Prospective evaluation of p53 as a prognostic marker in T1 transitional cell carcinoma of the bladder

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1 Original Article p53 AS A PROGNOSTIC MARKER IN BLADDER CANCER DALBAGNI et al. There have been several published reports that p53 is a useful prognostic marker for progression and survival in bladder cancer. Authors from the USA used p53 tissue typing by immunohistochemistry in a prospective cohort of patients with T1 bladder cancer, and did not find it to be a clinically useful prognostic marker. In a study from the same institution, investigators found that the concept that a minimal surgical margin in patients undergoing partial nephrectomy for renal cortical tumours should not be allowed in patients with a high malignant potential. Prospective evaluation of p53 as a prognostic marker in T1 transitional cell carcinoma of the bladder Guido Dalbagni, Dipen J. Parekh, Leah Ben-Porat, Michele Potenzoni, Harry W. Herr and Victor E. Reuter Department of Urology, Division of Epidemiology and Biostatistics, Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Accepted for publication 20 September 2006 OBJECTIVE To prospectively evaluate p53 overexpression as a predictor of survival in patients with a first diagnosis of T1 transitional cell carcinoma (TCC) of the bladder, as several reports implicate p53 as an important prognostic marker for progression and survival, but all previous studies were retrospective, giving conflicting and irreproducible results, rendering inappropriate any attempt at integrating p53 into clinical decision-making. tumours and 34 had associated carcinoma in situ. During the follow-up, 34 additional patients had a radical cystectomy and nine died from bladder cancer. The association of p53 overexpression with progression or survival was not significant. CONCLUSIONS p53 tissue typing by IHC in a prospective cohort of patients with T1 bladder cancer was not clinically useful as a prognostic marker in a contemporary series of T1 tumours. PATIENTS AND METHODS Patients with a first diagnosis of T1 TCC of the bladder were enrolled; p53 overexpression was assessed by immunohistochemistry (IHC) using both monoclonal antibody 1801 and DO7. The pathological stage and IHC score were assigned by one pathologist, and the markers were scored categorically. RESULTS Of the 89 patients who were evaluable, 53 had p53-positive tumours. The median follow-up for the survivors was 52 months. Eighty-two patients had high-grade tumours, using the World Health Organisation/International Society of Urological Pathology 1998 grading system. Fifty-eight patients had unifocal KEYWORDS bladder cancer, markers, T1, p53, survival INTRODUCTION Bladder cancer presents as superficial (nonmuscle-invasive) papillary tumours in 70% of cases [1]; two-thirds of superficial bladder tumours are confined to the mucosa (Ta) and a third invade the lamina propria (T1) [1,2]. Although both Ta and T1 tumours commonly recur, T1 cancers are potentially more lethal because 30 50% of tumours managed conservatively progress to muscle invasion or metastases, usually within 5 years [2]. It is not possible at presentation to accurately predict any subsequent invasion. However, multiple tumours, high-grade (G3) morphology, large 2006 BJU INTERNATIONAL 99, doi:1111/j x x 281

2 DALBAGNI ET AL. tumours (>3 cm), associated carcinoma in situ (CIS) and presence of tumour at the first follow-up cystoscopy after local treatment are associated with a greater risk of stage progression and reduced survival [1,2]. Other controversial adverse prognostic factors under investigation include vascular invasion, microvessel density, depth of submucosal invasion, molecular genetic alterations and expression of putative tumour markers [3 5]. One of these markers is the overexpression of the p53 protein product of the mutated Tp53 gene. Early studies showed that p53 mutations are common events in bladder cancer, and are associated with tumour stage and grade [6]. Following these earlier reports, we and others investigated the role of p53 as a prognostic marker and in a retrospective study we reported an association between p53 overexpression, progression and survival in patients with T1 TCC of the bladder [7]. Although these results have been confirmed by some investigators and refuted by others, p53 has never been validated prospectively [6]. The objective of the present study was to prospectively evaluate p53 as a prognostic marker in highrisk patients with T1 cancer of the bladder. PATIENTS AND METHODS Patients with a first diagnosis of T1 TCC of the bladder were enrolled; the pathological stage, grade and p53 status were assigned by one pathologist (V.E.R.). The tumours were staged according to the new TNM system of the International Union Against Cancer and graded according to the WHO/International Society of Urological Pathology 1998 grading system [8]. A re-staging TUR was used in all patients. Exclusion criteria included previous radiation to the pelvis, previous systemic chemotherapy, histology that was not TCC, previous history of a T1 tumour, or tumours of higher stage. The Institutional Review Board at the authors institution approved the protocol, and informed consent was obtained for all patients. For immunohistochemistry (IHC), sections from bladder tumours and normal bladder specimens were analysed using a standard avidin-biotin technique [9]. Briefly, 5-µm thick sections were deparaffinized and incubated at room temperature in 10% normal horse serum for 30 min. Tissue sections were incubated overnight at 4 C with the mouse monoclonal antibody PAb1801 (Ab2; Oncogene Science, Inc., Uniondale, NY, USA) at 200 ng/ml. This antibody recognises a denaturation-resistant epitope located between amino acids 32 and 79, which is present in both wild-type and mutant human p53 proteins [10]. The presence of a mutation confers on the gene product a half-life of four to 20 times longer than the wild-type protein. This increased half-life is partly due to conformational changes of the molecule and partly to its complexing with a heat-shock protein (hsc70) [11]. Thus, an increase in the steady-state level of the mutated protein leads to positive nuclear staining by IHC, whereas the short half-life of the wild-type product precludes its detection. There is a strong correlation between p53 nuclear overexpression, as detected by IHC, and the presence of a mutation as shown by DNA sequencing [12]. The monoclonal antibody DO7 (Oncogene Science) was also used. Deparaffinized tissue sections of a breast carcinoma known to contain mutant p53 protein were used as a positive control. As a negative control, we used a class-matched, nonspecific mouse monoclonal antibody (MIgS1-kp-1; PharMingen, San Diego, CA, USA) at the same final concentration. Tissues were subsequently incubated at room temperature with secondary biotinylated horse anti-mouse antibodies (Vector Laboratories, Inc., Burlingame, CA, USA) for 30 min at a 1 : 100 dilution, and then with avidin-biotin-peroxidase complexes (Vector Laboratories) at a 1 : 25 dilution for 30 min. Diaminobenzidine (6%) was used as the chromogen, and 1% modified Harris haematoxylin was used as the counterstain. All slides were reviewed and tumour cells that showed staining in >20% were considered to be positive for nuclear overexpression of p53 protein. The endpoints of the study were overall (OS) and disease-specific survival (DSS). Survival time was defined as the time from the initial diagnosis to the date of the last follow-up. Survival curves were estimated using Kaplan Meier method. The relationship between survival and sex, age, size, grade and vascular invasion was investigated using the log-rank test. The relationship between survival time and age was assessed using a Cox proportional hazard regression model. The incidence of progression to muscle invasion and the incidence of radical cystectomy was evaluated, and its relationship to p53 status of the tumour at initial diagnosis assessed using the competing-risk analysis from the modified chi-square test. RESULTS In all, 89 patients were enrolled (Table 1), including 71 men and 18 women (median age 66 years, range 39 93); 82 (92%) had highgrade tumours, 58 (65%) had unifocal tumours, 34 (38%) had associated CIS and seven had established vascular invasion in the tumour specimen. p53 overexpression, defined as >20% expression, was recorded for 53 patients (60%). Patients with p53-positive and p53-negative tumours were similar for grade, CIS and vascular invasion. The patients who had early cystectomy and those who were managed conservatively with or with no BCG were equally distributed among the p53- positive and p53-negative groups. The median (range) follow-up for the survivors was 52 (16 90) months; during the follow-up, 58 patients (65%) survived with no evidence of disease, 15 (17%) died from bladder cancer, seven (8%) were alive with evidence of disease, and nine (10%) died from other unrelated causes. The median (95% CI) 3- and 5-year OS rates were 81 (73 90)% and 68 (56 80)%, respectively, and the respective DSS rates were 87 (79 94)% and 79 (70 89)% (Fig. 1). In the univariate survival analysis, greater age, early cystectomy, multiple tumours, the presence of CIS, high grade, the presence of vascular invasion and overexpression of p53 (Table 2) were not associated with a poor outcome. There was no difference in OS and DSS (Fig. 2) between patients with and with no p53 overexpression. Ten patients from the surveillance group progressed to muscle-invasive disease. Delayed radical cystectomy was used in 26 patients with no evidence of progression to muscle invasion. From the competing-risk analysis, p53 status did not influence the rate of progression to muscle invasion (P = 7), nor the rate of delayed cystectomy (P = 7). There was no difference in the incidence of cystectomy in p53-positive patients, with 17 in the p53-positive group having a delayed cystectomy compared to nine in the p53- negative group (Table 3 and Fig. 3). DISCUSSION This is the first prospective evaluation of a prognostic marker in bladder cancer; p53 as a BJU INTERNATIONAL

3 p53 AS A PROGNOSTIC MARKER IN BLADDER CANCER TABLE 1 The patients characteristics stratified by p53 result (the p53 result for one patient was missing). Values are n (%) unless stated otherwise Characteristic Overall p53-positive p53-negative P Number of patients Gender Male 71 (80) 40 (75) 30 (86) 9 Female 18 (20) 13 (25) 5 (14) Age, years Mean (median) 65 (66) 66 (68) 62 (63) 9 Range Number of tumours Single 58 (65) 35 (66) 22 (63) 2 Multiple 31 (35) 18 (34) 13 (37) Tumour grade High 82 (92) 52 (98) 29 (83) 1 Low 7 (8) 1 (2) 6 (17) CIS Yes 34 (38) 24 (45) 10 (29) 3 No 55 (62) 29 (55) 25 (71) Vascular invasion Yes 7 (8) 3 (6) 4 (11) 3 No 62 (70) 37 (70) 24 (69) Study group Cystectomy 14 (16) 11 (21) 3 (9) 5 Surveillance, no BCG 38 (43) 20 (38) 32 (49) Surveillance, BCG 37 (42) 22 (42) 32 (43) TABLE 2 Univariate analysis of DSS DSS, %, median (95% CI) Variables N % censored 3-year 5-year P* Gender Male (78 95) 79 (68 90) 8 Female (73 100) 82 (63 100) Age (10 year groups): Hazard ratio (95% CI) 1.4 ( 2.2) 1 Study group Cystectomy (79 100) 81 (57 100) 2 Surveillance (77 94) 80 (70 90) Number of tumours Single (77 96) 79 (67 91) 1 Multiple (74 99) 82 (68 96) Tumour grade High (77 93) 78 (67 88) Low 7 7/7 7/7 7/7 CIS Yes (72 97) 76 (57 95) 5 No (79 97) 81 (69 92) Vascular invasion Yes 7 5/7 67 (29 100) 67 (29 100) 4 No (77 95) 79 (67 90) P53 result Positive (79 97) 75 (61 90) 6 Negative (71 97) 84 (71 97) *log-rank test; Cox regression model; One group had no events. FIG. 1. The DSS of all 89 patients enrolled; 15 died from disease. Proportion surviving Months FIG. 2. The DSS of the 53 patients (10 dead from disease) with tumours overexpressing p53, vs the 35 with a normal p53 (five dead from disease). One patient had no tissue blocks for analysis. Proportion surviving Negative (N = 35, 5 DOD) Postitive (N = 53, 10 DOD) Months prognostic marker hag no association with survival in patients with T1 TCC. It is well known that morphologically similar tumours presenting in an assigned stage can behave differently, and this seriously hampers the ability to accurately predict clinical behaviour in an individual case. Occult micrometastatic disease, inadequate tumour resection, recurrent and invasive tumours occur in a substantial proportion of patients, and are major causes of treatment failure and subsequent death. It is also becoming increasingly evident that morphological changes and their clinical manifestations are preceded by molecular and biochemical alterations. The proper identification of those alterations by using biological markers is needed for the appropriate selection of therapy. There are many reports of the retrospective evaluation of markers in small groups of patients [13,14]. Retrospective analyses have given conflicting and irreproducible results, rendering inappropriate any attempt to integrate those markers into clinical decision-making [5]. Conflicting results are partly due to the inherent flaws 2006 BJU INTERNATIONAL 283

4 DALBAGNI ET AL. of retrospective analysis, and partly to differences in the methods used [13,15]. In the present study we focused on a homogeneous group of patients with T1 TCC bladder cancer who were enrolled prospectively. The surgeons and the patients were unaware of the IHC results at the time of decision-making, to avoid any decision bias. Although our initial observation in a retrospective analysis of non-muscle-invasive TCC strongly supported p53 as an independent prognostic marker [7], we failed to detect a correlation between p53 overexpression and clinical outcome in the present prospective study. This group of patients differed significantly from the previous reported series of patients with T1 tumours. In that retrospective analysis, radical cystectomy was offered at the time of progression to muscle invasion, while in the present contemporary series, a radical cystectomy was used for uncontrollable disease that was not muscle-invasive, as defined by recurrent T1 and those refractory to BCG, as for muscle-invasive tumours. Twenty-six patients had a delayed radical cystectomy for non-muscle-invasive disease, while only nine had a delayed cystectomy for progression to a muscle-invasive tumour. One patient who progressed did not have a cystectomy. It is conceivable that earlier intervention with radical cystectomy improves the overall outcome and offsets the effect of p53-positive status on DSS. The disparity in the significance of markers reported between the retrospective and prospective studies might be mainly attributable to the change in the treatment system. p53 overexpression was previously reported to be associated with as greater rate of recurrence, progression and aggressive biological behaviour in patients with TCC of the bladder. All the previous studies were retrospective in a heterogeneous patient population with bladder cancer, leading to conflicting reports. The present study is the first to examine prospectively the role of a molecular marker in bladder cancer. Moreover, the role of p53 was evaluated in a homogeneous patient population with a primary diagnosis of T1 bladder cancer with no additional therapy. It is possible that the diligent and more aggressive approach of the contemporary management of T1 tumours offset the prognostic significance of p53 status. Median (95% CI) incidence Variable N events 3-year 5-year P* Overall Progression (7 25) 16 (9 29) Cystectomy (27 51) 37 (27 51) p53 results progression Positive 7 16 (7 33) 20 (10 40) 7 Negative 3 10 (3 32) 10 (3 32) p53 result cystectomy Positive (30 64) 43 (30 64) 7 Negative 9 29 (17 51) 29 (17 51) Incidence probability CONFLICT OF INTEREST None declared. REFERENCES P53 Negative Progression P53 Positive Progression P53 Negative Cystectomy without progression P53 Positive Cystectomy without progression Years from initial diagnosis 1 Millan-Rodriguez F, Chechile-Toniolo G, Salvador-Bayarri J, Palou J, Algaba F, Vicente-Rodriguez J. Primary superficial bladder cancer risk groups according to progression, mortality and recurrence. J Urol 2000; 164: Millan-Rodriguez F, Chechile-Toniolo G, Salvador-Bayarri J, Palou J, Vicente- Rodriguez J. Multivariate analysis of the prognostic factors of primary superficial bladder cancer. J Urol 2000; 163: Quek ML, Stein JP, Nichols PW et al. Prognostic significance of lymphovascular invasion of bladder cancer treated with radical cystectomy. J Urol 2005; 174: Goddard JC, Sutton CD, Furness PN, O Byrne KJ, Kockelbergh RC. Microvessel density at presentation predicts subsequent muscle invasion in superficial bladder cancer. Clin Cancer Res 2003; 9: TABLE 3 Incidence of progression and delayed cystectomy in surveillance patients *from competing-risk analysis with modified chi-square test. FIG. 3. The incidence of progression or cystectomy stratified by p53 status. 5 Malats N, Bustos A, Nascimento CM et al. P53 as a prognostic marker for bladder cancer: a meta-analysis and review. Lancet Oncol 2005; 6: Rabbani F, Cordon-Cardo C. Mutation of cell cycle regulators and their impact on superficial bladder cancer. Urol Clin North Am 2000; 27: Sarkis AS, Dalbagni G, Cordon-Cardo C et al. Nuclear overexpression of p53 protein in transitional cell bladder carcinoma: a marker for disease progression. J Natl Cancer Inst 1993; 85: Reuter VE, Epstein JI, Amin MB, Mostofi FK. The WHO/ISUP Consensus Classification of Urothelial (Transitional Cell) Neoplasms : continued discussion. Hum Pathol 1999; 30: Cordon-Cardo C, Finstad CL, Bander NH, Melamed MR. Immunoanatomic distribution of cytostructural and tissue-associated antigens in the human urinary tract. Am J Pathol 1987; 126: Banks L, Matlashewski G, Crawford L. Isolation of human-p53-specific monoclonal antibodies and their use in BJU INTERNATIONAL

5 p53 AS A PROGNOSTIC MARKER IN BLADDER CANCER the studies of human p53 expression. Eur J Biochem 1986; 159: Rivas CI, Wisniewski D, Strife A et al. Constitutive expression of p53 protein in enriched normal human marrow blast cell populations. Blood 1992; 79: Finlay CA, Hinds PW, Tan TH, Eliyahu D, Oren M, Levine AJ. Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life. Mol Cell Biol 1988; 8: Queipo Zaragoza JA, Ruiz Cerda JL, Palmero Marti L, Rubio Martinez LA, Vera Sempere F, Jimenez Cruz JF. [Prognostic value for progression of the regulating proteins of the cellular cycle in PT1G3 bladder tumours]. Actas Urol Esp 2005; 29: Mitra AP, Datar RH, Cote RJ. Molecular staging of bladder cancer. BJU Int 2005; 96: Lorenzo Romero JG, Salinas Sanchez AS, Gimenez Bachs JM et al. p53 Gene mutations in superficial bladder cancer. Urol Int 2004; 73: Correspondence: Guido Dalbagni, Department of Urology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. dalbagng@mskcc.org Abbreviations: CIS, carcinoma in situ; (O)(DS)S, (overall) (disease-specific) survival; IHC, immunohistochemistry BJU INTERNATIONAL 285

Influence of stage discrepancy on outcome in. in patients treated with radical cystectomy.

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