The role of URO17™ biomarker to enhance diagnosis of urothelial cancer in new hematuria patients—First European Data

Abstract Introduction and objectives Novel biomarker research is vital for the progression of safe and thorough diagnostic medicine. There is now a need to improve the diagnosis of bladder cancer via a noninvasive urine test while balancing the risks of harm from investigational procedures, such as cystoscopy and radiological tests, against the likelihood of malignancy. We evaluate the diagnostic accuracy and sensitivity of Uro17™ urinary biomarker for the detection of urothelial cancer in hematuria patients in a prospective blinded validation study. Uro17™ is an immunobiomarker which binds to the oncoprotein Keratin 17, which is involved in the replication cycle of malignant cells. This study compared cystoscopic and histological investigations against Uro17™ results in patients being investigated for symptoms of urothelial cancer. Materials and methods After receiving both local and national ethics/protocol approval, 71 patients were consented and recruited into the study. All patients were scheduled to undergo cystoscopic investigation, and following recruitment, a urine sample was collected. Urine samples were anonymized and processed as per standard cytology protocols and stained using Uro17™ immunobiomarker. The pathologists assessing the results were blinded to the patient and background history, and the results were compared to the biopsy histology. Results The full cohort of enrolled patients consisted of 71 participants included. There were 55 males and 16 females, with an average age of 70. Thirteen were current smokers, 42 ex‐smokers, and 16 nonsmokers. The malignancies detected included both muscle‐invasive (n = 6) and non‐muscle‐invasive tumors (n = 38), and tumors of all grades and carcinoma in situ. Uro17™ was shown to have an overall sensitivity of 100% and a specificity of 92.6%, with a positive predictive value of 0.957 and negative predictive value of 1. Uro17™ investigation was positive in every case of urothelial malignancy. Conclusions Our current data indicates Uro17™ is a highly sensitive noninvasive bladder cancer urine detection test that can improve the diagnosis of Bladder cancer. This can further improve diagnostic capabilities in primary care, reduce the number of referrals to Urology department, and reduce the number of unnecessary invasive procedures for new patients with a suspected urinary bladder cancer.

inflammatory markers and urinary tract infections. 8 There is a clear need for improved diagnostic methods, especially for patients with bladder cancer who may present without visible hematuria. 3 The gold standard for investigation of bladder cancer is cystoscopy, as this allows direct visualization of the urothelial lining of the urethra and bladder, however, this is uncomfortable for the patient and does not allow investigation of the upper tracts. 9 Cystoscopy is often paired with radiological scanning such as CT urography to assess for upper urinary tract disease. This is usually performed during the first encounter that the patient has with the urology service, resulting in a significant number of patients unnecessarily undergoing both radiation exposure and invasive procedures, which can lead to further complications such as urinary tract infections. 10,11 These cases of unnecessary investigations for bladder cancer result in significant costs, estimated to be over £100 million annually in the UK alone. 12 Following treatment for diagnosed bladder cancer, follow-up by means of cystoscopic surveillance is required for many years. For non-muscle-invasive bladder cancer (NMIBC), the 3 year total costs per person for surveillance alone are estimated to be over £4500, with additional costs for any recurrence or progression. 13 Risk grouping/stratifying models, probability nomograms, and artificial neural networks are all methods by which urologists have hoped to reduce the amount of unnecessary investigations that are performed and to help better focus resources on patients who truly require them. 14 However, these methods, no matter how much collective data are inputted, are generic and not patient specific, and will therefore result in some patients being incorrectly stratified.
As research into the molecular biology of bladder cancer has progressed, genetic markers have been identified in the tumorigenesis and progression of the disease. 15

| Aims and objectives
The aim of this trial was to investigate and measure the accuracy and precision of the urinary bladder cancer test, URO17™, which uses K17, when compared with the gold standard of rigid cystoscopy and bladder biopsy, in new diagnosis of bladder cancer in hematuria patients without a prior history of bladder cancer.

| Study design
The study was designed as a prospective blinded validation trial with

| Ethics approval
Local ethics approval gained through R&D departmental procedure.

| Inclusion and exclusion criteria
Inclusion: • Patients aged ≥18 years of age; • Patients under investigation for bladder cancer due to undergo investigative standard of care biopsy. Exclusion: • Patients aged <18 years of age; • Patients with catheter in situ; • Patients who are currently undergoing radiation therapy; • Proposed subject has no bladder (due to surgical removal); • Patients unable or unwilling to provide consent; • Patients currently on investigational drug trials.
As the inclusion criteria required patients to be undergoing cystoscopy & biopsy for histological analysis and comparison, and as our trust utilizes One Stop Clinic Services, the meant that patients had likely already undergone flexible cystoscopy prior to study recruitment. The inclusion criteria were developed this way to ensure that full histological analysis was performed thus reducing possible surgeon variability.

| Data collection
Following consenting and recruitment, a urine sample was collected from each participant and the following data were collected; gender; age; weight, height, and BMI; smoking status; previously diagnosis of bladder cancer (staging and treatments provided); urine sample first morning void status; urine sample volume; and urine sample hematuria by routine dipstick. Subsequent cancer diagnosis will be collected for participants for a period of 5 years via review of available patient medical records.

| Urine sample preparation
Urine samples were anonymized, stored at 4°C and underwent cen-

| Diagnostic test evaluation
The URO17™ staining results were analyzed by two blinded pathologists (SA and RS) simultaneously and results scored as per scoring system in Table 2. No patient information, including any previously diagnosed malignancy of treatments, was available to the pathology team.
Biopsy histology was performed through the SOC route with no evidence on pathology request systems that patients were part of the trial and were reported according to TNM staging and grading as per WHO classification. This histological data were collected by the research team once URO17™ results had been provided.

| URO17 results
URO17™ and white light cystoscopy + biopsy results are shown in Table 5. In patients with no previously diagnosed bladder cancer As per the pathology scoring system,   It is important to highlight the significance of these results in comparison to other urinary biomarkers for bladder cancer. 5,19,[29][30][31][32][33] We have confirmed that URO17™ has one of the highest sensitivity and specificity reported in identifying bladder cancer through noninvasive urine samples. Unlike other nucleic-based bladder cancer biomarker tests that are complex and expensive to run, URO17™

TA B L E 4 Malignancies detected
test is an immunocytochemical assay that utilize the same cytology samples that are used in urine cytology. The test is also easily adaptable to the standard IHC instrumentations and reagents that are readily available and familiar in most laboratories which will make the test very a cost-effective perform. Furthermore, having a same pathologist who usually interpret urine cytology samples also interpret URO17 test will provide efficient workflow and increased utility for urine cytology. While many will see URO17™ as yet another biomarker in a myriad of new noninvasive investigations for bladder cancer, this study confirms the original finding by Babu et al 25 and expands the application of URO17™ to hematuria patients. In the end, combinations of biomarkers may be the key to providing the most effective investigation where the application of URO17™ will play a major role, and continued research into this must be encouraged with the aim of improving patient diagnostics. We will be following all patients in the study up for 5 years to evaluate those patients who may develop a urological malignancy in due course with a current URO17™ result.
This current data suggest that URO17™ could be a sensitive and specific test to detect PUNLMP and both papillary and nonpapillary carcinomas which could potentially providing diagnostic utility in cases where it could help identify lesions that can be easily missed by traditional urine cytology. Furthermore, the data also showed that URO17™ test was able to detect BC in renal pelvis that was missed by urine cytology and cystoscopy which suggest that URO17™ test could be used to augment and increase the accuracy of cystoscopy and traditional urine cytology in monitoring patients for recurrence. 5,25 . 5 This trial was developed with the aim of testing the applicability of URO17™ as a real-world biomarker for urinary tract malignancy, and therefore, must include all types of patients and malignancy who undergo urological investigations. We constructed this prospective trial in such a way as to allow predictable clinical variabilities, such as urine sample size and sample hematuria, as well as to eliminate confounding factors, such as the skill of a pathologist who may detect non-biomarker-related signs in specimens if aware of the background of the patient. As the test uses standard immunostaining available to most pathology departments, we demonstrated that this test could be used by any department/laboratory with minimal difficulties or additional setup.
The next stage of investigation for URO17™ to be rolled out as a screening program would be a large scale, multisite thorough testing to demonstrate these promising results on a national scale. 34