues and employing a p53 gene–sequencing chip. We compared p53 gene status, mutation site, and protein status with time to recurrence.

Results The p53 gene and protein statuses show significant concordance, yet 35% of cases showed discordance. Exon 5 mutations demonstrated a wild-type protein status in 18 of 22 samples. Both the p53 gene and protein statuses were significantly associated with stage and clinical outcome. Specific mutation sites were associated with clinical outcome; tumors with exon 5 mutations showed the same outcome as those with the wild-type gene. Combining the p53 gene and protein statuses stratifies patients into three distinct groups, based on recurrence-free intervals: patients showing the best outcome (wild-type gene and unaltered protein), an intermediate outcome (either a mutated gene or an altered protein) and the worst outcome (a mutated gene and an altered protein).

Conclusion We show that evaluation of both the p53 gene and protein statuses provides information in assessing the clinical recurrence risk in bladder cancer and that the specific mutation site may be important in assessing recurrence risk. These findings may substantially impact the assessment of p53 alterations and the management of bladder cancer.

	INTRODUCTION

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The p53 gene and protein statuses both play a critical role in the regulation of the normal cell cycle, cell cycle arrest, and apoptotic response.^1-3 Alterations in the p53 protein, leading to a loss of its tumor suppressor function, have been reported previously by us and by others.^4-6 The p53 gene status has been examined in a number of malignancies, including cancers of bladder,⁷ breast,⁸ lung,⁹ ovary¹⁰ and colorectal cancer.¹¹ The wild-type p53 protein has a short half-life of 15 to 30 minutes.¹² However, missense p53 gene mutations result in a protein with a prolonged half-life,¹³ which is the basis of its nuclear accumulation that is detectable by immunohistochemistry (IHC). Nuclear accumulation of the p53 protein in bladder cancer has been associated with mutations in the gene, although substantial discordance has been demonstrated between the altered p53 protein status (nuclear accumulation) and mutant p53 gene status.^14-17 Nuclear accumulation of p53 is associated with a poor clinical outcome in invasive bladder cancer.^4,5,18 However, there is evidence that the wild-type p53 protein can also accumulate to detectable levels,¹⁹ in part because of aberrant expression of upstream regulators of p53 function. Further, the absence of nuclear accumulation of the p53 protein does not rule out a mutated p53 gene.^7,14,15 Few studies have examined the relationship between the p53 gene status and clinical outcome because of the difficulty and cost of sequencing.^7,20 The recent development of chip-based, p53 gene–sequencing technologies addresses this limitation. We had previously investigated p53 protein status in archival paraffin-embedded tissue specimens by IHC in a large cohort of patients with operable bladder cancer who were treated uniformly by radical cystectomy.⁴ In the current study, using the available tissue specimens from this same cohort, we queried the complete coding region of the p53 gene (exons 2 through 11) for mutations using the Affymetrix p53 GeneChip (Roche Molecular Systems, Pleasanton, CA) and examined the clinical significance of the discordance between p53 gene mutations and nuclear protein accumulation to compare the p53 gene status, the specific site of mutations, and the protein status with clinical outcome in patients with bladder cancer.

	PATIENTS AND METHODS

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Patient Population
This study included 150 patients who underwent en bloc radical cystectomy, pelvic lymphadenectomy, and urinary reconstruction for either invasive bladder cancer (n = 143) or recurrent high-grade noninvasive transitional cell carcinoma of the bladder that had become refractory to intravesical therapy (n = 7) at the University of Southern California (USC) Norris Comprehensive Cancer Center from April 1983 to December 1988. These patients are a subset of those in a previously published series.⁴ Patients who received neoadjuvant radiation or systemic chemotherapy were excluded from the present study. Tumor tissue was available in 180 eligible samples in which paraffin-embedded tumor blocks from the cystectomy specimens were still available, and adequate DNA for analysis was obtained from 150 samples, according to USC institutional review board approval number 02A043. After cystectomy, 23 (15%) of 150 patients received adjuvant chemotherapy, one (0.7%) patient each received either adjuvant radiation therapy alone or both adjuvant chemotherapy and radiation therapy.

Histologic grading (Bergkvist system grades 1 through 4), and pathologic staging (American Joint Committee on Cancer, 6th edition) were performed.^21-23 Pathologic-stage subgroups were defined as organ-confined (OC) and lymph node-negative (OC; pTa, pTis, pT1, or pT2; LN–); non–organ-confined (extravesical involvement) but lymph node-negative, with extravesical involvement (EV; pT3 or pT4; LN–); and lymph node-positive (LN+),²¹ as we have done previously.²⁴ Thirteen patients who either underwent salvage cystectomies or were considered not to have achieved a complete resection were included in the LN+ cohort.

Antibodies and IHC
p53 IHC was carried out using the pAb 1801 (Pharmingen BD Biosciences 14471A; Becton-Dickinson Biosciences, Franklin Lakes, NJ), as described previously.⁴ The p53 nuclear reactivity was classified into two categories: wild-type (p53-negative; < 10% of tumor-cell nuclei positive for p53 nuclear reactivity) and altered p53 (p53-positive; 10% of tumor cell nuclei positive for p53 nuclear reactivity).

DNA Extraction
Following identification of the tumor area from hematoxylin and eosin(HE)–stained, 5-µ sections, three to five 10-µ–thick sections were cut for DNA extraction, with microdissection performed when tumor areas represented less than 50% of the tissue. DNA extraction was carried out and quality was assessed, as described earlier.²⁵ The Affymetrix p53 GeneChip analysis was carried out as recently described.²⁶ Independent studies have confirmed that the mutation data by GeneChip is in agreement with the direct sequencing analysis.^10,27-29 The data were analyzed with the p53 GeneChip Mixture Detection Algorithm (Roche), with scores of 13 or greater considered indicative of mutations.

Statistical Analysis
The primary objectives of this study were to document the p53 mutations present in bladder cancer tumors and to evaluate the association between p53 gene and protein statuses. Contingency tables and the Pearson's ² test were used to evaluate the association of p53 protein and gene statuses with tumor grade, pathologic stage, and lymph node status. The kappa statistic and its associated test were used to assess the concordance between the gene and protein statuses (classified as wild-type or not). The secondary purposes of this study were to examine the joint association between p53 protein and gene statuses with outcome and to examine the association of the specific site of mutation with outcome. Because the LN+ patients had a uniformly poor outcome (76% ± 7% recurred by 5 years), these analyses were done twice: once, including all 150 patients and again, including only those patients with OC or EV (and LN–) disease. The primary measure of clinical outcome was the time to first recurrence of bladder cancer, which was calculated from the time of cystectomy to the date of the first documented clinical recurrence or to the date of last follow-up visit; patients who died before recurrence were censored at the time of death. Survival, defined as time from cystectomy to death of any cause, was also examined. Kaplan-Meier plots³⁰ were used to display the relationship between stage and p53 status, with time to progression. The Cox proportional hazards model was used to estimate the hazard ratios, based on p53 protein and gene statuses overall and stratified by the stage. P values in the tables for associations involving the time to recurrence are based on the global-score test, using the proportional hazards model; P values in the figures are based on the unstratified log-rank test.³¹ The assumption of proportional hazards in our Cox regression modeling was confirmed by the parallelism in log(-log(S(t))) graphs and by the examination of Schoenfeld residual plots. Further, plots of deviance residuals did not show any outliers or nonrandom patterns. Analyses were rerun after stratifying by adjuvant therapy (25 patients), which resulted in consistent patterns and P values; thus, results are presented with all patients combined. All reported P values are two-sided. Nominal P values are given; no adjustments were made for the number of P values calculated.

	RESULTS

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Relationship Between p53 Protein Status and p53 Gene Status
Mutations of the p53 gene were detected in 55 (37%) of 150 samples by GeneChip assay. Nuclear accumulation of the p53 protein was seen in 54 (36%) of 150 samples. Nuclear accumulation of the p53 protein by IHC showed significant concordance with p53 gene mutations (Table 1; P = .004): 69 (73%) of 95 samples with the wild-type p53 gene status demonstrated a wild-type p53 protein status, and 28 (51%) of 55 samples with a mutated p53 gene status showed protein alterations. The kappa estimate of concordance was 0.24 (P = .003), which indicated that, although the observed concordance was more than what would be observed by chance alone if there were no association between the gene and protein statuses, there was also substantial discordance.

Location and Frequency of p53 Mutations and Association of Specific Exonic Mutations With p53 Protein Status
Of the 55 tumors showing p53 exonic mutations, 52 (95%) showed mutations in the hot-spot region of the p53 gene (exons 5 through 8, the region that represents the DNA-binding domain). Seventy exonic mutations were observed in these 55 tumors, with 66 mutations located in the DNA-binding domain. Ten tumors had mutations in multiple exons, and all of these had mutations that involved either exon 5 and/or 8. Exon 5 (18 of 55 as a single mutation and 11 of 55 as one of multiple mutations) and exon 8 (14 of 55 as a single mutation and 6 of 55 as one of multiple mutations) harbored the greatest number of mutations (Table 2); in fact, 44 (80%) of 55 patients with p53 mutations had a mutation occurring in exon 5, exon 8, or both. Specific exonic mutations were associated with p53 protein status (Table 2). Sixteen (89%) of 18 single exon 5 mutations and one of one tumor with multiple mutations in exon 5 demonstrated a wild-type p53 protein status; 5 (36%) of 14 single exon 8 mutations demonstrated a wild-type p53 protein status. One (17%) of six single exon 7 mutations demonstrated a wild-type p53 protein status.

Association of p53 Protein Status and p53 Gene Status With Clinical Outcome
The overall p53 protein status was significantly associated with clinical outcome as an individual determinant (Fig 1A, P < .001), (5-year, recurrence-free survival [5RFS] for wild-type v altered protein, mean ± SE, 70% ± 5% v 32% ± 7%). Similarly, patients with p53 gene mutations were more likely to experience recurrence (Fig 1B), but the effect was not as strong (P < .004; 5RFS wild-type v mutated gene, 66% ± 5% v 44% ± 7%).

View larger version (15K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Association between p53 protein and gene status with clinical outcome. (A) Estimated probability of remaining recurrence-free, based on the Kaplan-Meier product-limit estimator, for patients classified according to p53 tumor-protein status, as assessed by immunohistochemistry: wild-type protein (nuclear accumulation of p53 protein detected in < 10% of tumor cells) versus altered protein (nuclear accumulation of p53 protein detected in 10% of tumor cells). P less than .001, based on the log-rank test. (B) Estimated probability of remaining recurrence-free, based on the Kaplan-Meier product-limit estimator, for patients classified according to p53 gene mutations: wild-type (no p53 mutations detected) versus mutated (any p53 mutation detected). P = .004, based on the log-rank test. Wt, wild-type; Mut, mutated.

View larger version (18K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. (A) Estimated probability of remaining recurrence-free, based on the Kaplan-Meier product-limit estimator, for patients classified according to the site of p53 gene mutations: no p53 mutations; a single mutation in exon 5; a single mutation in exon 8; or a single mutation in other exons, or multiple mutations (P = .001, based on the log-rank test). (B) Estimated probability of remaining recurrence-free, based on combined expression of p53 gene status and protein status with single exon-5 mutations added to the tumors with no mutations, for all patients combined (P < .001, based on log-rank test). Wt, wild-type; Mut, mutated.

	DISCUSSION

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To our knowledge, this is the first study in invasive bladder tumors to examine both p53 alterations detected by IHC and mutations in the p53 gene, through examination of the entire coding region, by using DNA extracted from paraffin-embedded tissue. In concordance with previous reports, we demonstrated that the majority of p53 mutations are located in the hot-spot region of the p53 gene. Further, we demonstrated that specific p53 exonic mutations are associated with p53 protein status. Although there is significant concordance between the p53 protein and the gene status, a substantial minority of samples show discordance. Both p53 protein and gene statuses are independent predictors of outcome in this study, and we have shown that their combination may be a more effective predictor of outcome than either one alone. Finally, this is the first study to show that the specific site of the p53 mutation may be important in predicting outcome in patients with bladder cancer.

The failure of the p53 protein to degrade, and thus its accumulation in the nucleus, can result either from contact mutations (which abolish the ability of the p53 protein to function as a transcription factor for the MDM2 gene, thereby reducing its own degradation^32-34) or from structural mutations (which cause nuclear aggregation because of protein unfolding³⁵). In bladder cancer, both molecular mechanisms appear to exist, which results in the nuclear accumulation of the p53 protein. In addition, the wild-type p53 protein can accumulate because of aberrant regulation and degradation.¹⁹

The finding that different mutation sites are associated with stages and outcomes is particularly interesting. Mutations in exons 5 and 8 were the most prevalent mutations found in this study. When the patients were stratified according to their stage, 58% of mutations seen in the OC and LN– category were contributed by exon 5, whereas 42% of mutations seen in the LN+ category were contributed by exon 8. Interestingly, nearly 90% of the samples with exon 5 mutations and a substantial proportion of tumors with the exon 8 mutation showed no nuclear p53 accumulation (wild-type protein status). It is noteworthy that mutations in exons 5 and 8 did not confer the magnitude of recurrence risk compared with mutations at other sites. Thus, specific site of mutations (namely, mutations in exons 5 and 8) may manifest with a p53 wild-type protein status and may not result in inactivation of p53 function at least in regard to recurrence, which results in a better clinical outcome.

Using the criteria of Ahrendt et al,⁹ a significant association with worse clinical outcome was seen in patients with either contact/structural/truncation or other mutations compared with patients with the wild-type p53 gene. However, the outcomes for patients with contact/structural/truncation versus those with other mutations were not significantly different from each other. This is comparable to the findings of Arhendt et al⁹ in non–small-cell lung cancer.

The rate of mutations found in our study is comparable to that in several other studies in bladder cancer.^36,37 Other studies have shown a lower prevalence of p53 mutations in bladder cancer,³⁸ but these studies have examined primarily noninvasive disease. It has been noted by us¹⁴ and by others,³⁹ that the prevalence of p53 mutations is related to the stage of disease, and studies of higher-stage patients have shown an increased prevalence of p53 alterations.¹⁷ In a recent study, Erill et al studied the p53 gene and protein.⁷ Their data suggest that genetic assays are necessary for the optimal determination of p53 alterations, particularly in tumors with a wild-type p53 protein status, and they recommend the inclusion of both p53 protein and mutation statuses into a predictive panel of tumor markers for bladder cancer. Our study supports both conclusions.

Although there is substantial evidence that p53 alterations are predictive of bladder cancer outcome, particularly in early-stage disease,^4,5,18 this remains an area of controversy despite many years of study. There is little doubt that the reasons for this include variations in patient treatment, stage, study design, and in assay type, validation, and performance. Further basis for this controversy may be the discordance of gene and protein statuses and the differential biologic effect of different p53 gene mutations, issues directly addressed in the current study. A very intriguing finding of this study is that certain mutations do not appear to have any effect on clinical outcome, which suggests that these mutations do not result in functional inactivation of the p53 protein, at least concerning tumor progression. Clearly, these results require confirmation. Because p53 alterations may affect the response to chemotherapy in patients with bladder cancer,⁴⁰ which is currently being tested in clinical trials, we may speculate that the site of the mutation could also influence the response to therapy; this idea needs further investigation.

	AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Employment: Lin Wu, Roche Molecular Systems, Pleasanton, CA; Nancy Patten, Roche Molecular Systems, Pleasanton, CA Leadership: N/A Consultant: Richard J. Cote, Roche Molecular Systems, Pleasanton, CA Stock: N/A Honoraria: N/A Research Funds: N/A Testimony: N/A Other: N/A

	AUTHOR CONTRIBUTIONS

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Conception and design: Ben George, Ram H. Datar, Lin Wu, Nancy Patten, Susan Groshen, Donald Skinner, Peter A. Jones, Richard J. Cote

Administrative support: Donald Skinner, Peter A. Jones

Provision of study materials or patients: Ben George, Ram H. Datar, Lin Wu, John Stein, Donald Skinner

Collection and assembly of data: Ben George, Ram H. Datar, Lin Wu, Nancy Patten, Stephen J. Beil, Susan Groshen

Data analysis and interpretation: Lin Wu, Jie Cai, Nancy Patten, Susan Groshen, Richard J. Cote

Manuscript writing: Ben George, Ram H. Datar, Lin Wu, Susan Groshen, Peter A. Jones, Richard J. Cote

Final approval of manuscript: Ben George, Ram H. Datar, Lin Wu, Jie Cai, Nancy Patten, Stephen J. Beil, Susan Groshen, John Stein, Donald Skinner, Peter A. Jones, Richard J. Cote

	NOTES

 
Supported in part by Grants No. NCI CA 70903, NCI CA 14089, and NCI PO1 CA 86871 from the National Cancer Institute.

B.G. and R.H.D. share first authorship.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted December 15, 2006; accepted July 19, 2007.

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