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Introduction

Bladder cancer is the fourth most common malignancy in men with an estimated 61,420 new cases and 13,060 deaths in the USA in 2006 [1]. The majority of tumours (70%) present initially as superficial non muscle-invasive tumours (Ta, T1, and carcinoma in situ). The probabilities of recurrence and progression in superficial bladder cancer at five years range from 31% to 78% and from less than 1% to 45% depending on grade and stage, respectively [2]. A close follow-up of the patient with superficial bladder cancer is needed to prevent progression to invasive, potentially lethal urothelial cell carcinoma (UCC).

Diagnosis and follow-up of bladder cancer is still based on cystoscopy and cytology. The limitations of both are well known. Cystoscopy remains an invasive and uncomfortable procedure and does not achieve the degree of diagnostic accuracy that urologists would like. For example, flat urothelial lesions like carcinoma in situ are frequently difficult to diagnose. For this reason, urine cytology is frequently used as an adjunctive test. It is still the most used urine test despite its poor sensitivity especially in low grade tumours. A multicenter study recently published by Grossmann et al showed an overall sensitivity for urine cytology of only 15.8% [3]. Moreover, cytology relies on a subjective and operator-dependent interpretation of visible findings and there is a considerable high inter- and intra- observer variability [4].

To overcome these limitations, many new urine-based marker tests for UCC have been developed.

The NMP22 Bladder Cancer Test has been extensively evaluated as a quantitative marker to detect bladder cancer. The implementation in clinical practice might be hampered because the test is not readily available. A simple in-office qualitative NMP22 test is now available since 2002, known as the NMP22® BladderChek® Test (Matritech GmbH, Freiburg, Germany) (Figure 1).

We investigated whether this point-of-care test has clinical utility in the diagnosis and surveillance of bladder cancer and compared its accuracy to detect cancer with that of urine cytology on voided urine (VU) and on bladder washout samples (BWO). A more important issue is the likelihood that a proportion of the false positive NMP22 test results may actually be true or subclinically positives overlooked by cystoscopy. We evaluated whether a ‘false’ positive BladderChek test has a prognostic value by performing follow-up cystoscopies for one year in these patients.

Material and methods

Patients

2 academic and 2 non-teaching hospitals prospectively enrolled 145 consecutive patients suspected for bladder cancer between June 2003 and July 2004. The patients were divided into two groups: 1) 103 patients had a previous history of bladder cancer and 2) 42 patients were investigated because of a suspicion for having bladder cancer because of symptoms (e.g. hematuria), but had no previous history of bladder cancer. Cytology could be evaluated in 90 patients on voided urine and in 55 on bladder washout samples, depending on hospital preferences. A voided urine specimen was collected just prior to cystoscopy and all samples were immediately evaluated by the NMP22®BladderChek® Test. The NMP22 assay was performed according to the instructions provided in the NMP22®BladderChek® Test kit.

Informed consent was acquired of all patients. Patients with an initial false positive NMP22®BladderChek® Test were followed with cystoscopies for one year.

NMP22®BladderChek®Test

The NMP22 point-of-care device uses a lateral flow immunochromatographic strip encased in a plastic cartridge to detect NMP22 qualitatively in the patient’s urine. The assay incorporates two different monoclonal antibodies, a capture antibody and a reporter antibody. The test device requires four drops of urine at room temperature and gives the result within 30 minutes. A colored band in the test position indicates a positive result (Figure 1). In order to increase the specificity of the test, patients with benign inflammatory conditions (e.g. cystitis), presence or history of foreign body (e.g. stent, nephrostomy tube), renal/ bladder calculi, bowel interposition segment (e.g. ileal conduit, continent diversion), other infiltrating foreign genitourinary cancer, irritation of the bladder (e.g. due to BCG or chemo therapy) were excluded from the study. Urine samples were taken before any instrumentation and invasive procedure or earliest 3 weeks after it.

Cytopathological analysis was carried out by cytopathologists of the different hospitals. The results were classified as negative, atypical, suspicious or positive. Staging and grading of the resected tumours was carried out by the pathologists of the different hospitals according to the TNM classification and the WHO/ISUP 1998 consensus classification [5].

Statistical analysis

The sensitivities of the NMP22®BladderChek® Test and urine cytology were calculated as the number of patients with true -positive test results divided by the total number of patients with histologically confirmed malignancy. Atypical cytological results were considered as negative and suspicious results were considered as positive. Specificity was defined as the percentage of patients with a negative test result who were not diagnosed with malignancy. In case of malignancy, histology was considered the gold standard, except in 3 cases where the tumour was not resected because of comorbidity reasons, but obviously malignant on cystoscopy. In absence of tumour the gold standard was the cystoscopy result. Corresponding 95% confidence intervals were calculated.

Results

A total of 145 patients (116 male and 29 female) were studied. The median age was 66.8 years (range 31.7 to 91.7) and 95.2% were Caucasians. 103 patients (71%) had a previous history of bladder cancer and 42 patients (29%) were suspected of having bladder cancer because of symptoms. Cytology was done in 90 patients on voided urine (VU) and in 55 on bladder washout samples (BWO). The NMP22® BladderChek® Test was only done in voided urine (all patients). Cystoscopy was positive or suspect in 27% (39/145) of patients. Thirty-five patients (24.1%) had malignancy (32 histologically confirmed, 3 tumours were not resected because of comorbidity reasons, but obviously malignant on cystoscopy). Four patients with a positive or suspect cystoscopy had no malignancy. Histopathology revealed 69% (22/32) TaG1/TaG2, 28% (9/32) TaG3/ ≥T1/ TIS tumours and one G1 (no TNM staging available), as shown in Table 1.

The overall sensitivities of the NMP22®BladderChek® Test, voided urine cytology and bladder washed urine cytology for the detection of bladder cancer were 71%, 54% and 64%, respectively. The specificities were 85%, 91% and 91%, respectively. The overall sensitivity, specificity, positive predictive value and negative predictive value for voided urine cytology, bladder washed urine cytology and the NMP22®BladderChek® Test are summarized in Table 2.

The sensitivities of the NMP22®BladderChek® Test and urine cytology (VU and BWO are pooled) according to tumour stage and grade are shown in Table 3.

and grade are shown in Table 3. The performance of the NMP22®BladderChek® Test seems to be slightly better in the patient group with no previous history of bladder cancer than in the group with previous history of bladder cancer; the sensitivities were 86% and 62%, respectively (Table 4).

Follow-up cystoscopies in the patient group with an initial false positive NMP22 test (n=15; 2 patients were lost to follow -up) revealed a tumour in 2 patients after 3-12 months (both after 6 months).

Discussion

Currently, the detection and surveillance of bladder cancer is still based on cystoscopy and cytology. Cystoscopy is invasive, uncomfortable, and expensive. Urine cytology lacks diagnostic efficacy due to its inter- and intra- observer variability and due to its low sensitivity, especially in low-risk tumours. Thus, it is evident that there is a need of a new non-invasive urinary marker for the detection and surveillance of urothelial carcinoma.

During the last years several new urine markers have been developed for urothelial carcinoma detection. A urinary marker used in the follow-up of non-invasive urothelial cancer should have a high sensitivity, because a false-negative test result places the patient at risk for progression to potentially lethal muscle-invasive urothelial carcinoma, especially the patients in the high-risk category [2]. An assay with a high sensitivity can, in case of a negative test result, be used to modify the current rigorous surveillance protocol of cystoscopy, especially in the low-risk group.

When a urinary marker is used as a screening device for the detection of malignancy in patients with risk factors or symptoms of bladder cancer, it should also have a high specificity and positive predictive value to avoid unnecessary cystoscopies.

The NMP22®BladderChek® Test provides point-of-care results within 30 minutes and the test minimizes operator variability. The assay is easy to perform, because it needs no laboratory equipment, it causes no morbidity and is relatively inexpensive.

The NMP22®BladderChek® Test has a better overall sensitivity and about the same specificity as cytology. We found that cytology on bladder wash out samples has a slightly better sensitivity and the same specificity than cytology on voided urine.

Our results show that the NMP22®BladderChek® Test has a higher sensitivity than cytology in detecting all different stages and grades of bladder cancer, only in grade 3 tumours cytology has a slightly better sensitivity, but the number of patients is too low to draw definitive conclusions, although the urinary marker should have a very high sensitivity for these tumours. In particular, in the low-risk group (Ta, G1-2) the BladderChek test has a much better sensitivity than cytology.

The NMP22 Bladder Cancer Test has been extensively evaluated as a quantitative marker to detect bladder cancer. A meta- -analysis based on 2,290 patients performed by Glas et al [6] showed a sensitivity of 67% (95%CI: 60%-73%) and a specificity of 78% (95%CI: 72%-83%) for the quantitative NMP22 Bladder Cancer test in the diagnosis of primary bladder cancer. Van Rhijn et al [7] assessed the sensitivity and specificity of the quantitative NMP22 Bladder Cancer test solely for bladder cancer surveillance. They found a sensitivity of 71% (range 47-100) and a specificity of 73% (range 55-98) based on 838 and 1203 patients, respectively.

A few studies using the qualitative NMP22®BladderChek® Test have been published at this time. Grossmann et al [3] evaluated the NMP22®BladderChek® Test in 1331 patients with an increased risk (smokers and patients with dysuria and hematuria), but without a history of bladder cancer. They reported a sensitivity and specificity of 55.7% and 85.7%, respectively. Moreover, the NMP22®BladderChek® Test detected 4 cancers that were not visualized during initial cystoscopy. They considered the NMP22®BladderChek® Test as a useful adjunctive to cystoscopy in the diagnosis of bladder cancer. Our results were even better, so we can confirm their conclusion.

Grossmann et al [8] evaluated the NMP22®BladderChek® Test also as a surveillance device. In 668 patients with a history of bladder cancer they applied the test for the detection of bladder cancer recurrence. They found a sensitivity and specificity of 49.5% and 87.3%, respectively, which is also in accordance with our results.

Moonen et al [9] found a slightly higher sensitivity for the NMP22®BladderChek® Test (57.1%) compared to cytology (42.9%), without a relevant loss in specificity (89.8% vs. 93.2%) in 73 patients with a previous history of bladder cancer. Negative tests in patients with Ta, grade 1 tumors were not scored as false negative to improve the overall sensitivity.

Tritschler et al [10] recently validated the diagnostic value of NMP22®BladderChek® Test by the highly sensitive photodynamic diagnosis. They found a sensitivity of 65% (26/40) and a specificity of only 40% (24/60) and concluded that the value of the test is limited because of its low specificity.

Unfortunately, the qualitative point-of care test does not allow the quantification of the NMP22 protein. A qualitative assay with a different cut-off value for patients with and without history of bladder cancer could possibly improve sensitivity and specificity in the different situations.

A problem in the interpretation of most urine tests is the frequent finding of false positive test results. Patients with bladder cancer have a substantially higher amount of NMP22 in the urine. However, because the NMP22 is released from dead urothelial cells, many benign conditions of the urinary tract can cause a false-positive result. In order to increase the specificity of the test, patients with benign inflammatory conditions have to be excluded from this office based test. However, it is likely that a proportion of false positive NMP22 test results may indeed be subclinically positives or true positives missed by cystoscopy, since cystoscopy, the reference standard for most marker studies, is of course also not fully sensitive. But, in our series most patients with a false-positive result underwent follow-up examination after 3-12 months (n=15) and only 2 of these patients developed bladder cancer at the follow-up cystoscopies. Our results are in accordance with Tritschler et al, they found only 3 tumours out of 27 false positive tested patients after 3-10 months of follow-up examination [10].

Lotan et al [11] recently created a decision analysis model to estimate the five year cumulative cancer-related costs and efficacy of screening a high-risk population (age >50 years, heavy smoking history, significant occupational exposure to toxins and dyes) for bladder cancer using a urine-based tumour marker. The model found that urine-based markers, such as the NMP22®BladderChek® Test are cost-effective in a high-risk population. Bladder cancer seems an ideal disease for screening a high risk population, because the risk factors are well known and an early detection of bladder cancer improves prognosis, quality of life and survival. The NMP22®BladderChek® Test may eventually expand beyond the urologic setting into the primary care setting for this screening of high-risk patients. However, prospective randomized trials testing the accuracy of bladder cancer detection in a completely asymptomatic high-risk cohort are indicated before bladder cancer screening can be recommended.

Conclusions

The NMP22®BladderChek® test has a better sensitivity and about the same specificity as cytology. The office based and non-invasive properties of this test make it a promising tool in the evaluation of bladder cancer, because immediate decisions on further investigations can be made and there is no delay as is the case for urinary cytology.

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Harm C. Arentsen
Department of Urology, Academic Medical Centre
University of Amsterdam
Meibergdreef 9 1105 AZ
Amsterdam, The Netherlands
phone +31 20 5666004
T.M.deReyke@AMC.UVA.NL