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Clinical Cancer Research Vol. 11, 4724-4732, July 1, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Assessment of the Transcriptional Activity of p53 Improves the Prediction of Recurrence in Superficial Transitional Cell Carcinoma of the Bladder

Anne-France Dekairelle1, Bertrand Tombal2,3, Jean-Pierre Cosyns4 and Jean-Luc Gala1,5

Authors' Affiliations: 1 Laboratory of Applied Molecular Technologies, Center for Human Genetics and 2 Laboratory of Cellular Physiology, Université catholique de Louvain; 3 Division of Urology and 4 Department of Pathology, Cliniques Universitaires Saint-Luc; and 5 Defense Laboratories Department, Belgian Armed Forces, Brussels, Belgium

Requests for reprints: Jean-Luc Gala, Laboratory of Applied Molecular Technologies, Center for Human Genetics, Université catholique de Louvain, Clos Chapelle-aux-Champs, 30-UCL/30.46, B-1200 Brussels, Belgium. Phone: 32-2-764-3165; Fax: 32-2-764-3166; E-mail: gala{at}lbcm.ucl.ac.be.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Purpose: To investigate the value of p53 functional analysis of separated alleles in yeast (FASAY) as a witness of p53/p21 pathway alteration and as a predictor of recurrence in superficial transitional cell carcinomas.

Experimental Design: p53 transcriptional activity was prospectively analyzed in 52 newly diagnosed transitional cell carcinoma using FASAY competent for the transactivation of p21 and bax promoters. TP53 and p21 gene expression was quantified by real-time PCR, and expression of corresponding proteins was assessed by immunohistochemistry. In addition to tumor stage and grade, the predictive value of FASAY, real-time PCR, and immunohistochemistry for tumor recurrence was assessed by Cox survival analysis.

Results: A total (p21 and bax) or partial (bax only) loss of transcriptional activity was observed in 15 of 52 (29%) and 4 of 52 (7.7%) cases, respectively, a partial loss being consistently associated with R283H mutation. p53 nuclear overexpression grossly overestimated (~40%) or underestimated (~10%) the true incidence of p53 transcriptional abnormalities, especially in Ta-T1 grade 1 to 2 tumors. Loss of p21 transactivation significantly correlated with decreased p21 gene expression and lack of expression of p21 (P = 0.001). FASAY had a better predictive value for recurrence than p53 immunohistochemistry (Cox hazard ratio, 6.57 versus 3.95; P = 0.0002 versus 0.019, respectively), whereas neither p21 immunohistochemistry (hazard ratio, 1.9; P = 0.29) nor TP53 or p21 gene expression were significant predictors of recurrence. The prognostic difference between FASAY and p53 immunohistochemistry was maintained in the subgroup of Ta-T1 grade 3 tumors.

Conclusions: FASAY is a valuable surrogate marker for assessing p53/p21 pathway alteration and predicts transitional cell carcinoma recurrence better than p53 immunohistochemistry.

Transitional cell carcinoma (TCC) of the bladder is the fourth most common malignancy in men and the eight most common cancer in women. Eighty percent of TCCs are initially superficial (TMN97: Ta-T1) and easily treated by transurethral resection of bladder tumor. Despite initial complete resection of the tumor, recurrence occurs in 30% to 90% of the cases (1, 2). Progression to muscle invasive and/or metastatic stages requires radical and/or systemic therapies in 15% to 20% of the cases. To decrease recurrence and progression rates, intravesical administration of chemotherapy and/or Bacillus Calmette-Guerin is considered as standard therapy (2, 3). Because these therapies are not devoid of local and systemic toxicity, their use should better be guided by individual risks for recurrence and progression (4).

Over the years, many attempts have been made to identify molecular markers that would supersede clinical features, such as tumor stage, grade, and multifocality (5, 6). Expression of TP53 gene has been one of the most frequently assessed genetic alterations in bladder TCC. In an overwhelming majority of studies, the prognostic significance of TP53 alterations in bladder TCC is relying on the identification of p53 nuclear overexpression by immunohistochemistry (7–15). Considering that the carcinogenic effect of TP53 mutations in tumor cells is mainly mediated by inactivation of p53 transcriptional activity, including loss of p21 expression, routine evaluation of this functional activity should be substituted to immunohistochemistry (16). The number of discordant results reported between immunohistochemistry and DNA analyses shows indeed that p53 nuclear protein accumulation cannot be considered as a reliable surrogate marker of p53 mutations and still less of its transcriptional activity (10, 13, 17–21). The routine use of a functional assay in yeast to selectively identify inactivating p53 mutations in bladder tumors seems therefore as a better approach but still needed confirmation of its predictive value on the outcome in superficial TCCs of the bladder (21, 22).

Here, the TP53 gene status of the tumor was determined by a functional analysis of separated alleles in yeast (FASAY) competent for the transactivation of p21 and bax promoters (22–24). Quantification of TP53 and p21 gene expression was carried out by real-time PCR, and quantification of p53 and p21 protein expression was carried out by immunohistochemistry. Data were compared and correlated with clinicopathologic features and clinical outcome.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 Conclusions
 References
 
Patients. After informed consent, 52 patients newly diagnosed with a bladder tumor were enrolled in the study. Patients with previous history of urinary infection, prostatic carcinoma, or in situ carcinoma of the bladder were not included. All patients underwent a complete transurethral resection of bladder tumor. In each case, a fragment of the tumor was obtained and processed for p53 transcriptional activity (see below). The remaining material tumor was formalin fixed and paraffin embedded for pathologic evaluation. In addition, a biopsy of the normal-looking surrounding bladder mucosa was obtained in 26 of 52 patients and processed for p53 transcriptional activity. These samples were used as paired controls in the quantification of gene expression by real-time PCR.

Anatomopathologic evaluation of the paraffin-embedded tumor specimens was done by a single pathologist (J.P.C.) according to TMN97 staging system for disease extension and WHO/International Society of Urological Pathology consensus for grade (25, 26).

All the patients received a single immediate postoperative instillation of epirubicin according to the European Organization for Research and Treatment of Cancer recommendation (3). All the patients, but 6 patients with muscle invasive disease, were then followed every 3 months by cytoscopy and urinary cytology. Bladder biopsy or transurethral resection of bladder tumor was done in the case of abnormal cystoscopy or class V cytology (27). A recurrence was defined as the diagnosis of at least one new tumor further classified Ta-T1 of any grade and a progression as a new lesion classified ≥T2.

Immunohistochemistry for p53 and p21 and scoring. Immunohistochemistry techniques were adapted from a previous report (28). Briefly, paraffin-embedded slides were dewaxed with Histosafe (Yvsolab, Beerse, Belgium), rehydrated, treated in citrate solution, and immunostained using the anti-p53 mouse monoclonal antibody DO-7 (Neomarkers, Fremont, CA) at a dilution of 1:300 or the anti-p21 mouse monoclonal antibody NCL-L-WAF-1 (Novocastra, Newcastle, United Kingdom) at a dilution of 1:30 for 16 hours at 4°C. The second antibody used was a ready-for-use anti-mouse EnVision-Peroxidase system (DAKO, Glostrup, Denmark) for p53 and PowerVision (ImmunoLogic, Duiven, the Netherlands) for p21 according to the manufacturer's protocols. A normal goat serum was used as negative control. Regarding p21 staining, a normal tissue biopsy was used as positive control. For p53 staining, cell lines showing a positive (DU145) and negative (LNCaP) p53 staining were used as positive and negative controls, respectively (29). Slides were examined under a light microscope. For p53, samples were stratified into three groups according to the percentage of positive nuclei [<10% (negative staining), ≥10% to <50% (intermediate staining), and ≥50% (positive staining); refs. 13, 21] and to the intensity of p53 nuclear staining stratified as weak or intense in the intermediate group (13). For p21, tumors with ≥10% nuclear staining were defined as positive (14).

Functional analysis of separated alleles in yeast. To detect inactivating mutations in TP53 gene, a FASAY assay was used as described (30, 31). In brief, the transcriptional activity of human p53 in tumor cells is assessed in Saccharomyces cerevisine where it activates a p53 target gene (Ade2). This reporter gene is under the control of a promoter that contains the p53-binding site from either ribosomal gene cluster (RGC), p21, or bax genes. Bladder biopsies and tumor samples stored at –80°C until mRNA extraction were thawed and thoroughly homogenized. The mRNA was extracted using a Dynabeads mRNA DIRECT kit (Dynal, Oslo, Norway) according to the manufacturer's recommendations. TP53 mRNA was reverse transcribed and part of the TP53 open reading frame comprising exons 4 to 11 was amplified by PCR. The reporter strains YIG-397, YPH-p21, and YPH-bax were grown and cotransformed with unpurified TP53 amplicons and a linearized yeast expression vector carrying the 5' and 3' ends of the TP53 open reading frame. Activation of the reporter by wild-type p53 results in white colonies, whereas mutant p53 produces pink or red colonies. Knowing that samples containing wild-type p53 can give a background of 5% to 10% red colonies due to PCR-induced errors and to the presence of an alternatively spliced TP53 mRNA, TP53 was considered as wild-type when <10% of red or pink colonies were detected (negative FASAY). Above this cutoff value, the FASAY was considered positive. The activity of the p53 mutant was determined by the color of at least 100 colonies per strain. A tissue sample carrying a well-characterized mutation and a blood sample from a Li-Fraumeni patient were both used as a positive control (32).

Sequence analysis. To characterize the inactivating mutations at the molecular level, the p53 plasmids were then recovered from red or pink yeast. Sequence analysis was done on an automated ABI 377 A apparatus (Applied Biosystems, Foster City, CA) using the Taq Dye Deoxy Terminator Cycle Sequencing kit from the same manufacturer and according to its instructions. The mutation was identified on both strands of plasmids from individual colonies. For each patient sample, 15 colonies (i.e., 5 per yeast promoter) were sequenced.

Quantification by real-time PCR Taqman assay. Gene-specific PCR primers and Taqman probes for human TP53, p21, and housekeeping gene Abelson (Abl) were designed using Primer Express Software version 1.5 (Applied Biosystems; Table 1) and purchased from Eurogentec (Ougrée, Belgium). Each probe was labeled with a fluorescent 5' reporter dye [6-carboxy-fluorescein (FAM)] and a 3' quencher [6-carboxy-tetramethyl-rhodamine (TAMRA)]. mRNA was extracted from frozen tissue samples using a Dynabeads kit according to the manufacturer's protocol and reverse transcribed by using random hexamers (Eurogentec) and SuperScript RNase H– reverse transcriptase (Invitrogen, Merelbeke, Belgium). Amplification was done using 2.5 µL cDNA, 12.5 µL Universal PCR Master Mix 2x (Applied Biosystems) in a total reaction volume of 25 µL. The Abl housekeeping gene and the genes of interest were amplified in parallel. The reaction was initiated at 50°C for 2 minutes and 95°C for 10 minutes followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 1 minute.


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  		Table 1. Primers and probes used in the real-time PCR

 
Data were recorded as cycle threshold (Ct) on a Taqman 7700 Sequence Detection System (Applied Biosystems) using analytic software from the same manufacturer. Each cDNA sample was amplified in triplicate and Ct values were averaged. The average Ct value for Abl was subtracted from the average Ct value for a gene of interest. This {Delta}Ct value obtained in pathologic tissues was then subtracted from a {Delta}Ct value obtained in normal tissues, giving a {Delta}{Delta}Ct value. As amplification efficiencies of the genes of interest and Abl were comparable, the amount of mRNA of each gene, normalized to Abl, was given by the relation 2–{Delta}{Delta}Ct. Positive (DU145) and negative (LNCaP) controls were those used for immunohistochemistry.

Statistical methods. The correlations among p53 FASAY, p53 immunohistochemistry, TP53 expression, p21 immunohistochemistry, p21 expression, and tumor stage and grade were tested by cross-tabulating data and applying the Spearman correlation test. Survival curves were built using a Kaplan-Meier test. Predictors of survival were identified using the Cox proportional hazard regression method. Statistical significance was set at P < 0.05. All analyses were done with the SPSS Statistical Package version 12.0 for Windows (SPSS, Inc., Chicago, IL).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 Conclusions
 References
 
Patient characteristics. There were 37 males and 16 females ages 43 to 89 years (median, 73 years). Diagnosis of TCC was confirmed in all patients but one with adenocarcinoma of the urachus. This patient was subsequently withdrawn from further evaluation. Tumor stages and grades are summarized in Table 2. The six patients diagnosed with muscle-invading tumor were scheduled for radical cystectomy.


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  		Table 2. Distribution of stage and grade in superficial TCCs of the bladder

 
Correlation between functional analysis of separated alleles in yeast and tumor stage and grade. A total or partial transcriptional inactivation of p53 (i.e., FASAY-positive) was found in 19 of 52 (36.5%) tumors, including 6 of 15 (40%) females and 13 of 37 (35%) males (Table 3). All the paired normal samples were FASAY negative. Fifteen p53-inactive mutants failing to transactivate the three yeast promoters and producing only red colonies were defined as nonfunctional. Four mutants presenting a transcriptional activity that was partial on RGC promoter (pink colonies), normal on p21 promoter (white colonies), and abolished on bax promoter (red colonies) were defined as partially functional (Tables 3 and 4). Sequence analysis of 15 yeast colonies obtained with a particular tumor consistently showed the same mutation (Table 4). A missense mutation was found in 17 (89%) mutants, all but one (G105D; patient 5) being already reported in the p53 database (IARC p53 mutation database version R9, December 2004: http://www-p53.iarc.fr/index.html; ref. 33). A compound heterozygous mutation was found in one of the two remaining cases (patient 16) and a 3-bp insertion in the other (patient 12). The R283H mutation was identified in all four mutants exhibiting partial binding activity (i.e., p21 transcribed but not bax). Considering the polymorphism at codon 72, a proline was found in 3 of 19 TP53 mutants (Table 4: patients 4, 6, and 15).


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  		Table 3. Patient characteristics

 

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  		Table 4. Activities in yeast of bladder tumor p53 mutations (p53 FASAY) and p53 mutant identification by sequence analysis

 
There was a strong correlation among FASAY, stage (Spearman R = 0.514; P = 0.000), and grade (Spearman R = 0.561; P = 0.000) of the tumors. The distribution of p53 FASAY mutation frequency by stage/grade is detailed in Table 5. Noteworthy, only 2 of 25 (8%) of Ta-T1 grade 1 to 2 carried a nonfunctional p53 versus 12 of 21 (57%) of the Ta-T1 grade 3.


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  		Table 5. Correlation between tumor and grade and p53 FASAY/p53 immunohistochemistry/p21 immunohistochemistry

 
Correlation between tumor stage and grade and p53/p21 immunohistochemistry. Immunostaining of p53 and p21 was done in 48 of 52 (92%) of the samples (Table 3). A positive p53 nuclear staining was found in 29 of 48 (60%) of the tumors. Staining distribution and intensity are detailed in Table 5. There was a strong correlation among p53 immunohistochemistry, stage (Spearman R = 0.418; P = 0.003), and grade (Spearman R = 0.386; P = 0.007) of the tumors. The rate of positive staining in Ta-T1 grade 1 to 2 tumors was 11 of 23 (47.8%).

Immunostaining of p21 was positive in 35 of 48 (72.9%) of the tumors. Repartition of p21 staining by stage/grade is detailed in Table 5. There was a strong correlation among p21 immunohistochemistry, stage (Spearman R = 0.555; P = 0.000), and grade (Spearman R = 0.480; P = 0.001) of the tumors. In Ta-T1 grade 1 to 2 tumors, the rate of positive staining was 22 of 23 (95.7%).

Correlation between functional analysis of separated alleles in yeast and p53/p21 immunohistochemistry. Fourteen of 31 (45%) samples with a functional protein showed a p53 nuclear immunohistochemistry staining, whereas 1 of 13 (7.7%) with a totally nonfunctional p53 was p53 immunohistochemistry negative. One partially functional p53 mutant (1 of 4, 25%) was also p53 immunohistochemistry negative. Among both falsely immunohistochemistry-negative p53 mutants, one (patient 16) carried a compound heterozygous mutation in exon 5 (P153T) and exon 8 (R283H) and the other one (patient 6) a mutation in exon 5 (L130F). In spite of these discrepant results, there was a good correlation between FASAY and p53 immunohistochemistry (Spearman R = 0.692; P = 0.000; Table 6).


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  		Table 6. Correlation between p53 FASAY and p53 immunohistochemistry/p21 immunohistochemistry

 
Thirty of 31 (96.8%) samples with a functional p53 were p21 immunohistochemistry positive, whereas 10 of 13 (76.9%) samples with a nonfunctional p53 were p21 immunohistochemistry negative. Among the four partially functional p53 mutants, two were p21 immunohistochemistry positive and two were immunohistochemistry negative. There was an excellent correlation between status of p53 determined by FASAY and p21 immunohistochemistry (Spearman R = 0.738; P = 0.000).

Correlation between functional analysis of separated alleles in yeast and quantitative PCR. TP53 and p21 quantitative gene expression was carried out in 23 paired tumor and normal tissue samples. For TP53, no correlation was found between FASAY and TP53 quantitative gene expression (Spearman R = 0.108; P = 0.659). Compared with control samples, TP53 gene expression in tumor samples was increased by a factor of 52.2 ± 82.4 (mean ± SD) in 13 of 17 (76.5%) patients with a functional p53, whereas it was also increased by a factor of 5.4 ± 4.3 (mean ± SD) in 4 of 6 (66.7%) patients with a nonfunctional p53. In the remaining patients with functional and nonfunctional p53, the TP53 gene expression in the tumor samples was lower than in the paired control samples (Table 3).

A significant correlation was found between FASAY and p21 gene expression (Spearman R = 0.468; P = 0.024): in comparison with paired control tissues, p21 gene expression was decreased by a factor of 2.4 ± 1.04 (mean ± SD) in 4 of 6 (66.7%) patients with a nonfunctional p53 and increased by a factor 16.4 ± 16.1 (mean ± SD) in 14 of 17 (82.4%) patients with a functional p53.

Prediction of tumor recurrence. Median follow-up was 27 months (lower and upper 95% confidence intervals, 24 and 30 months, respectively). Twelve recurrences were diagnosed and no progression (Table 3). Recurrence-free survival rates of 1 and 2 years are 83% and 69%, respectively (Fig. 1A).



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  		Fig. 1. A, Kaplan-Meier survival analysis for recurrence. Number of patients still in observation. B, Kaplan-Meier survival analysis for recurrence in the group of patients with Ta-T1 grade 3 tumors. Solid line, tumors with a functional (F) or partially functional (PF) p53; dashed line, tumors with a nonfunctional (NF) p53. Number of patients still in observation.

 
Cox regression analysis models were built to assess the predictive value of the following variables on tumor recurrence: grade 3, stage Ta versus T1, p21 immunohistochemistry (≥10%), p53 immunohistochemistry (≥10% or ≥50% positive), and p53 FASAY (totally functional or totally nonfunctional). The results are detailed in Table 7. The presence of a nonfunctional p53 was the best predictor of recurrence in univariate analysis followed by grade 3. On the other hand, p21 immunohistochemistry was not a predictor of tumor recurrence. In the subgroup of 21 patients with a Ta-T1 grade 3 tumor, there was still a quasi-significant correlation (P = 0.094) between the rate of recurrence and a positive FASAY (Cox hazard ratio, 3.95; 95% confidence interval, 0.8-19.7). Recurrence was indeed observed in 7 of 11 (63%) of the patients with a nonfunctional p53 and in 2 of 10 (20%) of the patients with a normal or partially functional p53 (Fig. 1B). In contrast, p53 immunohistochemistry, with or without enforced cutoff values, did not allow such discrimination in the same subgroup of patients.


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  		Table 7. Cox regression analysis for recurrence: univariate models

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Conclusions
 References
 
Despite significant advances in the understanding of tumor biology, evaluation of the risk of recurrence of bladder TCC still relies on the interpretation of clinical features, including tumor's stage and grade, multifocality, and presence of carcinoma in situ (34). Consequently, there is a major interest in identifying molecular features indicating a higher risk of recurrence or progression (6, 35, 36). In this respect, p53 has been one of the most widely studied predictor of outcome (9, 10, 15). However, unlike the many observations with other tumors, the prognostic significance of nuclear expression of p53 in bladder TCC remains controversial (7, 8, 12, 14, 37–39). To some extent, this uncertainty may result from known limitations of immunohistochemistry. Although immunohistochemistry is ubiquitously used to detect p53 mutations (7–15), the technique carries several known interferences, including nonsense mutations and activation of wild-type p53 by xenobiotics or activated oncogenes as well as from methodologic biases resulting from variations in sample preparation, technique of fixation, anti-p53 antibody specificity, or variable positive cutoff (7, 8, 10, 13, 17–21). To improve immunohistochemistry results, several methods have been developed, such as quantitative fluorescence image analysis (40). Despite being not yet routinely carried out in clinical laboratory, they should be considered in forthcoming comparisons with the p53 functional assay.

The critical biochemical function of p53 in carcinogenesis and tumor progression relies on alterations of the transcription of target genes controlling cell growth and apoptosis (16). Therefore, the use of a functional assay in yeast to selectively identify inactivating p53 mutations in bladder tumors, first reported by Pfister et al. in 1999, seems to be a more valuable alternative to immunohistochemistry (16, 21, 22). In this study, the transcriptional activity of p53 was compared with p53 and p21 at the protein and gene expression levels. There was a significant correlation among p53 mutations, lack of p21 transcriptional activity, and lack of p21 staining, but our data did not confirm the prognostic value of p21 staining for bladder cancer progression as reported for more invasive TCCs (15, 39). There were significant correlations between p53 immunohistochemistry and tumor stage and grade as well as between p53 immunohistochemistry and p53 transcriptional abnormalities. However, there was no correlation between p53 mutations and TP53 gene expression.

The proportion of p53 alterations detected by FASAY in Ta-T1 tumors in the present series was similar to the proportion reported by Pfister et al., 36.5% (19 of 52) versus 34% (16 of 47), respectively (22), as was the percentage of positive FASAY in invasive TCCs [83% (5 of 6) versus 100% (4 of 4), respectively], grade 2 tumors (17% versus 18%, respectively), and grade 3 tumors (79% versus 61%, respectively). However, we also compared FASAY and p53 immunohistochemistry. The rate of p53-positive immunohistochemistry without detectable mutation and p53-negative immunohistochemistry in tumors with p53 mutation confirmed the superiority of FASAY for detecting the p53 alterations (10, 13, 15). It is of note that discrepant results were mostly found in the group of Ta-T1 grade 1 to 2 tumors, p53 mutation being suspected based on a positive p53 immunohistochemistry in 13 patients but confirmed by FASAY in only 2 patients, including 1 with a partially functional p53. Discrepancy between immunohistochemistry and FASAY was striking in a specimen carrying a single premature stop codon in exon 8 but intense p53 staining (20, 21). Nevertheless, we confirmed a strong p53 staining in 4 of the 6 (66.7%) assessable tumors with a mutation in exon 8 (13).

In the current study, the mutational spectrum of p53, assessed on the nearly entire TP53 open reading frame, confirmed that the mutation rate is age independent (13). All the mutations, except the G105D, were already listed in the p53 mutation database (IARC p53 mutation database version R9, December 2004: http://www-p53.iarc.fr/index.html; ref. 33). Interestingly, four partially functional mutants were characterized by the same R283H mutation, giving a partial transcription of the consensus RGC promoter (pink colonies), a normal transcription of p21 promoter (white colonies), and a loss of transcription of the bax promoter (red colonies). Accordingly, the prevalence of R283H in superficial bladder TCCs and its prognostic value deserves further evaluation. In addition, assessment of the polymorphic codon 72 showed a proline (72P) and arginine (72R) alleles in 3 and 16 samples, respectively. The weak proportion of 72P mutants in our series of patients did not support a significant contribution of polymorphism 72P to recurrence, in opposition to an earlier report (41).

However, the most interesting and original result presented here is the added predictive value of FASAY test for tumor recurrence in a homogenous population of patients with superficial bladder TCCs. Compared with other classic predictors of recurrence, a positive FASAY test seemed indeed to be the strongest predictor of recurrence and superseded tumor grading and stage as well as p53 immunohistochemistry staining (for cutoff 50%). As already mentioned, p21 immunohistochemistry and p53 immunohistochemistry (for cutoff 10%; ref. 8) or p21 and TP53 gene expression were not significant prognostic factors for recurrence. Whereas grade 3 was a strong predictor of relapse, a positive FASAY maintained also its ability to better identify patients at risk of recurrence in the Ta-T1 grade 3 subgroup. However, unlike Lopez-Beltran et al. (7), we did not confirm the value of p53 immunohistochemistry, with or without enforced cutoff values, as a predictor of recurrence in the same subgroup of patients. Considering the unpredictable and highly variable natural history of Ta-T1 grade 3 tumors, these results highlight the interest of FASAY test as an adjunct in this subset of patients.


   Conclusions
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusions
References
 
FASAY seems as an optimal and robust screening assay that is readily done, requires only tiny amounts of tumor cells, and allows a sensitive and specific detection of p53 transcriptionally inactive mutants while ruling out biologically silent polymorphisms. The assay is rapid and reproducible. Mutations altering totally or partially the p53 transcriptional activity are easily recognized as illustrated with the recurrent R283H mutation. FASAY results are also significantly correlated to tumor stage and grade and p53/p21 immunohistochemistry. Altogether, these features compare favorably with TP53 sequence analysis and immunostaining, making FASAY an attractive surrogate marker for assessing p53/p21 pathway alterations. Finally, the FASAY significant predictive value for recurrence in Ta-T1 tumor, including the group of grade 3 tumors, is an essential observation. Indeed, adequate predictions of recurrence after transurethral resection in noninvasive bladder TCCs are still lacking and would have practical therapeutic implications.


   Acknowledgments
 
We thank Ph. Camby (Department of Pathology) and H. Delhez and M. Heusterspreute (Laboratory of Applied Molecular Technologies) for expert assistance, Prof. J. Lebacq (Department of Physiology) for careful reading of the article, Prof. P.J. Van Cangh (Chairman of the Division of Urology) for support during the study, and Th. Frebourg (University Hospital, Rouen, France) and R. Iggo (Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland) for providing the yeast.


   Footnotes
 
Grant support: Communauté Française de Belgique Action de Recherche Concertée grant 04/09-317, Loterie Nationale de Belgique, and Fonds National de la Recherche Scientifique grant 7.4536.04.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: A-F. Dekairelle and B. Tombal contributed equally to this work.

Received 1/19/05; revised 4/ 8/05; accepted 4/12/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 

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L. M. Irenge, A. Robert, and J.-L. Gala
Quantitative Assessment of Human {beta}-Globin Gene Expression In Vitro by TaqMan Real-Time Reverse Transcription-PCR: Comparison with Competitive Reverse Transcription-PCR and Application to Mutations or Deletions in Noncoding Regions
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