Pretreatment p53 Nuclear Overexpression as a Prognostic Marker in Superficial Bladder Cancer Treated with Bacillus Calmette–Guérin (BCG)

European Urology

European Urology 45 (2004) 475–482

Pretreatment p53 Nuclear Overexpression as a Prognostic Marker in Superficial Bladder CancerTreated with Bacillus Calmette^Gue¤rin (BCG) Fabien Sainta,b,*, Marie-Aude Le Frere Beldab,c,1, Rodrigo Quintelaa,b,1, Andras Hozneka,b, Jean-Jacques Patarda,b, Jacqueline Bellotc, Zivko Popovb, Elie S. Zafranic, Claude C. Abboua,b, Dominique K. Chopina,b, Sixtina Gil Diez de Medinab a

Department of Urology, Hoˆpital Henri Mondor, 94000 Cre´teil, France Groupe d’Etude des Tumeurs Urologiques (GETU) and EMI-INSERM 99/09, Hoˆpital Henri Mondor, 94000 Cre´teil, France c Department of Pathology, Hoˆpital Henri Mondor, 94000 Cre´teil, France b

Accepted 14 November 2003 Published online 13 December 2003

Abstract Introduction: Altered p53 gene product correlates with the stage and grade of bladder tumor, but its value as a predictor of BCG response has been disappointing. In order to revisite the prognostic value of pretreatment p53 nuclear overexpression for the BCG response, we studied a large cohort of consecutive patients with superficial bladder cancer treated with BCG. Methods: From 1988 to 2001, 102 patients with a history of multifocal, recurrent, and/or high-risk papillary transitional cell carcinoma or carcinoma in situ, were treated for the first time with BCG. p53 immunostaining was performed on paraffin-embedded tissues using monoclonal antibody DO7 and an automated immunostainer. Special attention was paid to the conditions of tumor fixation. p53 overexpression was defined as more than 20% tumor cells with p53-stained nuclei. Results: Immunostaining was significantly higher for Ta/T1 G3
Cis (p < 0001), tumoral substage T1b (p ¼ 0:001), grade 3 (p ¼ 0:0001), and Cis (p ¼ 0:02). Times to recurrence, progression and cancer death were shorter among patients with p53 overexpression (p ¼ 0:03; p < 0:0001; p ¼ 0:0003). In multivariate analysis, p53 overexpression was an independent predictor of recurrence (p ¼ 0:0003) [RR ¼ 0:15; 95%CI, 0.06 to 0.42]. Conclusion: Pretreatment p53 nuclear overexpression in superficial bladder tumors is associated with a high risk of disease recurrence, progression and cancer death after BCG therapy. Applying antibody DO7 with an automated immunostainer and stringent fixative conditions, p53 nuclear immunostaining yields clinically relevant information and may be a useful tool for selecting patients with superficial bladder cancer who might be resistant to BCG. # 2003 Elsevier B.V. All rights reserved. Keywords: Bladder cancer; p53; Tumor suppressor gene; BCG therapy

1. Introduction The use of intravesical instillation of Bacillus Calmette–Gue´rin (BCG) has markedly improved the out*

Corresponding author. Present address: Service d’Urologie, CHU d’Amiens, Avenue R. Laennec, Salouel, 80054 Amiens Cedex 1, France. Tel. þ33-3-22-45-59-41; Fax: þ33-3-22-45-57-91. E-mail address: [email protected] (F. Saint). 1 These two authors made the same contribution to this work.

come of intermediate and high-risk superficial bladder cancer [1,2]. However, early BCG failure is associated with a worse prognosis and survival might be better in patients treated by first-line cystectomy compared to those with invasive disease after failed BCG therapy [3–5]. It has been independently reported that p53 nuclear accumulation correlates with advanced bladder tumor stage and grade [6,7]. However, the prognostic value of

0302-2838/$ – see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.eururo.2003.11.018

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F. Saint et al. / European Urology 45 (2004) 475–482

the p53 status of superficial bladder tumors treated with BCG remains controversial [8–12]. Despite progress in antigen retrieval techniques, technical variations in p53 immunohistochemistry may result in antigen or epitope denaturation and masking, leading to conflicting results and interpretation [13]. In order to assess the prognostic value of p53 immunostaining, for the response to BCG, we analyzed retrospectively and prospectively pretreatment p53 overexpression in a group of 102 patients with superficial bladder cancer who were treated with BCG for the first time. Special attention was paid to the conditions of sample fixation, and the staining procedure was standardized. Correlations were sought between immunostaining status and recurrence, progression and disease-specific survival.

2. Methods 2.1. Protocol From 1988 to 2001, a total of 102 patients (96 men and 6 women), with a mean age of 64.6 years (range 36.8–88) and a history of papillary transitional cell carcinoma, with or without carcinoma in situ (Cis), were enrolled in this study. Tumors were classified by stage according to the TNM classification [14], and by grade according to the World Health Organization criteria [15]. Patients with mixed-stage tumors were always classified according to the highest stage observed. All Cis were analyzed as high-grade tumors. The tumors studied were two primary/secondary Cis; 16 Ta (superficial non-invasive papillary tumors) or T1 (lesions invading the lamina propria) grade 3 tumors with Cis; 22 Ta or T1 grade 3 tumors; 62 Ta or T1 grade 1 or grade 2 tumors. All patients were followed at our Department of Urology. Tissues were obtained during endoscopic resection in every case, and pretreatment urinary cytologic results were recorded. The initial tumors, sampled immediately before BCG treatment, were used for this purpose. Patients underwent complete resection, and the muscle layer was always assessable. Second look TUR was systematically conduced for stage pT1bG3 and when resection was considered partial or when muscle was not assessable. Transitional cell carcinomas and Cis of the bladder were treated for the first time with six weekly instillations of intravesical BCG, three to six weeks after the last transurethral resection (150 mg of BCG IMMUN F1 strain, Institut Pasteur, Paris, France (n ¼ 44; from 1988 to 1996) or 81 mg of BCG Connaught Immucyst1 strain Aventis-Pasteur, Lyon, France) (n ¼ 58; from 1996 to 2001), in 50 ml of saline instilled via a 14 F catheter). Four weeks after the last instillation, urinary cytology and cystoscopic examination with randomized sites of mapping cold biopsies (area adjacent to the tumor, opposite bladder wall, bladder dome, trigone, and prostatic urethra) were performed. If these examinations were negative, patients treated by the IMMUN F1 strain protocol were simply monitored while patients treated with Immucyst1 strain received maintenance therapy (three-weekly instillations, given at 3, 6, 12, 18, 24, 30 and 36 months). Patients were instructed to retain the solution for approximately 2 hours. Intradermal BCG inoculation was not performed. Patients were followed up by cystocopy and urinary

cytologic examination every 3 months for the first year, and twiceyearly thereafter, in the absence of recurrence or progression. Responses were assessed on the basis of histological and cytological criteria and were classified as complete if urinary cytology and bladder-wall biopsy were negative. Tumor recurrence was defined as positive transurethral resection and/or biopsy, without progression of stage. Tumor progression was defined as progression of tumor stage, muscle infiltration, metastasis or tumor recurrence requiring additional therapy or cystectomy [2]. 2.2. p53 immunohistochemistry After transurethral resection, specimens were immediately fixed in 10% formalin (from 1988 to 1996; n ¼ 32), or fixed in AFA (2% formalin, 70% ethanol absolut, 5% acetic acid, and 18% distillated water; CARLO ERBA Reactifs, Val de Reuil, France) (from 1996 to 2001; n ¼ 70) for up to 18 hours and then paraffin-embedded. Paraffin-embedded sections were hematoxylin-eosin (HE) stained and analyzed by two pathologists (MALFB and JB) and classified according TNM and WHO criteria. Sample treated with AFA fixative were prospectively sectioned, stained with hematoxylin eosin and p53-immunostained the day after paraffin embedding. Samples fixed with formalin were p53-immunostained retrospectively on freshly cut slides without storage. Immunostaining was always done with an automated immunostainer (Ventana Medical Systems Inc., Tucson, AZ, USA) according to the manufacturer’s instructions. Sections (5 mm) of paraffin-embedded specimens were deparaffinized and rehydrated through graded ethanol solutions. To block endogenous peroxidase activity, sections were treated for 3 minutes with 0.3% hydrogen peroxide in methanol. Before applying the primary antibody, antigen retrieval was performed by heating the slide in a microwave oven in citrate buffer (10 mM; pH 6.7) for a total of 42 minutes (12-minutes at 750W and three ten-minute periods at 350W, evaporated buffer being replaced between heating periods). Sections were then incubated with monoclonal antibody p53-DO7 (DAKO, France) at 1:20 dilution in Ventana’s proprietary buffer for 60 minutes at room temperature. This was followed by incubation with horse antimouse secondary antibodies for 30 minutes. The sections were then washed thoroughly in phosphate-buffered saline, and the peroxidase signal was developed in 0.05% 3,30 -diaminobenzidine (DAB) and 0.01% hydrogen peroxide in phosphate-buffered saline. For negative controls, the primary antibody was substituted with distilled water; strongly positive slides of transitional cell carcinoma with documented p53 nuclear overexpression were used as positive controls. One examiners (MALFB) blindly scored nuclear p53 immunoreactivity in tumor cells by manually counting the number of stained nuclei. Five hundred cells in the most strongly positive section were counted by light microscopy (400 magnification), and a p53 labeling index (LI) was calculated as the percentage of positive nuclei among all scored nuclei. p53 overexpression was defined as more than 20% tumor cells displaying stained nuclei, according to Lacombe et al. [16], leading to categorization of patients as p53-negative or -positive. For statistical analysis, tumors were divided into these two categories. 2.3. Statistical analysis Statistical analyses were done using StatView software version 4.5 (Statistical Analysis System Institute, Cary, NC, USA). The following tumor and host variables were analyzed: age, sex, stage, tumoral substage T1a (lesion invading the superficial lamina propria)/T1b (lesion invading the deep lamina propria), high-risk tumors (Ta/T1 G3
Cis) versus intermediate-risk tumors (Ta/T1 G1G2), grade, multifocality, tumor size, associated carcinoma in situ (Cis), previous recurrence, recurrence during the first six

F. Saint et al. / European Urology 45 (2004) 475–482

months of follow-up, pre-BCG cytology, BCG strain and schedule, and p53 overexpression. Intercorrelations between age, tumor size, and p53 overexpression were tested using the non-parametric Mann–Whitney–Wilcoxon test. Intercorrelations between sex, stage, tumoral substage T1a/T1b, HR/IR, grade, multifocality, associated Cis, previous recurrence, recurrence during the first six months of follow-up, pre-BCG cytology, BCG strain and schedule, and p53 overexpression were tested by using Fisher’s exact test. All reported p values are 2-sided (significant at p < 0:05). The log-rank test was used to identify correlations between the incidence of tumor recurrence, progression and death and p53 overexpression, as well as clinical and pathological variables including age, sex, stage, tumor substage T1a/T1b, grade, multifocality, tumor size, associated Tis, previous recurrence, recurrence during the first six months of follow-up, BCG strain and BCG schedule. Time to recurrence, progression, and the disease-specific survival time were analyzed using the Kaplan– Meier method. The cutoff date for patients without recurrence, progression, or cancer death was the date of the last cystoscopy. The Cox proportional hazards model was used for multivariate analysis of parameters predictive of tumor recurrence and to estimate relative risks [17]. In

477

particular, stepwise regression (maximum partial likelihood ratio method) was used to select independent prognostic factors.

3. Results The average age of the patients was 64.6 years (range 36.8–88). Median follow-up was 40 months (range 4.4–164.5 months). Fifty tumors were multifocal (49%) and 52 were unifocal (51%). Of the 102 individuals evaluated, 36 (35.3%) experienced disease recurrence and 13 (12.7%) disease progression; 5 (4.9%) died of bladder cancer. Seventy-eight patients (76.5%) were p53-negative and 24 (23.5%) p53-positive. The relations between pretreatment p53 status and clinical and pathological variables are summarized in Table 1. The two patient groups were similar in terms of age, pre-BCG cytology, multifocality, previous TCC, tumor size, and the BCG

Table 1 Standard and pathological variables characteristics and relation to p53 overexpression (assessed by Fisher exact test () and by Mann-Whitney test (D)) Patients characteristics

p53 n (%)

p53þ n (%)

p value ()

High risk (n ¼ 39) (Ta/T1 G3
Cis) Intermediate risk (n ¼ 63) (Ta/T1 G1G2)

21 (53.8%) 57 (90.5%)

18 (46.2%) 6 (9.5%)

p < 0.0001

T1a (n ¼ 53) T1b (n ¼ 17)

45 (84.9%) 7 (41.2%)

8 (15.1%) 10 (58.8%)

p ¼ 0.001

Grade

G1–G2 (n ¼ 64) G3 (n ¼ 38)

58 (90.6%) 20 (52.6%)

6 (9.4%) 18 (47.3%)

p ¼ 0.0001

Cytology before BCG

Positive (n ¼ 64) Negative (n ¼ 22) Unknown (n ¼ 17)

46 (71.9%) 19 (86.4%)

16 (28.1%) 3 (3.6%)

NS

Multifocal tumors

Yes (n ¼ 50) No (n ¼ 52)

37 (74%) 42 (80.8%)

13 (26%) 11 (21.2%)

NS

22.9 [2–70]

21.4 [2–70]

NS(D)

Stage

Tumor size/mm (mean/[range]) Associated Cis

Yes (n ¼ 18) No (n ¼ 84)

10 (55.5%) 68 (81%)

8 (44.5%) 16 (29%)

p ¼ 0.02

Previous tumor before BCG

Yes (n ¼ 35) No (n ¼ 67)

30 (85.7%) 48 (71.7%)

5 (4.3%) 19 (28.3%)

NS

BCG strain

Pasteur (n ¼ 44) Connaught (n ¼ 58)

38 (86.4%) 40 (69%)

6 (13.6%) 18 (31%)

NS

BCG maintenance schedule

Yes (n ¼ 51) No (n ¼ 51)

36 (70.6%) 42 (82.4%)

15 (29.4%) 9 (17.6%)

NS

Recurrences

Yes (n ¼ 36) No (n ¼ 66)

26 (72.3%) 52 (78.8%)

10 (27.7%) 14 (21.2%)

NS

Yes (n ¼ 19) No (n ¼ 17)

10 (52.6%) 16 (94.1%)

9 (47.4%) 1 (5.9%)

Progression

Yes (n ¼ 13) No (n ¼ 89)

5 (1 HR, 4 IR) (38.5%) 73 (20 HR, 53 IR) (82%)

8 (6 HR, 2 IR) (61.5%) 16 (12 HR, 4 IR) (18%)

p ¼ 0.001

Death by cancer

Yes (n ¼ 5) No (n ¼ 97)

1 (20%) 78 (80.4%)

4 (80%) 20 (19.6%)

p ¼ 0.002

Recurrences Before 6 months After 6 months

HR: High risk tumor; IR: Intermediate risk tumor.

p ¼ 0.01

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F. Saint et al. / European Urology 45 (2004) 475–482

strain and schedule. Pretreatment p53 nuclear overexpression was identified in 24 patients, and was more frequent in high-risk tumors (TaG3 and T1G3 with or without Cis (Ta/T1 G3
Cis); 18 of 39 cases, 46.2%) than in intermediate-risk tumors (Ta/T1 G1G2; 6 of 63 cases, 9.5%) (p < 0001). p53 nuclear overexpression was identified in 18 of 70 (25.7%) bladder tumors from patients with T1 disease. The prevalence of p53 nuclear overexpression was higher in stage T1b than in stage T1a (p ¼ 0:001) [58.8% (10/17) versus 15% (8/53)], and higher in grade 3 than in grades 1 and 2 (p ¼ 0:0001) [47.3% (18/38) versus 9.3% (6/64)]. Nuclear p53 overexpression was also higher in tumors with associated Cis (8 of 18, 44.5%), than in tumors without Cis (16 of 84, 19%) (p ¼ 0:02). Of the 78 p53negative patients, 26 (33.3%) had a disease recurrence, compared to 10 (41.7%) of the 24 patients with p53positive tumors (NS). Recurrence occurred earlier in patients with p53-positive tumors (90% before 6 months) than in patients with p53-negative tumors (38.5% before 6 months) (p ¼ 0:01). Of the 78 p53-negative patients, 5 (6.4%) had disease progression, compared with 8 (33.4%) of the 24 p53positive patients (p ¼ 0:001). One (1.3%) of the 78 p53-negative patients died of bladder cancer, compared with 4 (16.7%) of 24 p53-positive patients (p ¼ 0:002). The mean times to recurrence, progression and death by cancer in p53-positive patients were respectively, 7.7 months (range 1.2–11.8), 19 months (range 6.3– 33.3), and 41 months (range 26.6–55). The corresponding times in p53-negative patients were respectively, 20.4 months (range 3.5–73), 22 months (range 10.9– 49.5), and 17.7 months. Median time to progression after recurrence was 13 months (range 1.4–36.6), and median time to cancer

death after progression was 19 months (range 4.8– 24.3) (Table 2). Log-rank univariate analysis showed that the risk of disease recurrence was significantly higher in patients with intermediate-risk tumors, positive pre-BCG cytology, previous TCC, no BCG maintenance therapy, and p53 overexpression (p ¼ 0:04; p ¼ 0:03; p ¼ 0:05; p ¼ 0:001; p ¼ 0:03). Likewise, the risk of disease progression was significantly higher in patients with Cis (with or without papillary tumors), recurrence during the first six months of follow-up, and p53 overexpression (p ¼ 0:002; p ¼ 0:03; p < 0:0001). Finally, the risk of cancer death was significantly higher in patients with high-risk tumors and p53 overexpression (p ¼ 0:002; p ¼ 0:0003). To identify the most informative combination of independent prognostic factors, variables that predicted recurrence in univariate analysis, including p53-positivity, HR/IR tumors, and BCG maintenance therapy, were subjected to multivariate analysis in a stepwise Cox proportional hazards regression model. Intermediate-risk tumors, BCG maintenance therapy, and p53 positivity remained independent predictors of recurrence as follow: intermediate-risk versus highrisk tumors (p ¼ 0:004) (relative risk ½RR ¼ 4:26; 95% confidence interval [CI], 1.56 to 11.65), a single six-weekly course of BCG versus BCG maintenance therapy (p ¼ 0:0001) (RR ¼ 6:01; 95%CI, 2.43 to 14.88), and p53 negativity versus p53 positivity (p ¼ 0:0003) (RR ¼ 0:15; 95%CI, 0.06 to 0.42). Figs. 1–3 illustrate actuarial disease-related survival curves according to recurrence, progression, and disease-specific survival, respectively, and p53 overexpression. Informative prognostic factors for tumor progression in univariate analysis were not subjected

Table 2 p53 nuclear expression, pathological characteristics at initial stage, time to tumor recurrence and progression, and cancer death of patients with tumor progression Patient No.

Stage before BCG

p53 IHC (%)

1 2 3

T1b G1 T1a G2 T1b G3

39 76 50

4 5 6 7 8 9 10 11 12 13

T1a G3 T1a G3 and Tis Ta G3 Ta G2 T1a G3 and Tis T1a G2 Ta G1 T1a G2 T1a G2 T1a G3 and Tis

29 72 86 35 75 0 0 3 0 0

Time to recurrence (month)

Time to progression (month)

Progression type

Treatment of progression

Death by cancer (month)

5.6 1.2 7

7 33.3 9

T3G3N0M0 T3G2NþM0 T2N0M0

26.6 51.8 –

11.8 9.3 – 7 6 – 12.9 6 16.2 –

18.5 30.7 6.3 23 25.9 14.3 49.5 16.4 18.6 10.9

T1G1N0M0 T2G2N0M0 T2G3N0M0 T0N0Mþ T4G3N0M0 T0N0Mþ T1G1N0M0 T2G3N0M0 T1G2N0M0 T4G3NþMþ

Cystectomy Cystectomy þ systemic chemotherapy Concomitant radiotherapy þ systemic chemotherapy Cystectomy Cystectomy Iterative resection Systemic chemotherapy Cystectomy Systemic chemotherapy Cystectomy Cystectomy Cystectomy Cystectomy þ systemic chemotherapy

– 55 – – 30.7 17.7 – – – –

Proportion of patients without recurrence

F. Saint et al. / European Urology 45 (2004) 475–482

1

to multivariate analysis because of the small number of events.

p53 negative-tumors (n=78)

,8 p53 positive-tumors (n=24)

,6

4. Discussion

,4 Patients censured ,2

p=0.03 (unadjusted) p=0.0003 (adjusted)

0 0

20

40

60

80

100

120

140

160

Months of follow-up

Proportion of patients without progression

Fig. 1. Kaplan–Meyer curve illustrates recurrence-free interval in patients with p53 immunostaining below (Group A; n ¼ 24) and above (Group B; n ¼ 79) 20%. Analysis showed that p53-positive group had significantly higher disease-recurrence rate (p ¼ 0:03; unadjusted) (p ¼ 0:0003; adjusted).

1

p53-negative tumors (n=78)

,8 ,6 p53-positive tumors (n=24) Patients censured

,4 ,2 p<0.0001 (unadjusted) 0 0

20

40

60

80

100

120

140

160

180

Months of follow-up

Fig. 2. Kaplan–Meyer curve illustrates progression-free interval in patients with p53 immunostaining below (Group A; n ¼ 24) and above (Group B; n ¼ 79) 20%. Analysis showed that p53-positive group had significantly higher disease-recurrence rate (p < 0:0001; unadjusted).

1

Proportion of patients surviving

479

p53-negative tumors (n=78) ,8

,6 p53-positive tumors (n=24)

Patients censured

,4

,2 p=0.0003 (unadjusted) 0 0

20

40

60

80

100

120

140

160

180

Months of follow-up

Fig. 3. Kaplan–Meyer curve illustrates disease-specific survival interval in patients with p53 immunostaining below (Group A; n ¼ 24) and above (Group B; n ¼ 79) 20%. Analysis showed that p53-positive group had significantly lower disease-specific survival rate (p ¼ 0:0003; unadjusted).

The most frequent molecular events in human cancer, including that of bladder transitional cell carcinoma, are mutations of the p53 gene, leading to neoplastic transformation and tumor progression by loss of p53 functions [18]. Mechanisms other than p53 gene mutation, such as MDM2 phosphorylation, can also lead to p53 accumulation and might explain why some authors have suggested that protein evaluation is superior to gene mutation analysis as far as clinical outcome is concern [19]. Physiologically, wild type p53 acts as a regulator of cell cycle through DNA repair or apoptosis [20]. Wild-type p53 down-regulates endogenous vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) production, thereby limiting tumor growth by hindering the neovascularization induced by overproduction of these angiogenic factors [21]. Loss of this important regulatory function allows neovascularization to proceed uninhibited, and allows tumor development and progression. Immunohistochemical analysis has extensively evaluated p53 expression as a prognostic factor for bladder cancer progression [7,22]. p53 overexpression was correlated with advanced tumor stage and grade [6], and was forwarded as a significant negative predictor of survival [7]. More recently, p53 nuclear accumulation in bladder carcinomas cells was found to correlate with the mitotic index and vascular invasion [23]. However, the issue of p53 expression in superficial bladder cancer and response to therapy is controversial. Conflicting findings have been reported with respect to superficial bladder cancer progression. Correlation between p53 status and the progression rate and/or the disease-specific survival of patients with superficial bladder cancer was reported in many subsequent studies [24–27] Nevertheless, some investigators came to different conclusions and failed to confirm that p53 overexpression was an independent predictor of either disease progression or patient survival [28–30]. Patients with recurrent or high-grade superficial bladder cancer, with or without Cis, are frequently treated with adjuvant intravesical BCG therapy. Several investigators have evaluated the prognostic value of nuclear p53 immunoreactivity before and after BCG therapy. A correlation between pretreatment p53 overexpression and disease progression after BCG therapy was found in one study [16]. Other authors found no such correlation (Table 3) [8–12]. Nevertheless, using a

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F. Saint et al. / European Urology 45 (2004) 475–482

Table 3 Clinical trials published evaluating relation between pre-BCG p53 immunostaining and BCG response Authors [ref]

Year

Patients Tumor (n) stage

Lacombe [18]

1996

98

Ovesen [10] Lebret [11] Pages [14] Zlotta [12] Peyromaure [13] Present study

1997 1998 1998 1999 2002

60 35 43 47 29 102

Fixative used before embedding

Microwave oven heating

p53 antibodies

Dilution or concentration

Positive Median Independent staining follow-up prognostic threshold (or mean) value tested (%)

Ta, T1, Tis

Non-specified

Non-specified

PAb 1801

1/1200

20

44

Tis T1G3 T1, Ta Ta, T1, Tis T1G3 Ta, T1, Tis

Non-specified Formalin Formalin þ Bouin Non-specified Non-specified Formalin þ AFA

Yes Non-specified Yes Yes Yes Yes

PAb 1801 mAb DO7 mAb DO7 mAb DO7 mAb DO7 mAb DO7

200 ng/ml 1/100 1/50 1/100 1/50 1/20

20 0–65 10 0–20 20 20

48 51.3 29 (27.4) 36.7 40

yeast functional assay, it was found that BCG treatment was less likely to be successful in patients with mutated p53 [31]. In additions, some authors have shown that p53 overexpression in patients whose tumors failed to respond to BCG therapy correlated with disease progression and poor survival [8,16,32]. To date whether p53 tumor status is an independent predictive factor of BCG response remains still a debate. In the present study, we used a highly standardized immunostaining procedure to analyzed the correlation between pretreatment p53 overexpression and the response to BCG in a large cohort of patients with long-term follow-up. p53 status was found to correlate with the time to recurrence, progression and death by cancer even if for progression and death by cancer a lack of statistical power might be due to the small number of patients within both groups. Several points might explain these differences. First, variability of published results may be related to technical considerations (including nature of materiel, fixative used, tissue section storage, antigen retrieval methods, antibodies and threshold utilized). All studies has been done retrospectively, using paraffin embedded tissues and several fixatives overtime such as various preparation of formalin and Bouin’s fixatives [10–12]. Bouin’s fixative can result in over-fixation, antigen or epitope denaturation or masking, inefficient antigen retrieval, and, as a results, false-negative immunostaining [33]. These fixatives were used in some studies [9,12] while others did not specify their protocol [8,10,11]. These differences in fixative protocols might explain failure to correlate p53 immunostaining and BCG response. For the past years, pathologists of our institution have compared several fixatives in an attempt to obtain the best compromise between morphology, p53 protein immunohistochemistry and DNA analysis for bladder cancer specimens. In 1996, AFA emerged as the best choice, and adopted for p53

Yes (progression) No No No No No Yes (recurrence)

immunostaining. Ethanol produces rapid fixation and prevents cross-linking associated with formalin [34]. But ethanol supplementation is not used in most laboratories due to higher cost. Problems related to the storage condition of cut tissue sections, and their frequent use in the research environment, may influence immunostaining efficiency [13]. In our study, 70% of the sample immunostaining were prepared routinely and prospectively, and all paraffin tissue section were cut the day of immunostaining to avoid problems related to storage. These conditions seem to be critical for the interpretation and variability of immunostaining results, but are rarely mentioned in published reports [9,11,12,16]. Antigen retrieval methods might also contribute to the variability of immunostaining results [34]. In most published studies, relating p53 immunostaining to BCG response, antigen retrieval was performed by heating the slides in a microwave oven [10–12], but for some of them, the protocol used was not described [8,12] or even not mentioned in other reports, ruling out any valid comparison [9,16]. It remains to be determined whether certain antigen retrieval protocols efficiently restore immunoreactivity in paraffin slides, and excessive heating can results in excessive background staining, compromising the interpretation of final results. Some other variability in p53 immunostaining can be observed depending on the antibody used [12,16]. Most published series that did not find any correlation between p53 overexpression and tumor recurrence/ progression after BCG therapy involved mAb DO7 antibody, whereas a study using PAb 1801 antibody showed correlation with tumor progression [16]. Our study is the first providing to show a correlation between p53 overexpression and recurrence, progression and cancer death, using mAb DO7 antibody. It is noteworthy that the two antibodies recognize

F. Saint et al. / European Urology 45 (2004) 475–482

neighboring epitopes on the amino-terminal domain of the protein [26]. Nevertheless, differences may exist, and there is probably a need to define adjusted dilution with the optimized antigen retrieval methods for p53 immunostaining. Moreover, the cut off used to define p53 overexpression is probably also of prime importance. We chose a cutoff value of 20%, as used by Lacombe, and came to the same conclusion as these latter authors, even though we used a different antibody [16]. Most of other authors used this cutoff, but others have used 10% [12], 15% [30], or more than 20% [9]. The interpretation of immunohistochemical results may vary among centers [35]. McShane and coworkers demonstrated that intra-laboratory reproducibility was good. Interlaboratory agreement was also good for specimens exhibiting either minimal or, on the contrary, abundant nuclear immunostaining, but was poorer for specimens with intermediate staining (10 to 15% of tumor cells) [35]. Such finding, again, indicates the need for caution when comparing results from different laboratories, and focus on the necessity to select an uniform threshold for binary interpretation. It is noteworthy that a cutoff above 20% yields good intra-laboratory reproducibility [35]. Another variability relates to the fact that certain mutations of p53 may be present in the absence of p53 accumulation and/or tumor heterogeneity [23,36]. In addition, tumor heterogeneity may account for small percentage of truly p53 positive cells and these tumors would be considered negative using immunohistochemistry. Finally, differences in patient population, regarding a previous history of bladder cancer, the treatment protocol and tumor characteristics (substage, associated Cis, and multifocality) may account for discrepancies among published results. Two-thirds of published studies of p53 as a prognostic markers for the response to BCG involved fewer than 50 patients. It is well estab-

481

lished that the power of statistical test (1-b), is related to the number of patient. When this number is low the power of the test decreases as the risk b (to declare nonsignificant some results while in fact the difference exist) increases [37]. Only large cohort and long follow-up with heterogenate tumor stage succeded to establish differences in time to tumor recurrence or progression and p53 immunostaining. This settle reinforce firstly the fact that definitive conclusions are of no value for small patients cohort and short follow-up, and secondly that classical pathological staging is unsuitable to predict BCG response, and that p53 is a relevant parameter to consider.

5. Conclusion This study strongly suggests that pretreatment p53 overexpression by immunohistochemistry, in a stringent standardized automated protocol, is predictive of tumor recurrence, progression and death by cancer after BCG instillation in superficial bladder cancer. Moreover p53 status yield significant prognostic information for tumor recurrence in addition to stage. A well-powered prospective trial, using a validated protocol for technical reproducibility, will be needed to determine the value of p53 immunostaining and to identify the subgroup of patients that might be poor BCG responders.

Acknowledgements This work was in part funded by Association Claude Bernard, Hoˆ pital Henri Mondor, Assistance Publique des Hoˆ pitaux de Paris (AP-HP), Universite´ Paris XII, the INSERM, the Fondation de l’Avenir pour la Recherche Me´ dicale Applique´ e, the Fondation pour la Recherche Me´ dicale, and the Fondation de France.

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