Interference From Haematuria Renders the Urinary BTA Test Not Specic Enough for Diagnosing Bladder Cancer: Evidence From Clinical Practice

Background The urinary bladder tumour antigen (BTA) stat test has already been used for the diagnosis and monitoring of bladder cancer (BC). However, more evidence is needed regarding its ecacy and utility in the clinic. In this study, we investigated the inuence of haematuria on the performance of the BTA stat test in a clinical cohort. Methods Urine samples from 836 subjects, including 50 healthy volunteers, 553 patients with benign urologic disorders, 124 patients with histologically proven BC, and 109 patients with other histologically proven urologic cancers, were analysed by the BTA stat test and urinalysis. We detected the sensitivity and specicity of the BTA stat test in each group, and analysed the effect of haematuria on the specicity. Results Our data showed that 58.06% of patients had haematuria in the BC group. Haematuria with benign prostatic hyperplasia (BPH), renal hamartoma (RH) and urolithiasis were identied in 39.01%, 42.86% and 66.49% of patients with benign urologic disorders, respectively. Haematuria was identied in 48.72% of prostatic cancer patients and 67.74% of renal cancer patients. The overall sensitivity of the BTA stat test was 90.32%. The sensitivity was 97.22% in BC patients with haematuria and 80.77% in BC patients without haematuria. The overall specicity in healthy individuals, patients with benign urologic disorders and other urologic cancers was 50.84%. In all patients with haematuria, the specicity of the BTA stat test was 15.82%, while it was 72.6% in patients without haematuria. Conclusions Haematuria has a signicant inuence on the BTA stat test. Our study suggested that the BTA stat test is not an ideal diagnostic tool for BC. test may be prone to detect particularly high false positives in haematuria patients. We analysed patients with haematuria symptoms in a clinical setting and found that the specicity of the BTA stat test was only 15.82% for all groups of patients with haematuria. The meaning of these data is that when only patients with haematuria symptoms who were suspected of having BC were considered, the specicity of diagnosing BC with the BTA stat test was only 15%. In addition, the proportion of BC patients with haematuria is very small. Previous studies have shown that only 0.7–1.3% of patients with haematuria actually have BC [13, 15, 16]. In this case, if the BTA stat test has such a high false-positive rate for haematuria, it will lack further directivity, and its clinical utility and benet will be low. The cost of screening includes the cost of labelling each patient and the cost of evaluating patients with false-positive results. Adding any test to the clinical assessment will increase costs; thus, the cost-effectiveness needs to be balanced by the benets. Optimal screening strategies require identifying a method with high specicity and reasonable sensitivity in a population with signicant disease prevalence [27]. The specicity of the marker plays a key role because more patients with false-positive results correlates with needing additional “unnecessary” tests. Our results suggest that the BTA stat test cannot achieve ideal effective specicity and that using it to screen and monitor for BC may lead to “unnecessary” costs. Of course, further studies can compare and analyse the specic costs involved in the clinical practice of qualitative urinary tract testing. However, there are no available data on this problem. Hence,


Background
Bladder cancer (BC) is the ninth most common cancer disease worldwide, and the incidence is particularly high in men [1]. Early BC has a better prognosis, but the progression of the disease greatly increases the rate of metastasis and mortality. The data showed that BC is likely to recur in 50-70% of patients within 5 years [2]. Approximately 15-25% of recurrent BC can be expected to progress to a higher tumour stage [3], and 50% of patients diagnosed with muscle-invasive BC succumb to the disease within ve years [4]. Therefore, early screening and long-term close follow-up with repeated examinations are essential.
Currently, cystoscopy and urine cytology are the best tests for the diagnosis of BC [5]. However, there are some limitations in practical clinical applications. Although cystoscopy is the gold standard, it is an invasive test and is not suitable as the rst choice for early screening and long-term follow-up. On the other hand, urine cytology has ideal speci city, but its sensitivity varies depending on the grade of the tumour, as urine cytology has low sensitivity in low-grade tumours. In addition, the interpretation of urine cytology results is highly dependent on the skill of the examiner, which varies greatly in different studies and observations [6]. Therefore, nding and evaluating a non-invasive, inexpensive, highly sensitive and speci c test for BC is important. Molecular markers have been proposed for the detection of bladder tumours within recent years. However, to be widely used, the speci city and sensitivity of these molecular markers need to be tested more in clinical practice.
The bladder tumour antigen (BTA) stat test is a qualitative enzyme immunoassay that detects a bladder tumour-associated antigen in urine. This antigen has been recognized as a human complement factor H-related protein (hCFHrp) and inhibits the complement pathway to cause cytolysis in cells with a resulting selective advantage for the tumour [7]. Previous studies have suggested that the sensitivity of the BTA stat test is superior to urine cytology, and it has been approved by the US Food and Drug Administration for diagnosing and monitoring of BC [8,9]. However, the speci city of the BTA stat test is often disturbed by some factors, such as infection and intravesical treatment [10][11][12]. Few studies have mentioned the impact of haematuria, and relevant clinical data are limited. In the clinic, haematuria is a symptom of BC that cannot be ignored. The initial diagnosis of BC most often occurs after an episode of gross or microscopic haematuria. Over 80% of patients with BC have some degree of haematuria [13,14]. On the other hand, there are many causes of haematuria other than BC, including many benign urologic disorders. Studies have shown that only 0.7-1.3% of patients with haematuria actually have BC [13,[15][16]. Even between 9 and 18% of normal individuals also have some degree of haematuria [17]. Hence, if haematuria has a serious effect on the speci city of the BTA stat test, using it to screen and monitor for BC can lead to unnecessary investigation and anxiety.
In this study, we analysed the speci city and sensitivity of the BTA stat test in BC. The effect of haematuria on the speci city of the BTA stat test in patients with BC, patients with benign urologic disorders, patients with other urologic cancers and healthy volunteers was also determined. Our study aims to evaluate the utility of the BTA stat test in the diagnosis of BC in clinical practice.

Patients
We recruited patients with BC, urinary benign diseases and other urinary cancers who visited the Urology Department of the First A liated Hospital of Chongqing Medical University from January 2018 to December 2019 (Table 1). Patients enrolled completed related inspections as needed. All diagnoses were con rmed by two urologists. Cystoscopy biopsy was the gold standard for BC diagnosis. Benign diseases of the urinary system were diagnosed according to the corresponding diagnostic criteria. Other urinary cancers were diagnosed based on the results of the corresponding pathological examination. In addition, we recruited a group of healthy volunteers without any clinical evidence of disease. All participants underwent BTA stat test and urinalysis, and none of the participants had mechanical manipulation of the urethra or bladder within the last two weeks before specimen collection. Participants who ultimately did not belong to one of the diagnostic categories or refused further tests to con rm a diagnosis were excluded. Except for the urinary tract infection (UTI) group in the benign urologic disorders group, patients with active UTIs in the other groups, indicated either by symptom reports or by pyuria, were excluded. Except for the urolithiasis group in the benign urologic disorders group, patients with a recent history of urolithiasis in the other groups were excluded.

BTA stat test
The BTA stat test (Polymedco, NY, USA) is a one-step qualitative assay. We assayed all samples according to the manufacturer's instructions [18]. Three drops of urine are placed on the BTA stat test device, which contains a small lateral ow immunochromatographic assay. The urine reacts with colloidal gold-conjugated anti-bladder tumour-associated antigen antibody to form an immune complex. Then, when the immune complex ows through the detection area, it binds to another anti-bladder tumour-associated antigen antibody and forms a visible colour band. If there is no hCFHrp in the urine, no visible line will form in the detection area. Regardless of the presence or absence of hCFHrp in the urine sample, the control area can bind to the detection antibody to form a visible line. Therefore, when both target and control zones form two visible lines, the reading is positive; when only the control line is formed, the reading is negative. The absence of a visible control line means that the test is invalid, and the test needs to be repeated.

Statistical analysis
Statistical analysis was performed using SPSS software, version 20.0 (Chicago, IL, USA). Analysis of the study population characteristics used descriptive statistics. Qualitative variables were expressed as proportions. Sensitivity is de ned as the ratio of the number of true positive test results and the number of subjects with con rmed BC. Speci city is de ned as the ratio of the number of true-negative test results and the number of subjects without BC. Exact 95% con dence intervals (CIs) indicate the precision of the sensitivity and speci city [8].

BTA stat test in patients with BC
Urinary specimens of 124 patients with histologically proven BC were examined prior to therapy. Histologically, 118 patients had transitional cell carcinoma (TCC), ve had squamous cell carcinoma (SCC) and one had undifferentiated carcinoma. Of these patients, 112 patients were BTA-positive, and 12 were BTA-negative with the BTA stat test. According to the histologic stage, 62 patients had a pTa tumour; 24 patients had a pT1 tumour; 28 patients had a pT2 tumour; and 10 patients had a higher tumour (

Effect of haematuria on the BTA stat test
In patients with benign urologic disorders, patients with haematuria account for a relatively high proportion. Haematuria in patients with BPH, RH and urolithiasis was 39.01%, 42.86% and 66.48%, respectively. In urologic cancers, 58.06% of BC patients were accompanied by haematuria, and 48.72% of prostatic cancer patients and 67.74% of renal cancer patients were also accompanied by haematuria (Fig. 1). Haematuria had a signi cant effect on the speci city of the BTA stat test in each group. In benign urologic disorders with haematuria, the false-positive rate of the BTA stat test was 211 of 238 patients, which was signi cantly higher than that of 80 of 315 patients without haematuria who had the same conditions. The false-positive rate of the BTA stat test increased correspondingly in patients with BPH, RH and urolithiasis, as a high proportion of these patients had haematuria (Fig. 1). The speci city of this test decreased to 8.94%-31.58% in patients with BPH, renal cyst, BAT, RH and urolithiasis associated with haematuria. When we removed the patients with haematuria from this analysis, and the speci city of the BTA stat test increased to 73.21-98.08%. In patients with prostatic cancer and renal cancer, the false-positive rates of the BTA stat test were 43 of 78 and 16 of 31 patients, respectively. In these patients with haematuria, the speci city of the BTA stat test decreased to 28.95% and 42.86%, while the speci city of the BTA stat test in patients with prostatic cancer and renal cancer were both 60% after removing patients with haematuria from this analysis. In all groups of patients with haematuria, the speci city of the BTA stat test was 15.82%, and the overall speci city of the BTA stat test was 70.5% in patients without haematuria (Table 4).

Discussion
The high recurrence rate of BC requires long-term regular follow-ups [1]. Non-invasive urine testing is ideal for both the patient and the healthcare system [19]. Among non-invasive urine tests, urine tests with molecular biomarkers are promising tools for diagnosing BC. However, these tests are still not well established in daily clinical routine and in the standard diagnostic workup of BC. Further evaluation of the usefulness of these biomarkers in complex clinical situations is needed before they are recommended for widespread clinical use.
The BTA stat test is a molecular urine test for BC that that is currently subject to the most attention. The BTA stat test speci cally recognizes bladder tumour-associated antigen (hCFHrp) in urine through monoclonal antibodies. This antigenic protein is considered to be isolated from the urine of patients with BC but cannot be detected in the urine of most healthy individuals [7]. Previous studies have compared the sensitivity of the BTA stat test and cytology detection in patients with BC diagnosed by cystoscopy, and the sensitivity of the BTA stat test was found to be superior to urine cytology and bladder irrigation cytology [20][21][22]. However, its effectiveness in real-world clinical situations, especially its speci city under different interference situations, needs further observations. In this study, we investigated the sensitivity and speci city of the BTA stat test in patients who were diagnosed with BC, and more importantly, we investigated the in uence of haematuria on the performance of the BTA stat test in a clinical cohort.
Our overall sensitivity results were similar to those published by Dov Pode et al. for the sensitivity of the BTA stat test in detecting bladder tumours [18]. In a European multicentre study of 107 patients with a nal diagnosis of BC con rmed by cystoscopy and biopsy, the overall sensitivity of the BTA stat test was 72% [23]. In the Rüdiger Heicappell et al. study, they reported sensitivities for grade I, II, and III disease to be 59.1%, 50%, and 77.3%, respectively [7]. By contrast, we demonstrated higher sensitivities of 80%, 70 and 97.75% for PUNLMP, low-grade and high-grade tumours, respectively. These results may be related to different tumour grading methods. More importantly, our analysis of the overall sensitivity included patients with haematuria, which may show higher sensitivity due to false positives. When patients with haematuria were removed from this analysis, the overall sensitivity of the BTA stat test was 80.77%. If you analyse only patients with haematuria, the overall sensitivity of the BTA stat test was 97.22%. In our study, the speci city of the BTA stat test was 100% in healthy volunteers, and this result was within the ranges reported by other authors, thus excluding the possibility of technical or analytical errors [10]. Although the speci city of the BTA stat test is high in healthy individuals, disappointing results have nevertheless occurred in patients with other urologic diseases. In a multicentre US study, the speci city of the BTA stat test in patients with BPH, urolithiasis and UTI was 88.5%, 50% and 76%, respectively [24]. These variations may be explained by inherent in uencing variables such as infection, trauma, and calculi [10,23]. Our study showed even lower speci city; the speci city of the BTA stat test decreased to only 31.89% and 15.52% in patients with urolithiasis and UTI, respectively. In patients with prostatic cancer and renal cancer, the speci city of the BTA stat test was only 44.87% and 48.39%, respectively. This may be because we did not exclude patients with haematuria from our total enrolment.
Our results suggested that haematuria leads to high positive rates. On the one hand, the majority of patients with BC will present with haematuria [13,14]. Considering that hCFHrp is a serum factor, the BTA stat test can possibly concomitantly detect serum proteins that cause haematuria. Another study supports our conclusion; the Makito Miyake group built an experimental haematuria model. They added whole blood to BTA-negative urine samples and found that spiking BTA-negative urine samples with as little as 1 µl whole blood /10 ml urine was enough to produce a positive BTA stat test result [25]. On the other hand, haematuria is the most common symptom in urology patients. In many benign or tumorous urologic diseases, haematuria is often the only manifestation [26]. Even 9-18% of normal individuals also have some degree of haematuria [17]. This fact means that the BTA stat test may be prone to detect particularly high false positives in haematuria patients. We analysed patients with haematuria symptoms in a clinical setting and found that the speci city of the BTA stat test was only 15.82% for all groups of patients with haematuria. The meaning of these data is that when only patients with haematuria symptoms who were suspected of having BC were considered, the speci city of diagnosing BC with the BTA stat test was only 15%. In addition, the proportion of BC patients with haematuria is very small. Previous studies have shown that only 0.7-1.3% of patients with haematuria actually have BC [13,15,16]. In this case, if the BTA stat test has such a high false-positive rate for haematuria, it will lack further directivity, and its clinical utility and bene t will be low. The cost of screening includes the cost of labelling each patient and the cost of evaluating patients with false-positive results. Adding any test to the clinical assessment will increase costs; thus, the costeffectiveness needs to be balanced by the bene ts. Optimal screening strategies require identifying a method with high speci city and reasonable sensitivity in a population with signi cant disease prevalence [27]. The speci city of the marker plays a key role because more patients with false-positive results correlates with needing additional "unnecessary" tests. Our results suggest that the BTA stat test cannot achieve ideal effective speci city and that using it to screen and monitor for BC may lead to "unnecessary" costs. Of course, further studies can compare and analyse the speci c costs involved in the clinical practice of qualitative urinary tract testing. However, there are no available data on this problem.

Conclusions
In patients with haematuria, there is a relatively low prevalence of BC. Our research shows that the BTA stat test appears to have a high false-positive rate and low speci city in patients with haematuria. Hence, using the BTA stat test to screen and monitor for BC is not ideal. Our data support the relative inadequacy of the BTA stat test as a diagnostic tool for BC.