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Journal of Clinical Oncology, Vol 25, No 34 (December 1), 2007: pp. 5341-5344 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.13.1904
p53: It Has It All, But Will It Make It to the Clinic As a Marker in Bladder Cancer?Institut Municipal d'Investigació Mèdica, Universitat Pompeu Fabra, Barcelona; Centro Nacional de Investigaciones Oncológicas, Madrid, Spain Tp53 is the most extensively studied cancer gene. As I shall try to outline here, the study of the function and dysfunction of its product, the p53 protein, covers almost any imaginable aspect of cell biology. It has it all! The history of p53 has been ever evolving. The Tp53 gene, isolated after its discovery in 1979, was shown to cooperate with oncogenes in in vitro transformation assays.1 p53 protein levels were shown to be often higher in tumor cells than in normal cells. Both of these observations were consistent with the notion that Tp53 was an oncogene. However, in 1988, the history of p53 underwent a remarkable twist when it was demonstrated that the genes initially isolated did not correspond to the normal (wild type) sequence but were, instead, mutated. Soon afterwards, it was shown that wild-type Tp53 is able to suppress tumor cell growth.2 It also became rapidly clear that one reason why the protein accumulates in human tumors is that wild-type p53 has a very short half-life (approximately 20 to 30 minutes), whereas mutant p53 proteins often have a much longer half-life (approximately 24 hours). Because p53 works as a tetramer, accumulation of mutant p53 can actually inhibit the function of the wild-type protein acting as a dominant negative. Thus, Tp53 turned from an oncogene into a tumor suppressor gene. Inactivation (loss of function) of Tp53 was subsequently shown to occur mainly through point mutation or allele loss. The demonstration of inherited Tp53 mutations in patients with Li-Fraumeni syndrome supported a tumor suppressor role,3 but its oncogenic shadow continued for many years. Strong support for an oncogenic role has emerged from the recent development of knock-in mice, in which wild-type Tp53 is replaced by a murine gene carrying mutations commonly found in human cancers. The histologic spectrum of tumors in these mice is similar to that of Li-Fraumeni syndrome, whereas Tp53 null (knock-out) mice have a different tumor spectrum.4 Furthermore, tumors from genetically engineered mice harboring these mutations progress more rapidly than those with null Tp53 alleles.5 These findings strongly support the notion that oncogene activity of p53 (that is, gain of function) results from novel functional properties of the mutant proteins. Thus, p53 has got it all—tumor suppressor and oncogene properties! For a long time, Tp53 was thought to be a unique gene, not belonging to a family. In additional historical twists, p63 and p73 were discovered as Tp53-homologous genes sharing biologic properties, such as induction of apoptosis, with p53, thus introducing a novel level of complexity. p53 binds to p63 and p73 and can interfere with their normal function.6 Somatic mutations in p63 and p73 do not contribute to human cancers, and dysregulation of their function occurs mainly at the level of mRNA splicing. Only recently has evidence of p53 mRNA splicing been acquired.7 Whether altered splicing contributes to the role of p53 in cancer requires additional investigation. The identification of the wild-type sequence of Tp53 and its tumor suppressor properties allowed dissection of its physiologic role (Table 1). p53 works as a transcription factor that activates the expression of target genes.8 In the absence of stress, cellular p53 does not act as a transcriptional activator because protein levels are too low. In response to a variety of cellular stresses (genome damage being one of the foremost), wild-type p53 can be activated by multiple post-translational mechanisms. Stress results in the activation of the ataxia telangiectasia–mutated kinase, which phosphorylates Chk2, a kinase that, in turn, phosphorylates p53.8 Phospho-p53 is stabilized, accumulates in cells, and can reach sufficient levels to regulate its target genes. p53 can then activate different transcriptional programs depending on the cell type, context, and type of stress, including a transient cell cycle arrest program that allows recovery from the causative stress, in part through engaging DNA repair pathways, and subsequent cell cycle progression; an apoptotic program that leads to cellular suicide to avoid the accumulation of genomic damage; and a senescence program that involves permanent cell cycle arrest, thus preventing the accumulation of cell clones harboring genomic damage. Therefore, loss of function of p53 heavily compromises processes that are crucial for the avoidance of tumor formation. The differential mechanisms leading to activation of one or another program are not yet completely unraveled. In addition, p53 can activate apoptosis through nontranscriptional mechanisms at the mitochondrial level.
The identification of the wild-type Tp53 sequence also led to the recognition that, in human tumors, somatic mutations occur in a wide variety of codons of the gene.9,10 Most of them are point mutations, leading to an amino acid substitution (missense mutations); a fraction of mutations lead to a premature stop codon and a truncated nonfunctional protein. The most common missense mutations cluster in exons 5 to 8, coding for the DNA binding domain of the protein required for its transcriptional regulatory activity. A catalogue of these mutations has been established at the International Agency for Research on Cancer (http://www-p53.iarc.fr/) and at Institut Curie (http://p53.free.fr/) in France, providing a detailed compilation of mutants in relationship to tumor type and clinical-pathologic characteristics, among others (Fig 1). These resources are extremely useful tools for p53 biologists despite some limitations that are well described in both databases. Although a handful of hotspot codons account for 30% of all mutations, the remaining 70% display a wide distribution, and more than 1,000 different mutations have been reported. This fact, together with the demonstrated notion that mutant p53 proteins can acquire novel functions,12-14 renders the interpretation of Tp53 mutation (and p53 overexpression) data very complex because different mutants display distinct functional properties. Most mutations in the DNA binding domain lead to the synthesis of dominant-negative proteins. Therefore, classifying human tumors as Tp53 wild type or mutant is an oversimplification.15 At the same time, this heterogeneity provides the basis for a more thorough analysis of the biologic and clinical significance of each mutation.
This leads to the issue of p53 molecular analysis. Because p53 nuclear overexpression is often associated with gene mutations, it has commonly been taken as a surrogate marker of mutations. p53 overexpression can be assessed using archival material and simple, cheap immunohistochemical assays in most pathology laboratories. In a review and meta-analysis of p53 overexpression relative to bladder cancer outcome, we found published data from more than 10,000 bladder tumors.16 Current evidence from this analysis supports the notion that p53 overexpression is associated with bladder tumors having a higher stage or grade and worse prognosis. By contrast, the p53 databases contain sequence information from approximately 1,200 bladder tumors (Fig 1), with no clear evidence for an association with outcome.17 This is so even if it has long been known that immunohistochemistry is not a good surrogate for mutation analysis.17 Overall, mutations can be detected in approximately one third of tumors without p53 nuclear overexpression, and they are undetectable in one third of tumors overexpressing p53.18 The mechanisms involved in this variability are indeed diverse and complex.15 In this issue, George et al19 report on the role of p53 alterations in predicting outcome of patients with bladder cancer undergoing radical cystectomy. Patients and tumors in this study are a subset of those included in a prior seminal report on the value of p53 nuclear overexpression in predicting bladder cancer progression.20 Patients receiving neoadjuvant radiation or systemic chemotherapy were excluded from the analysis; 30 of 180 tumors could not be analyzed because of inadequate quality of DNA. A major contribution of this study is that both Tp53 gene status as well as p53 protein expression were assessed in the tumors of 150 patients. Furthermore, unlike most studies examining Tp53 gene mutations, all coding exons were interrogated using a specifically designed chip. Certainly, these are important contributions. The study had two major aims. The first was to assess the association between gene mutations and protein status. As in other studies, the concordance of both analyses was far from perfect. George et al19 found that 51% of Tp53 mutant and 27% of Tp53 wild-type tumors showed protein accumulation. These findings are particularly relevant because the full coding sequence of Tp53 was analyzed. Overall, these findings are in agreement with those in the literature.21,22 Both gene mutations and protein overexpression were found to occur more commonly in tumors of higher stage, even within this group of advanced bladder cancers. Most of the mutations occurred in hotspot exons and hotspot codons, and 10 of 55 Tp53 mutant tumors harbored multiple mutations. These findings are also in general agreement with the literature and are similar to those recently reported by our group in T1 grade 3 bladder tumors.18,21 Remarkably, George et al19 found that tumors with exon 5 mutations more commonly displayed normal p53 immunostaining, in contrast with the remaining Tp53 mutant tumors. Exon 5 mutations were also more commonly found in bladder-confined tumors. There is not a precedent for such observations in the bladder cancer literature. The second aim of the study was to examine the added value of information on Tp53 mutations and protein status when analyzing association with outcome. In this regard, patients with abnormal findings in both assays had the worst prognosis, those with normal findings in both assays had the best prognosis, and those with one abnormal and one normal finding had an intermediate probability of recurrence and survival. p53 molecular information was an independent predictor only among patients without lymph node involvement. After stratifying for adjuvant therapy, consistent significant patterns of association were found. On the basis of these findings, the authors propose that both types of assays should be performed to better establish the prognosis of patients with advanced bladder tumors. Although the findings of this study are of great interest and potential relevance, there is not sufficient evidence presently to change clinical practice. The patients included were recruited at a single study center, most of them more than 20 years ago, and little information is provided on the subsequent management, an issue that is important regarding survival as an end point. Furthermore, some of the novel and most interesting associations reported rely on subgroup analyses. Despite the fact that this is one of the largest reports on Tp53 and protein status in muscle-invasive tumors, the number of patients in each subgroup, according to level of organ and lymph node involvement and Tp53 mutations by exon, is small, so the statistical conclusions become less robust. These findings should be replicated in independent series before they can be applied in clinical practice. Preferably, prospective, multicenter clinical trials, carried out in the context of uniform patient management, should be performed. This task is not easy. Cote et al23 have paved the path to determine, in a prospective clinical trial, whether stratification by Tp53 status can impact on the management of patients with advanced bladder cancer. This ongoing study is based on the relationship between p53 alterations and response to chemotherapy. The results of this important work are eagerly awaited. The slow progress made in the application of knowledge on p53 molecular status in the management of patients with bladder cancer, and other tumors, deserves some thought. Thirteen years have elapsed since the publication of the report by Esrig et al20 describing the accumulation of nuclear p53 and tumor progression in bladder cancer. The lack of definitive evidence supporting the use of p53 molecular analyses in the clinic, resulting from many different factors discussed in detail elsewhere,16 has rendered a substantial fraction of the medical community skeptical about the value of p53 alterations.15 The variability in the results of p53 immunohistochemical assays in different laboratories is one concern. The p53 chip, which allows exhaustive and standardized interrogation of gene mutations, has not yet found wide implementation, probably because of cost, need for technical improvements, and lack of evidence that it provides independent additional information. Finally, competitors have come onto the stage. Techniques allowing the exploration of thousands of potentially interesting markers using high-throughput methods have evolved rapidly. Microarrays have allowed the identification of putative expression signatures of stage, outcome, and carcinoma in situ bladder cancer.24,25 Recent multicenter studies have validated, or helped to reshape, the original signatures as well as reduced the complexity and cost of the assays.26 Multicenter prospective studies are now underway to test the independent value of such signatures. So, what are the take home messages? Although there is not enough evidence to use any molecular analysis of p53 (either at the gene or protein level) in the clinic, the present findings should stimulate more work on p53 in bladder cancer. Studies should aim at determining whether mutations in specific Tp53 exons and in specific codons bear distinct prognostic or predictive value.13,27 Because of the mutational diversity, this will require large multicenter consortia. Tp53 mutations may impinge on resistance to therapy, and detailed information on treatment and therapeutic response should be gathered. If we are to continue running prognostic marker studies as we have until now, progress will remain slow,16,28-30 and only the few (or very few) markers with strong prognostic impact will enter clinical practice. If we want to rapidly bring in the clinic markers that are less strong, yet robust, we need to design better studies. The difficulties in design and conduct and the failures in replication are similar in prognostic marker and genetic association studies.31 I will use the latter example to provide some insights into how we can improve. Many association studies of low-penetrance genes/alleles have failed to be replicated.31 However, in the first half of this year, a dozen reports on whole genome association studies assessing a few hundred thousand markers in a variety of common diseases have been published, showing a remarkable level of consistency.32 They were all conducted as large multicenter studies with built-in replication in independent case series, and they all used strict statistical criteria.33 We need to move forward in this direction when assessing prognostic markers. An additional pressure to move fast if p53 molecular studies are to reach the clinic is that, unlike 10 or 20 years ago, competitors also appear fast, either as new markers or as improved assays for old markers. Unless we perform studies with markers of outcome in a more efficient manner, interesting important assays and markers will not make it to the clinic and much may be lost in translational research. AUTHOR'S DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The author(s) indicated no potential conflicts of interest.
ACKNOWLEDGMENTS I thank Xavier Mayol, PhD, and Núria Malats, MD, PhD, for their valuable comments. REFERENCES 1. Eliyahu D, Raz A, Gruss P, et al: Participation of p53 cellular tumour antigen in transformation of normal embryonic cells. Nature 312:646-649, 1984[CrossRef][Medline] 2. Finlay CA, Hinds PW, Levine AJ: The p53 proto-oncogene can act as a suppressor of transformation. Cell 57:1083-1093, 1989[CrossRef][Medline] 3. Malkin D, Li FP, Strong LC, et al: Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250:1233-1238, 1990 4. Lang GA, Iwakuma T, Suh YA, et al: Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell 119:861-872, 2004[CrossRef][Medline] 5. Caulin C, Nguyen T, Lang GA, et al: An inducible mouse model for skin cancer reveals distinct roles for gain- and loss-of-function p53 mutations. J Clin Invest 117:1893-1901, 2007[CrossRef][Medline] 6. 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Hernandez S, Lopez-Knowles E, Lloreta J, et al: FGFR3 and Tp53 mutations in T1G3 transitional bladder carcinomas: Independent distribution and lack of association with prognosis. Clin Cancer Res 11:5444-5450, 2005 19. George B, Datar RH, Wu L, et al: p53 gene and protein status: The role of p53 alterations in predicting outcome in patients with bladder cancer. J Clin Oncol 25:5352-5358, 2007 20. Esrig D, Elmajian D, Groshen S, et al: Accumulation of nuclear p53 and tumor progression in bladder cancer. N Engl J Med 331:1259-1264, 1994 21. Kelsey KT, Hirao T, Schned A, et al: A population-based study of immunohistochemical detection of p53 alteration in bladder cancer. Br J Cancer 90:1572-1576, 2004[CrossRef][Medline] 22. López-Knowles E, Hernández S, Kogevinas M, et al: The p53 pathway and outcome among patients with T1G3 bladder tumors. Clin Cancer Res 12:6029-6036, 2006 23. Cote RJ, Esrig D, Groshen S, et al: p53 and treatment of bladder cancer. Nature 385:123-125, 1997[Medline] 24. Dyrskjot L, Thykjaer T, Kruhoffer M, et al: Identifying distinct classes of bladder carcinoma using microarrays. Nat Genet 33:90-96, 2003[CrossRef][Medline] 25. Dyrskjot L, Zieger K, Kruhoffer M, et al: A molecular signature in superficial bladder carcinoma predicts clinical outcome. Clin Cancer Res 11:4029-4036, 2005 26. Dyrskjot L, Zieger K, Real FX, et al: Gene expression signatures predict outcome in non-muscle-invasive bladder carcinoma: A multicenter validation study. Clin Cancer Res 13:3545-3551, 2007 27. Olivier M, Langerod A, Carrieri P, et al: The clinical value of somatic TP53 gene mutations in 1,794 patients with breast cancer. Clin Cancer Res 12:1157-1167, 2006 28. McShane LM, Altman DG, Sauerbrei W, et al: REporting recommendations for tumor MARKer prognostic studies (REMARK). Nat Clin Pract Oncol 2:416-422, 2005[CrossRef][Medline] 29. Kyzas PA, Denaxa-Kyza D, Ioannidis JP: Quality of reporting of cancer prognostic marker studies: Association with reported prognostic effect. J Natl Cancer Inst 99:236-243, 2007 30. Munro AJ, Lain S, Lane DP: P53 abnormalities and outcomes in colorectal cancer: A systematic review. Br J Cancer 92:434-444, 2005[Medline] 31. Colhoun HM, McKeigue PM, Davey Smith G: Problems of reporting genetic associations with complex outcomes. Lancet 361:865-872, 2003[CrossRef][Medline] 32. Altshuler D, Daly M: Guilt beyond a reasonable doubt. Nat Genet 39:813-815, 2007[CrossRef][Medline] 33. NCI-NHGRI Working Group on Replication in Association Studies, Chanock SJ, Manolio T, et al: Replicating genotype-phenotype associations. Nature 447:655-660, 2007[CrossRef][Medline]
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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