Competitive amperometric immunosensor for determination of p53 protein in urine with carbon nanotubes/gold nanoparticles screen-printed electrodes: A potential rapid and noninvasive screening tool for early diagnosis of urinary tract carcinoma.

Since p53 protein has become recognized biomarker for both diagnostic and therapeutic purposes in oncological diseases with particular relevance for bladder cancer, it is highly desirable to search for a novel sensing tool for detecting the patient's p53 level at the early stage. Here we report the first study on the development and validation of a novel disposable competitive amperometric immunosensor for determination of p53 protein at subnanomolar levels, based on p53 immobilization on gold nanoparticles/carbon nanotubes modified screen-printed carbon electrodes. The assay protocol requires the use of single anti-p53 mouse monoclonal antibody (DO-7 clone), able to recognize both wild-type and mutant p53. The developed immunosensor as well as the protocol of the electrochemical immunoassay were optimized by means of an experimental design procedure to assess the suitability of the device to be validated and applied for the determination of p53 in untreated and undiluted urine samples. It was found that the developed competitive immunodevice was able to achieve wide linear range detection of wild-type p53 from 20 pM to 10 nM with a low detection limit of 14 pM in synthetic urine samples, suggesting the sensor's capability of working in a complex sample matrix. The excellent performance results also in terms of selectivity, trueness and precision, coupled with the advantages of an easy preparation and low-cost assay in contrast to other methods which require very complex, time-consuming and costly nanostructured architectures, makes the developed competitive immunosensor an analytically robust diagnostic tool, valuable for implementation of screening and follow-up programs in patients with urologic malignancies.

Immunocytochemical staining for p53 and Ki-67 helps to characterise urothelial cells in urine cytology.

OBJECTIVE: The presence of atypical cells in urine cytology is unsatisfactory for both cytologists and clinicians. The objective of this study was to test whether p53 and Ki-67 immunostaining could improve urothelial carcinoma (UC) detection on urinary cytology. METHODS: A total of 196 urine samples were analysed, 142 from the bladder, 41 from the upper tract and 13 from ileal bladder replacement. Cytology results were expressed as normal (N) (n = 81), atypia cannot exclude low-grade UC (ALG) (n = 25), suspicious for high-grade UC (SHG) (n = 39) and high-grade UC (HG) (n = 51). Actual diagnoses were confirmed by histopathological analysis, cystoscopic examination or follow-up for at least 1 year. Immunocytochemistry performed on CytoSpin™ slides allowed the determination of the percentage of positive cells with p53 and Ki-67. RESULTS: The median percentage values [first to third quartile] of p53 and Ki-67 were 0 [0-5] and 0 [0-1] for N cytology, 5 [0-40] and 2 [1-10] for ALG, 10 [0-30] and 6 [3-25] for SHG, and 30 [10-80] and 20 [10-30] for HG, respectively. Statistically higher values were observed for both tests (P < 0.001) in positive cytologies (ALG, SHG and HG). The optimal cut-offs were 5% for p53 and 3% for Ki-67. The sensitivity and specificity for the detection of all UC were 86.4% and 76.7% for cytology alone, 81.3% and 93.2% for cytology and p53, 75.7% and 88% for cytology and Ki-67, and 68.9% and 97.5% for cytology, p53 and Ki-67, respectively. CONCLUSION: Using p53 and/or Ki-67 in addition to cytology increases the specificity without penalising the sensitivity.

TP53 and FGFR3 Gene Mutation Assessment in Urine: Pilot Study for Bladder Cancer Diagnosis.

AIM: To assess, in a prospective clinical research study, a new non-invasive and reliable test to accurately detect tumor protein 53 (TP53) and fibroblast growth factor receptor-3 (FGFR3) mutations in cells in urine. MATERIALS AND METHODS: TP53 mutations were analyzed using the functional analysis of separated allele in yeast (FASAY) method, which allows functional analysis of the P53 protein, and FGFR3 mutations were assessed with the SNaPshot system, detecting the eight most frequent point-mutations of this gene. Chi-square test or Fisher's exact test were used to compare TP53 and FGFR3 mutations in the tumors according to tumor stage and grade. RESULTS: TP53 and FGFR3 mutations in bladder tumors increased and decreased respectively with increasing tumor stage and cellular grade (p<0.05 and p<0.001, respectively). A total of 103 tumor/urinary sediment couples were analyzed. TP53 or FGFR3 mutations were observed in 76 tumors. The sensitivity for the detection of this type of mutation in urine was 46%, the specificity was 81%, the positive predictive value was 94% and the negative predictive value was 37%. CONCLUSION: Our original data confirmed the feasibility of TP53 and FGFR3 mutation detection in urine sediment. These measurements, together with urine cytology, may increase tumor detection. The sensitivity of the TP53/FGFR3 phenotype test in the urine was less than 50% and was not able to replace standard cystoscopy in the diagnosis of bladder tumors.

Phlda3, a urine-detectable protein, causes p53 accumulation in renal tubular cells injured by cisplatin.

Measurable indicators of renal injury are required for the assessment of kidney function after toxicant challenge. In our previous study, pleckstrin homology-like domain, family A, member 3 (Phlda3) was a most greatly up-regulated molecule downstream from p53, culminating with kidney tubular injury. This study investigated the positive feedforward effect of Phlda3 on p53 in an effort to explain the largest increase of Phlda3 in injured tubules and the potential of its urine excretion. qRT-PCR assays confirmed a rapid and substantial increase in Phlda3 messenger RNA (mRNA) in the kidney cortex of mice treated with a single dose of cisplatin. Cisplatin overexpression of Phlda3 was verified by gene set analyses of three different microarray databases. In the immunohistochemistry, Phlda3 staining intensities were augmented in the tubules as kidney injury worsened. Moreover, the urinary content of Phlda3 was increased after cisplatin treatment, as were those of other kidney injury markers (Kim-1 and Timp-1). By contrast, cisplatin failed to increase Phlda3 mRNA in the liver despite hepatocyte necrosis and ensuing increases in serum transaminase activities. In NRK52E tubular cells, siRNA knockdown of Phlda3 enhanced the ability of cisplatin to increase p-Mdm2 presumably via Akt, enforcing the interaction between Mdm2 and p53. Consistently, a deficiency in Phlda3 abrogated p53 increase by cisplatin, indicating that Phlda3 promotes p53 accumulation. Phlda3 overexpression had the opposite effect. In addition, treatment with cyclosporine A or CdCl2, other nephrotoxicants, increased Phlda3 mRNA and protein levels in NRK52E cells, as did cisplatin treatment. Overall, Phlda3 may cause p53 accumulation through a feedforward pathway, facilitating tubular injury and its urine excretion.

[Preliminary study of p53 and FGFR3 gene mutations in the urine for bladder tumors]. / Étude préliminaire des mutations des gènes p53 et FGFR3 sur le culot urinaire des tumeurs de la vessie.

INTRODUCTION: Two major pathways are described in bladder carcinogenesis: one for invasive or high grade tumors characterized by alteration of the p53 tumor suppressor gene and the other for non-invasive tumors or low grade involving mutations FGFR3. The objective of our study was to validate the research in the urine of mutations in these two genes in patients with a bladder tumor. PATIENTS AND METHODS: In our preliminary study, we investigated 36 patients the FGFR3 and p53 mutations in tumors and urine collected during endoscopic resection. The p53 mutations were sought in FASAY, which allows a functional analysis of the protein P53. The FGFR3 mutations were sought in SNaPshot that searches the eight most frequent mutation points of this gene. RESULTS: For 24 patients (66% of cases), we found at least one of the two mutations in the tumor. This mutation was present in the urine in 15 patients (sensitivity=62.5%). In only one patient, we found a mutation in the urinary sediment that did not exist in the tumor (specificity=91.7%). CONCLUSION: The search for mutations of p53 and FGFR3 in the urine was a simple and non-invasive assay, which seems superior to urinary cytology for the detection of bladder tumors, raising hopes of an interest in this bio-assay for surveillance of bladder tumors and screening risk populations.

A locked nucleic acid clamp-mediated PCR assay for detection of a p53 codon 249 hotspot mutation in urine.

Hepatocellular carcinoma (HCC) has a 5-year survival rate of <10% because it is difficult to diagnose early. Mutations in the TP53 gene are associated with approximately 50% of human cancers. A hotspot mutation, a G:C to T:A transversion at codon 249 (249T), may be a potential DNA marker for HCC screening because of its exclusive presence in HCC and its detection in the circulation of some patients with HCC. A locked nucleic acid clamp-mediated PCR assay, followed by melting curve analysis (using the SimpleProbe), was developed to detect the TP53 249T mutation. In this assay, the locked nucleic acid clamp suppressed 10(7) copies of wild-type templates and permitted detection of 249T-mutated template, with a sensitivity of 0.1% (1:1000) of the mutant/wild-type ratio, assessed by a reconstituted standard within 2 hours. With an amplicon size of 41 bp, it detects target DNA sequences in short fragmented DNA templates. The detected mutations were validated by DNA sequencing analysis. We then tested DNA isolated from urine samples of patients with HCC for p53 mutations and identified positive TP53 mutations in 9 of 17 samples. The possibility of using this novel TP53 249T assay to develop a urine or blood test for HCC screening is discussed.

p53 immunodetection of liquid-based processed urinary samples helps to identify bladder tumours with a higher risk of progression.

p53 could help identify bladder tumour cases with a risk of progression from superficial to invasive disease. Semiautomatic, liquid-based cytology (LBC) techniques offer an opportunity to standardise molecular techniques. The aim of our study was to investigate whether LBC could improve p53 immunolabelling, and to assess whether urinary p53 could have a prognostic value. Immunoreactivity for p53 was studied in 198 urine samples after treatment with the Cytyc Thinprep processor. After antigen retrieval, cells were labelled with a monoclonal antibody that recognises both wild-type and mutant form of the p53 protein (Clone DO-7, Dako), 1/1000. Positivity for p53 was assessed in 17.2% of the cases. High-grade (G3) tumours were positive in 74.1% of the cases. Comparatively, low-grade (G1-2) urothelial carcinomas were positive in 23.5% of the cases. During a median follow-up period of 26 months, recurrence was observed in 52.9% of the cases with p53 overexpression, and in only 10.9% of negative cases (P < 0.001). The progression rate was 35.3% of p53-positive cases vs 5.5% of p53-negative cases (P < 0.001). Progression-free survival was significantly shorter in patients with p53 accumulation (P = 0.007). In a multivariate analysis stratified on grade and stage, p53 was an independent predictor of overall survival (P = 0.042). The results show that using Thinprep LBC, p53 immunolabelling of voided urothelial cells allows most high-grade tumours to be detected and may help identify cases with a higher risk of recurrence and progression.

Genetic analysis of Tp53 from urine sediment as a tool for diagnosing recurrence and residual of bladder carcinoma.

OBJECTIVES: Longitudinal study of Tp53 mutation in urine sediments of 26 patients with mutated primary transitional cell carcinoma (TCC) of the urinary bladder at different time periods after transurethral resection of the bladder (TURB), i.e. before and after the first TURB, prior to the control resection and before treatment of a recurrence. METHODS: DNA of the critical Tp53 exons 5-8 was anaylzed by temperature gradients (TGGE) and sequence. RESULTS: (1) In 11 of 12 patients (91.7%) mutation reoccurred with the detection of recurrence of the disease. The mutation frequency in patients without any recurrence was 1 in 8 (12.5%) after a follow-up period of 4-16 months. (2) In 7 of 10 patients, the mutation was no longer detectable in the urine sediment after TURB. (3) The mutation frequency at the control resection 6 weeks after the first TURB was 5 in 7 (71.4%) in patients found to have residual and 1 in 7 (14.2%) in the tumor-free patients. (4) In 9 of 10 samples identical mutations were found by sequence in the recurrent tumor. These results show a significant correlation between the detection of a Tp53 mutation in the urine sediments and tumor recurrence or residual. CONCLUSIONS: (1) Tp53 mutations in the urine sediment could be a useful indicator of tumor recurrence or tumor residual in patients ( approximately 40%) with primary mutated bladder cancer tissue. (2) These results support the monoclonal seeding theory. (3) The finding of identical mutations at different times indicate that the tumor was never totally removed.

Urine cytology. It is still the gold standard for screening?

Urine cytology remains the gold standard for bladder cancer screening. It is the test against which all others are compared when evaluating potential bladder tumor markers. The answer to whether urine cytology possess the optimal combination of sensitivity and specificity to retain consideration as the best screening device depends on the goals of the clinical practice. Urine cytology has excellent specificity with few false-positive cases. Its overall sensitivity is poor, but this drawback is explained for the most part by poor criteria for identifying well-differentiated, low-grade TCC. The natural history of such lesions is the occurrence of multiple superficial recurrences in 70% to 80% of patients, with only a minority (10% to 15%) progressing to muscle invasive or metastatic disease. Because patients with low-grade TCC are at low risk for progression, they are monitored primarily for the development of a subsequent tumor. One might argue that the detection of new low-grade lesions is of secondary importance to the early detection of disease progression. The performance characteristics of urine cytology in this regard are much improved. Urine cytology often results in the identification of high-grade malignant cells even before a cystoscopically distinguishable gross lesion is present. Routinely diagnosing grade I TCC may be clinically irrelevant. Ancillary techniques to improve the sensitivity of urine cytology have been insufficiently additive to have much clinical value. Several promising bladder tumor markers have been investigated as potential screening tools and are summarized in Table 3. BTA, nuclear matrix proteins, and fibrin/fibrinogen degradation products share lower specificities than urine cytology and may have high rates of false positivity. Telomerase is highly sensitive and highly specific but is not readily available as a point-of-service test. Hyaluronidase and hyaluronic acid are promising prognostic markers, but hyaluronidase does not detect grade I TCC. Early results from studies of this marker await verification. Combining some of these new markers may optimize their performance status, allowing the advantages of one test to correct the shortcomings of another. Likewise, their combination with urine cytology may prove beneficial. Although adding urine cytology has not increased the sensitivity of some point-of-service tests, few studies have addressed the effect on specificity. Until an obvious winner is declared in the race to find a bladder tumor marker, urine cytology will remain the gold standard screening method because of its comfortable familiarity.

[Usefulness of P53 oncoprotein in urinary wash cytology: experience in patients with superficial bladder carcinoma]. / Utilidad de la oncoproteína p53 en citología urinaria de arrastre: experiencia en pacientes con carcinoma vesical superficial de vejiga.

OBJECTIVES: 1) To determine p53 expression in urinary wash cytology by immunohistochemistry in patients with superficial transitional cell carcinoma of the bladder and controls; 2) to correlate p53 in urinary wash cytology and anatomopathological characteristics of the bladder tumors analyzed; 3) to determine the utility of p53 expression in urinary wash cytology as a prognostic factor; and 4) to identify a subgroup of patients with superficial tumors of worse prognosis in order to improve control of the evolution of the tumor and treatment. METHODS: From 1993 to 1998, 141 cases were studied; 32 controls comprised group I and 109 (38 primary and 71 recurrence) patients with transitional cell carcinoma of the urinary bladder comprised group II. RESULTS: In group II, 29.5% were positive for p53 in urinary wash cytology, while no positive cases were found in group I. A total of 104 valid data were analyzed, which showed a higher percentage of p53-positive cases in grade III tumors (44.4%). Statistical analysis showed the percentage of p53-positive cases increased with tumor grade in a linear trend (p = 0.17). The recurrence rate in the p53-positive was 20% greater than in the p53-negative cases. Tumor progression was three times higher in the p53-positive than in the p53-negative patients. CONCLUSIONS: The application of biomolecular knowledge to cytology is a useful complement in follow-up of patients with superficial transitional cell carcinoma of the bladder and offers additional parameters to distinguish benign and malignant cells. Immunohistochemical determination of p53 in urinary wash cytology identifies patients with superficial tumors with a worse prognosis.

Does p53 immunostaining improve diagnostic accuracy in urine cytology?

The frequent change of the transitional cell carcinoma of the urinary tract accounts for the fact that cytological abnormalities in urinary specimens are often not sufficient to enable a definitive diagnosis of malignancy. The purpose of this work was to evaluate the possible use of p53 protein in increasing the diagnostic accuracy of urinary cytology. The expression of p53 was investigated by immunocytochemistry in two groups of urinary specimens, one cytologically positive and the other cytologically negative for cancer. Immunostaining was carried out using a monoclonal antibody to p53. In the positive group, in which bladder cancer was confirmed by cystoscopy and biopsy (31 cases), positive reaction for p53 was found in 55% of the cases (17 cases). In the negative group (92 cases), presence of cancer was histologically ascertained in 64 cases and in this group 15 cases (23.4%) showed positive p53 staining. In the remaining 28 cases of this group, where TCC was not present, 7 cases showed p53 positivity in non-neoplastic urothelial cells. This result shows that, while immunocytochemical detection of p53 in urinary specimens may be used for prognostic evaluation of patients with bladder cancer, it does not contribute to the diagnostic accuracy in cases with morphologically inconclusive or negative cytology. The sensitivity and specificity of the method in detecting bladder carcinoma were 23.5 and 75%, respectively.