Clinical implications of p53 mutation analysis in bladder cancer tissue and urine sediment by functional assay in yeast

Beata Schlichtholz1,3, Malgorzata Presler1 and Marcin Matuszewski2

1 Department of Biochemistry and 2 Department of Urology, Medical University of Gdansk, Gdansk, Poland

3 To whom correspondence should be addressed Email: bsch{at}amg.gda.pl


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study we correlate the p53 gene mutations in tumour tissue with urine sediment using a functional assay in yeast, and relate the p53 status to the outcome in a group of patients with transitional cell carcinoma of the bladder. The p53 mutations were found in three of 30 (10%) Ta/T1 tumour tissue samples and in two of 20 (10%) corresponding urine sediments. In the stage T2–T4 tumour p53 mutations were found in tumour tissues and urine sediments in 13 of 31 (42%) and in seven of 18 (39%) samples, respectively. In 80% (8/10) of cases, the p53 mutations found in tumour tissue were re-detected in urine sediment. Median follow-up was at 20 months. Disease recurred in 18 of the 61 patients (30%) with a median time of 5 months. In Ta/T1 tumours the frequency of recurrence was 37% (11/30) compared with 23% (7/31) of T2–T4 tumours. The 3-year overall survival (OS) was 82% (50/61). The p53 status was significantly associated with stage (P = 0.0077, two-sided Fisher's exact test), grade (P < 0.001) and lymph node involvement (P = 0.027). There was an association between the p53 mutations and shorter OS (P = 0.033; log-rank test); however in a multivariate analysis adjusted for stage, grade, lymph node status and age the p53 mutation was not an independent predictor of survival. There was no correlation of the p53 status with decreased disease-free survival (P = 0.8; log-rank test). The data presented indicate that the yeast functional assay is a useful method for p53 gene mutation analysis in tumour tissue and p53 mutation can be re-detected in urine sediment, but further validation of the assay in non-invasive screening for p53 mutations is needed.

Abbreviations: DFS, disease-free survival; OS, overall survival; TCC, transitional cell carcinoma


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bladder cancer is the second most common genitourinary malignancy after prostate cancer. In 1999, there were approximately 7100 new cases of bladder cancer in Poland, accounting for 8.6% of new cases in men (the fourth cancer in men) and 2% in women, and was responsible for ~3% of all cancer-related deaths (1). Epidemiologically transitional cell carcinoma of the bladder represents 90–95% of cases in Europe, while 5% represents mostly squamous cell carcinoma. Most superficial bladder carcinomas grow non-invasively and have a high frequency of recurrence of ~50% (2) whereas up to 15% progress to a more advanced stage. Cystoscopy and voided urine cytology are the most accepted techniques of urinary system neoplasm detection. The limitations of urinary cytology and invasiveness of cystoscopy have led to studies searching for the new bladder tumour markers that allow for non-invasive detection of bladder carcinomas. The p53 tumour suppressor gene plays a key role in suppression of neoplastic transformation by cell cycle arrest and/or by apoptosis. Alterations of the gene p53 are common in bladder cancer, occurring in ~50% of transitional cell carcinoma and are seen more frequently in high-grade invasive tumours (3,4). Moreover, loss of p53 function has been associated with progression to invasive disease and decreased survival (5).

In the present study we determined the p53 status in tumour tissue and paired voided urine by functional assay in yeast followed by sequencing. A large number of tumours have been examined using this method, including breast (6), upper aerodigestive tract (7), hepatocellular carcinoma (8) and acute myeloid leukaemia (9), but very few bladder carcinomas have been studied (10). Additionally the presence of exfoliated cancer cells in voided urine provides a good opportunity to test whether a yeast-based functional assay can be used for non-invasive detection of the p53 gene mutations in bladder carcinoma.

The aim of our study was: (i) to investigate patients with low- and high-stage transitional cell carcinoma (TCC) of the bladder for the presence of p53 mutations using functional assay in yeast; (ii) to compare the p53 mutations in tumour tissue with these from urine sediments; (iii) to further evaluate feasibility and sensitivity of the p53 gene functional analysis to detect bladder cancer cells in urine; (iv) to determine the association between the p53 status and clinicopathological parameters including age, sex, pathologic stage, tumour grade and lymph node involvement. Multivariate survival analysis was performed considering p53 as an independent prognostic factor.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumour samples and exfoliated cells
The Department of Urology at the Medical University of Gdansk provided a total of 61 bladder tumours and 50 matched voided urine samples, 30 biopsies of bladder tumours were obtained by transuretal resection of the bladder while 31 at cystectomy. The patients' median age was 62 years (ranging from 32 to 85). Tissues collected included obvious tumour tissue that was confirmed by subsequent histopathological diagnosis at the Pathology Department, Medical University of Gdansk. Histological staging and grading was performed according to the fifth edition of the TNM classification (11). The clinicopathologic characteristics of patients are summarized in Table I. Samples were frozen immediately in liquid nitrogen, and stored at –80°C. Exfoliated cells were collected from 50 ml of naturally voided urine from 50 patients with bladder carcinoma. The urine was stored at 4°C until centrifugation, followed by a washing and lysis procedure. Survival was determined from the date of primary therapy. Follow-up was available for 61 patients.


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Table I. Clinicopathological characteristics of 61 bladder cancers

 
cDNA synthesis
Isolation of total RNA from frozen bladder tumour tissue, adjacent non-neoplastic tissue, served as normal bladder tissue, and urine sediment was performed using RNA extraction kit (RNeasy Mini Kit; QIAGEN, USA) and stored at –20°C. In case of urine sediments sufficient amounts of RNA were obtained from 38 patients. Reverse transcription was performed on 1 µg RNA using Omniscript Reverse Transcriptase (QIAGEN, USA) and Oligo d(T)15 Primer (Promega, USA) in a total reaction volume of 20 µl, following the manufacturer's instruction.

PCR
p53 cDNA was amplified using the following two sets of primers P3 and P17 or P4 and P16 as described previously by Waridel et al. (7). cDNA (2 µl) was subjected to PCR in a 25 µl reaction, using ProofStart DNA Polymerase (QIAGEN, USA) with the following conditions: 1x PCR buffer, 1x buffer Q, 1.5 mmol/l MgCl2, 200 µmol/l of dNTPs, 10 pmol of each primer. Following an initial incubation of 5 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at 63°C and 1 min at 72°C were performed. The amplification was performed in a Model PTC-200TM Thermal Cycler (MJ Research, Watertown, MA, USA). PCR products were analysed on a 1.5% agarose gel.

Homologous recombination in yeast
The PCR product was used in a yeast-based assay (12). For gap repair pFW35 and pFW34 yeast expression vectors were used (7). The Saccharyomyces cerevisiae strain yIG397 that allows for the evaluation of p53 transactivation function was originally constructed by Richard Iggo (12). The strain yIG397 with an ADE2 gene open reading frame downstream from three copies of a p53-responsive DNA sequence found at the ribosomal gene cluster was co-transformed using PEG-based method with linearized expression vector and various p53 cDNA as described previously (12). Transformants were selected on leucine-deficient media containing limiting amounts of adenine (5 mg/l). Colony colour was assessed after 3 days growth at 30°C. The mutants were scored as described previously (13). Red colonies, indicative of the p53 mutations, represented on average 69% of colonies for tumour and 50% for urine sediment-isolated RNA. Normal bladder tissue gave always <10% of red colonies.

Recovery of p53 plasmids from yeast and DNA sequencing
Plasmid DNA was extracted by rapid DNA isolation method described by Hoffman and Winston (14). Mutations were identified on both strands of plasmids from individual colonies by direct sequencing of p53-PCR products. p53 plasmids recovered from yeast were amplified with the same set of primers as for p53 cDNA amplification prior to homologous recombination. PCR products were purified by QIAqick Gel Extraction Kit (QIAGEN) and sequenced directly by BigDye terminator chemistry followed by capillary electrophoresis with an ABI 310 Genetic Analyzer according to the supplier's instruction (Applied Biosystems).

Statistical analysis
Based on available clinical and molecular data, the following associations were examined in patients, the presence of the p53 mutation in tumour specimen versus sex, age, grade and stage. Univariate analysis using two-sided Fisher's exact test and multivariate analysis using logistic regression were performed. In addition, two prognostic outcomes were examined: disease-free survival (DFS), defined as the time from primary therapy to first recurrence including censored times and OS, defined as the time from primary therapy to death (from any cause) or last follow-up. The last follow-up evaluation was performed in October 2003. Total length of the study at the time of analysis was 44 months, and median follow-up was 20 months (range 2–44 months). In bivariate association clinical variables were dichotomized as follows: age at diagnosis, ≤65 versus age >65 years; tumour grade low versus moderate or high grade, stage Ta/T1 versus T2–T4.

Univariate survival curves were constructed by the method of Kaplan–Meier, and the significance of differences estimated by the log-rank test. Cox proportional hazard regression analysis was used to examine the prognostic value of each variable. Patient's age, grade, stage, lymph node and p53 status were entered into Cox regression. For all analyses, a P value of <0.05 was accepted as significant and the analyses were performed with the software STATISTICA version 6 (StatSoft, Tulsa, USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Functional analysis of the p53 mutations in paired tumour and urine in bladder cancer
We tested mRNA of tumour specimens from 61 bladder cancer patients for the presence of p53 mutations. In addition, the same analysis was done in corresponding voided urine specimens from 38 patients. To determine the transcriptional activity of p53, a reporter assay was performed using a reporter construct containing the p53-responsive element upstream ADE2 reporter, as described by Flaman et al. (12). The PCR products corresponding to the 5' and 3' parts of p53 cDNA were co-transformed into yIG397 along with either the linear form of pFW-34 or pFW-35 expression vectors.

In general the p53 mutations were found in 16 of 61 patients (26%) in tumour specimens and in nine of 38 (24%) corresponding urine sediments. In 80% (8/10) of the cases, the p53 mutations found in tumour tissue were re-detected in urine sediment. In non-muscle invasive Ta/T1 stage tumours we found p53 mutations only in three of 30 (10%) tumour tissues and in two of 20 (10%) urine sediments. In contrast, in muscle invasive tumours we found p53 mutations in 13 of 31 (42%) tumour tissues and seven of 18 (39%) urine sediments. In normal bladder tissue p53 mutations were not found (0/10). Additionally, all positive cases were analysed by sequencing of the p53 transcripts. All specimens that showed the presence of red colonies in the yeast assay were confirmed to have mutation by sequence analysis. As shown in Table II, in exons 4–11 of the p53 gene we found 15 missense mutations, three nonsense mutations, two deletions and two insertions including splice side mutation. Furthermore, in three cases we found multiple mutations in the p53 gene with one complex mutation. In case of splice site mutation at the intron 7–exon 8 border we observed inclusion of the last 3 bp of intron 7 into the coding sequence resulting in the insertion of a stop codon. It is worth noting that this is the first time that a mutation at codon 110 in TCC tissue has been reported (Table III).


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Table II. Characteristics of tumours containing p53 mutations

 

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Table III. Comparative analysis of the incidence of p53 mutants isolated from patients with transitional cell carcinoma of the bladder with the IARC database

 
In our study, the status of the codon 72 polymorphism of 12 tumours was determined. The arginine residue at codon 72 was observed in nine out of 12 (75%) tumours with the p53 mutations. This may support the observation of Martin et al. that mutant alleles containing R72 are preferentially selected during carcinogenesis (18). They found that R72 p53 mutants had enhanced ability to bind p73 protein (a homologue of the p53 protein) thus, neutralizing p73-induced apoptosis.

In most cases the genetic change in urine DNA was identical to that identified in the tumour. However, in one case (BT34) the p53 mutation found in the tumour biopsy (R273H) was different from the p53 mutation found in the corresponding urine (G279E). Moreover, in case BT25 we found one additional mutation in urine sediment at codon 181 compared with tumour tissue with the only mutation at codon 174. In only one case with the mutation in the tumour tissue did we not find any mutation in the corresponding urine sediment.

Correlation of p53 mutations with clinicopathological markers
A correlation analysis between the frequency of the p53 mutations and (TNM/UICC) stage and grade (WHO) was performed. The association was found between the presence of p53 mutation and stage (P = 0.0077, two-sided Fisher's exact test), grade (P < 0.001) and lymph node involvement (P = 0.027). There was no association between the p53 mutation and patient sex and age. Multivariate analysis using a logistic regression model demonstrated that grade was a dominant factor associated with the p53 gene mutation (OR 6; 95% CI 1.55–23.6; P = 0.01).

Patient survival analysis
Survival data were available for all cases studied. The 3-year OS was 82% (50/61). At the end of analysis, 15% of patients died of disease and 3% died disease-free. Kaplan–Meier overall survival (OS) curves for bladder cancer patients showed a statistically significant association between the presence of p53 mutations and poor outcome (P = 0.033; log-rank test) (Figure 1). Univariate analysis revealed significant correlation for age (P = 0.021; log-rank test), grade (P = 0.029), stage (P = 0.003) and lymph node status (P = 0.0002). Furthermore, multivariate Cox hazard regression model was used to investigate several variables at a time. In multivariate analysis, the p53 mutation (P = 0.85) was not an independent predictor of tumour survival when assessed with tumour grade, tumour stage, lymph node status and age.



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Fig. 1. Cancer specific OS for bladder cancer stratified by the p53 status. P = 0.033, log-rank test.

 
DFS from time of surgery was 70%. Of 61 patients, 18 (30%) developed local recurrence with a median time of 5 months (ranging: 1–24). In Ta/T1 tumours the frequency of recurrence was 37% (11/30) compared with 23% (7/31) of T2–T4 tumours.

There was no relationship between the p53 mutations and decreased DFS (P = 0.8; log-rank test). On univariate analysis, sex (P = 0.7), grade (P = 0.3) and stage (P = 0.17) were not significant factors.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we focused on functional analysis of the p53 mutations using yeast assay. Functional analysis is based on inability of the p53 mutants to transactivate the target sequence in yeast and specifically detects functionally important mutations thus, avoiding analysis of polymorphisms and silent mutations. Moreover, it overcomes the problem of contamination of tumour tissue with normal tissue making this assay particularly useful for large-scale screening. This assay allowed us to detect the p53 mutations not only in tumour tissue but also in urine sediment. We detected the p53 mutations in 42% of T2–T4 tumour tissues and in 39% of urine sediments and demonstrated that the p53 mutation analysis based on the yeast assay can be a useful tool for the detection of muscle-invasive bladder cancer. In contrast to the study of Curigiliano et al. (19) who found a p53 mutation in 41% stage T1 tumour samples by denaturing gradient gel electrophoresis, we found mutations only in 10% stage Ta/T1 tumours, and the p53 mutation pattern found in urine sediment was not always identical to those found in tumour tissue. It should be noted, however, that we determined only functionally important mutations that can result in lower frequency of p53 mutations in our study. The presence of additional mutations in two urine sediments compared with tumour tissue can be a result of either (i) upper urinary track lesions or (ii) possible polyclonal expansion of tumour. This is not consistent with the concept of clonal origin of bladder cancer (20); however, recently some studies have provided evidence for polyclonal origin of recurrent bladder tumours and the existence of more than one tumour clone especially in early-stage bladder carcinoma (21,22). One case in which the p53 mutation could not be identified in the urine sediment had low-stage tumour. Absence of p53 mutation in this case may reflect: (i) a lack of exfoliated cancer cells in the urine, (ii) high ratio of normal cell to tumour cells in the specimen or (iii) a presence of heterogeneous clones in the tumour and urine specimens. The 80% (8/10) concordance between the p53 mutation in the tumour tissue sample and urine sediment, comparable with 84% (16/19) in the study by Prescott et al. and higher compared with 25% (2/8) agreement in the study by Dahse et al. indicates that urine may be as efficient as tumour tissue analysis, however, further evaluation is needed (23,24).

In this study we identified eight p53 mutations that have not been found in transitional cell carcinoma of the bladder based on the IARC database analysis (Table III). The most noteworthy of these mutations was a splice site mutation at intron 7/codon 261 that generated a new stop codon, which can induce nonsense-mediated mRNA decay (25). Premature stop codons were observed in three more cases, which suggests that in our group of patients mRNA decay and/or translational repression of nonsense-containing mRNA may occur (26). Thus, providing new evidence for the utility of a yeast functional assay for p53 mutations screening over immunohistochemical analysis.

Most of the mutations (20 out of 22) were located in the core domain of p53 (Table III). We found predominantly missense mutations; however, nonsense and splice site mutations were observed as well. Interestingly, in three cases we found very rare multiple mutations. In our group of patients endogenous mutations were very rare, we found only one C to T transition at the cytidine phosphate guanosine site at codon 248, which suggests spontaneous deamination of 5-methylcytosine. The prevalence of exogenous mutations is not surprising as cigarette smoking and occupational exposure to arylamines are thought to account for more than half of all cases of bladder cancer, with smoking being the more important risk factor. It is also suggested that there is a certain pattern of mutation in smoking patients (27,28). There are also weak hot spots of transversion mutations at codon 241, 249 and 280, which may suggest the action of carcinogenic adducts. Interestingly in our study we found in two cases G>T transversion at codons 157 and 273 suggesting that a tobacco carcinogen may be responsible for these mutations (29).

Using a functional assay in yeast we demonstrated that this method allows the identification of multiple mutations in a single gene in a single patient's cDNA sample. Three multiple mutations found in our group of patients were predominantly point mutations. In two cases (BT15 and BT9) the second mutation always led to premature translation termination. The presence of nine multiple p53 mutations in bladder cancer patients was reported by Taylor et al. who found 32 p53 mutations out of 64 bladder tumours (30). One of these tumours had five mutations at four codons (89, 101, 132 and 214). Although this tumour was from a patient exposed to arylamine, the frequency of multiple mutations was high for both exposed and unexposed patients.

In general, the p53 mutation is associated with a higher frequency of progression to a more advanced stage and a higher rate of death from bladder cancer. The unfavourable prognostic effect of p53 mutations on bladder cancer patient survival has been shown by many authors. However, data from the literature concerning the prognostic relevance of the p53 mutations on OS and DFS are contradictory. In our study we found an association between the p53 mutation and stage, high histological grade and lymph node involvement. Patients with p53 mutations showed statistically significant shorter survival (P = 0.033), but in a multivariate analysis the p53 mutation was not an independent predictor of OS when assessed with tumour grade, stage, lymph node status and age. There was no correlation of DFS with the p53 status. Low incidence of p53 mutations in Ta/T1 tumours may suggest that p53 alterations in superficial bladder cancer are related to a more aggressive phenotype and a higher risk of recurrence.

In conclusion, our results provide support for the use of a yeast-based assay for p53 mutation screening in tumour tissue and indicate that urine may be as efficient as tumour tissue analysis. However, further evaluation should be carried out, as the voided urine is not an optimal source of RNA extraction. A key advantage of this type of analysis is detection of functionally important p53 mutations and high specificity. Furthermore, this study assessed the prognostic value of p53 mutations in relation to OS and DFS in TCC. Although the p53 mutation can be found in urine specimens by yeast functional assay, the use of this type of analysis to aid the non-invasive detection of tumours needs further validation.


    Acknowledgments
 
We gratefully thank T.Soussi for the yeast strain yIG397 and yeast expression vectors. We also thank J.Swierczynski for helpful comments and discussion on the manuscript. This work was supported by the State Committee for Scientific Research Grant KBN 6 P05A 092 20.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 7, 2004; revised July 7, 2004; accepted August 2, 2004.





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