Advertisement

The Role of Bladder Epicheck Test In Follow-Up of Patients with Non-Muscle Invasive Bladder Cancer

Published:March 16, 2022DOI:https://doi.org/10.1016/j.clgc.2022.03.009

      Highlights

      • New non-invasive diagnostic tool are needed for management of NMIBC.
      • Urinary cytology has some limitation due to confounding factors such as inflammation and due to interobserver variability.
      • Methylation test have been already shown their sensitivity and their specificity in patients with NMIBC.
      • This test could overcome the limits of urinary cytology and could be incorporated in the management of patients in follow-up for NMIBC.

      Abstract

      Introduction

      EpiCheck is a new urinary test that analyses DNA methylation biomarkers in order to identify high-risk urothelial cancer

      Materials and methods

      A prospective single centre study was performed. We analysed Epicheck results in a population of 231 patients in follow-up for non-muscle invasive bladder cancer. The primary endpoint was to evaluate sensitivity and specificity of Epicheck in detecting any type of bladder cancer recurrence. The secondary endpoint was to evaluate specificity and sensitivity of Epicheck in patients with high-risk recurrence and in patients recently treated with endovesical therapy (< 3 months).

      Results

      Negative predictive value (NPV) for cytology was 83 % while for bladder Epicheck it was 89 %, while positive predictive value (PPV) was 67 % and 73 % for cytology and Epicheck respectively. Considering only high grade non muscle invasive bladder cancer the sensitivity of Epicheck was 91 % and for cytology was 81 %, specificity was 85 % and 83 % and negative predictive value of Epicheck outreached 96 % compared to 92 % of cytology. Among patients with an ongoing or recent endovesical treatment it appears that sensitivity of Epicheck was 88% % compared to 73 % of cytology, specificity was 97 % and 85 % and NPV was 92 % compared to 82 % for cytology.

      Conclusion

      The EpiCheck (test showed very high diagnostic values, higher than the currently, gold standard. The test might clinically improve the BCa management in terms of, reduced number of inconclusive/suspicious reports of cytology and endoscopy, reduced number of further examinations, reduced associated patient and economic

      Keywords

      Abbreviations:

      NMIBC (Non muscle invasive bladder cancer), BCG (Bacillus of Calmette e Guerìn), EMDA (Electromotive drug administration), MMC (Mitomycin C)

      Introduction

      Non muscle invasive bladder cancer (NMIBC) is a disease with a well-known risk of recurrence that requires a strict follow-up after initial treatment.
      Urological guidelines recommend the use of urinary cytology, cystoscopy and radiological evaluation of upper urinary tract during the follow-up
      • Babjuk M
      • Burger M
      Compérat EMet al European association of urology guidelines on non-muscle-invasive bladder cancer (TaT1 and Carcinoma In Situ) - 2019 update.
      .
      Many efforts have been made to reduce the number of invasive procedures such as cystoscopy and to increase the use of non-invasive diagnostic tools for non-muscle invasive bladder cancer, both for the economic burden of the disease on the health service and both for a better clinical compliance to the follow-up for the patients
      • Leal J
      • Luengo-Fernandez R
      • Sullivan R
      • et al.
      Economic burden ofbladder cancer across the European Union.
      .
      Several urinary biomarkers have been tested and evaluated as adjunctive tool for diagnosis and follow-up of patients with NMIBC, but considering together sensibility, specificity and costs compared to urinary cytology, none of these biomarkers is routinely used in clinical practice and outside clinical studies
      • Lotan Y
      • O'Sullivan P
      • Raman JD
      • et al.
      Clinical comparison of noninvasive urine tests for ruling out recurrent urothelial carcinoma.
      .
      DNA methylation is a process well studied in many cancers including urothelial neoplasia.
      It alters gene expression involving typically oncogenes and oncosuppressors; this process has been already related to progression of disease in non-muscle invasive bladder cancer
      • Gonzalo S
      Epigenetic alterations in aging.
      • Beukers W
      • Kandimalla R
      • Masius RG
      • et al.
      Stratification based on methylation of TBX2 and TBX3 into three molecular grades predicts progression in patients with pTa-bladder cancer.
      .
      The Bladder EpiCheck test (Nucleix Ltd) is a urinary biomarker based on DNA methylation changes associated with bladder cancer in a panel of 15 genomic biomarkers generating a score (range 0-100) that has been recently studied with good results in terms of sensitivity and specificity, particularly for high-risk cancers.
      We analyzed the role of this biomarker in a heterogenous population of patients with non-muscle invasive bladder cancer.

      Materials and Methods

      From January 2018 to July 2019, we evaluated data from 231 consecutive patients with a history of non-muscle invasive bladder cancer (NMIBC).
      A urine sample was collected to perform both the voided urinary cytology and the Bladder EpiCheck test at the time of the endoscopic control.
      The routine follow-up of patients includes a radiological study of upper urinary tract that was made with CT scan in case of high-grade disease and with kidneys and bladder ultrasound in case of low-grade disease.
      Out of 231 patients analyzed,148 patients have had previous endovesical therapy and 101 received local treatment in the previous 3 months: the endovesical therapy was Bacillus of Calmette Guerìn (BCG) for 71 patients Mytomicin C (MMC) for 24 patients and electromotive drug administration (EMDA) of MMC for 6 patients (Table 1).
      Table 1Characteristics of the Patients
      SEX OF PATIENTSMale 146

      Female 85
      AGE73.5 years (range 45-93 years)
      NMIBC STAGE AND GRADEHigh grade - T1G3   74 pts
        - TaG3     15 pts
        - T1G3 + Cis   13 pts
        - CIS      39 pts
      Low Grade - TaG1-2   75 pts
        - T1 G1     8 pts
      ENDOVESICAL THERAPY IN THE LAST 3 MONTHS  - BCG     71 pts
        - MMC     24 pts
        - EMDA     6 pts
      In our department indeed, for a disease not responsive to BCG in patients not suitable for radical cystectomy considering comorbidities or will of the patients, device assisted MMC is a second option
      • Racioppi M
      • Di Gianfrancesco L
      • Ragonese M
      • et al.
      ElectroMotive drug administration (EMDA) of Mitomycin C as first-line salvage therapy in high risk "BCG failure" non muscle invasive bladder cancer: 3 years follow-up outcomes.
      .
      Patients with a positive cystoscopy were candidates to histologic evaluation of bladder tumor. Patients in which only the cytology was positive for high grade urothelial carcinoma or suspected (HGUC or SHGUC) according the Paris system for reporting urinary cytology, were candidates to random bladder biopsies and prostatic urethra biopsies in case of male patients.
      Two different expert dedicated pathologist with at least 10 years of experience evaluated the urinary samples collected at the follow-up visit.
      One pathologist evaluated only the cytology and all the biopsies performed in patients with a suspected recurrent disease. Another pathologist evaluated the Epicheck samples and he was blinded to the results of cytology and of any possible biopsy.
      A patient was defined negative when cytology, cystoscopy and histology were negative.
      The first endpoint of the study was to evaluate the sensitivity, specificity, negative and positive predictive value of Epicheck.
      Then we compare these results with the values of the standard of care for NMIBC management such as urinary cytology.
      The secondary endpoint was to evaluate Epicheck test results in terms of sensitivity and specificity in patients recently (< 3 months) treated with endovesical therapy.
      Statistical analysis was performed using the Pearson chi-square test. A “P” value <.05 was considered significant.

      Cytology

      The samples were centrifugated for 10 minutes at 2000 revolutions per minute. The resulting pellets were resuspended in Thin Prep PreservCyt solution and were processed using the TP 5000 System (Hologic Inc.)
      Cytological evaluation was performed using Papanicolaou staining procedure and the diagnosis was formulated according to the Paris System for Reporting Urinary Cytology classifying the cytological specimens in Negative for High Grade Urothelial Carcinoma (NHGUC), atypical urothelial cells (AUC), Suspicious for High Grade Urothelial Carcinoma (SHGUC), positive for High Grade Urothelial Carcinoma (HGUC)
      • Rosenthal DL
      • Wojcik EM
      • Kurtycz DFI
      The Paris System for Reporting Urinary Cytology.
      .

      Bladder EpiCheck test

      For the Bladder EpiCheck test (Nucleix Ltd), the urine sample was centrifugated twice at 1000g for 10 minutes at room temperature. DNA extracted using Bladder EpiCheck DNA extraction kit was digested using a methylation sensitive restriction enzyme, which cleaves DNA at its recognition sequence if it is unmethylated. The samples were prepared for the PCR assay using the Bladder EpiCheck test kit and the results were analyzed using the Bladder EpiCheck software. For the sample that pass the internal control validation, an EpiScore (a number between 0 and 100) was calculated and an Episcore ≥ 60 indicates a positive result, while a score < 60 indicates a negative result.

      Results

      The mean age of the patients was 73.5 years (range 45-93 years); 146 were male and 85 females.
      148 patients had a history of high grade non muscle invasive bladder cancer, of whom 90 patients had a history of solitary or multiple HG papillary carcinoma (T1-Ta), while 58 cases had a history of carcinoma in situ carcinoma (Cis) primary or concurrent. 83 patients had a history of papillary low-grade disease (G1-G2) according the 1973 and the 2004 World Health Organization (WHO) classification.
      During the period of the study 71 patients had a bladder cancer recurrence: 17 patients have a low-grade recurrent disease, 27 patients have a papillary high-grade recurrence with a concurrent CIS in 8 cases; 3 patients have a T2 disease and 24 have a solitary CIS (Table 2).
      Table 2Results
      STAGE AND GRADE OF RECURRENCE- TaG1-218 pts
      (18 Low Grade; 53 High grade)- T1G319 pts
      - T1G3 + Cis8 pts
      - CIS24 pts
      - T23 pts
      EPICHECK POSITIVE (> 60)78 pts
      CYTOLOGY POSITIVE73 pts
      For the final analysis 22 Patients were excluded because data regarding Epicheck results were missing, whether for problems in the collection of the sample or in the examination.
      78 patients have a positive Epicheck, with an episcore ≥ 60 and 57 patients with a positive Epicheck had a NMIBC recurrence of any grade.
      Regarding cytology 73 patients have a positive cytology and 49 patients with a positive cytology had a NMIBC recurrence of any grade.
      Overall sensitivity for cytology was 69 % and for bladder Epicheck 80 %; specificity was 82 % for cytology and 84 % for the Epicheck.
      NPV for cytology was 83 % while for bladder Epicheck it was 89 %, while PPV was 67 % and 73 % for cytology and bladder Epicheck respectively.
      Specifically considering only high grade non muscle invasive bladder cancer the sensitivity of Epicheck was 91 % (48 patients with a positive Epicheck had a high-grade recurrence) and for cytology was 81 %, (43 patients with a positive cytology had a high-grade recurrence) specificity was 85 % and 83 % and negative predictive value of Epicheck outreached 96 % compared to 92 % of cytology.
      Regarding the secondary endpoint, among patients with an ongoing or recent (< 3 months) endovesical treatment with BCG or MMC it appears that sensitivity of Epicheck in this specific subset of patients was 88% % compared to 73 % of cytology, specificity was 97 % and 85 % and NPV was 92 % compared to 82 % for cytology.
      In the group of patients with CIS the sensitivity and specificity for cytology were 78% and 74%, and for Bladder EpiCheck 87,5% and 88,1%, respectively.
      Negative predictive value for CIS was 94.3 % for cytology and 97.5 % for Epicheck.

      Discussion

      NMIBC is a disease with a high impact on quality of life of patients and a significant economic burden considering their rate of recurrence and the need of long follow-up with cystoscopy.
      Despite its objectivity, even cystoscopy has not a sensitivity and specificity of 100 % in detecting high risk recurrent disease, particularly if we consider flat lesion such as carcinoma in situ that sometimes could be missed at cystoscopy, much more if no adjunctive tools such as NBI or PDD are available
      • Daneshmand S
      • Patel S
      • Lotan Y
      • et al.
      Efficacy and safety of blue light flexible cystoscopy with hexaminolevulinate in the surveillance of bladder cancer: a phase III, comparative, multicenter study.
      Urinary cytology represents a fundamental exam for NMIBC follow-up, low cost, non-invasive and easy to perform. However, its sensitivity and specificity are not as high as required, particularly for low-risk disease and its results could be affected by a lot of benign condition such as infections or stones. Finally, it is an exam operator dependent and therefore it requires experience and a dedicated pathologist.
      A lot of biomarkers have been tested and some of them approved for the diagnosis and follow-up of NMIBC
      • Soria F
      • Droller MJ
      • Lotan Y
      • et al.
      An up-to-date catalog of available urinary biomarkers for the surveillance of non-muscle invasive bladder cancer.
      ,
      • Schmitz-Dr€ager C
      • Bonberg N
      • Pesch B
      • et al.
      Replacing cystoscopy by urine markers in the follow-up of patients with low-risk non-muscleinvasive bladder cancer?-An International Bladder Cancer Network project.
      but to date, none of these is extensively used in clinical practice given their cost and that they do not overcome cytology in terms of sensitivity and negative predictive value.
      In particular none replaced cystoscopy.
      Therefore, a marker or a test with a high negative predictive value, especially for high-risk bladder cancer, would be very useful in order to reduce the number of cystoscopies and to possibly extend the interval between the endoscopic diagnostic exams. Indeed, missing a low-grade recurrence would not affect the history of the disease, considering the raising evidence that even an active surveillance is suitable for this low-risk recurrences
      • Hurle R
      • Lazzeri M
      • Vanni E
      • et al.
      Active surveillance for low risk nonmuscle invasive bladder cancer: a confirmatory and resource consumption study from the BIAS project.
      .
      Bladder Epicheck test has shown good specificity; this is of utmost importance considering that low specificity could lead to a high number of endoscopic procedures for false positive results.
      The results of our study showed that Epicheck is a reliable test compared to cytology in terms of sensitivity, specificity and negative predictive value in patients in follow-up for NMIBC. The data obtained even confirmed the fact that particularly in high risk NMIBC sensitivity of Epicheck could outperform urinary cytology.
      In the validation study of Epicheck the reported sensitivity for the test was 90 % and specificity 83 % and NPV 97 % in a cohort of 222 patients under surveillance forn NMIBC of whom 40 with a recurrent disease (including Ta, T1, T2 and Cis)
      • Wasserstrom A
      • Frumkin D
      • Dotan ZA
      • et al.
      Molecular urine cytology—Bladder EpiCheck is a novel molecular diagnostic tool for monitoring of bladder cancer patients.
      .
      In the multicentric study of Witjes et al. indeed reported data of sensibility, specificity and NPV of Epicheck in a population of 440 patients with a history of NMIBC under routine surveillance.
      • Witjes JA
      • Morote J
      • Cornel EB
      • et al.
      Performance of the Bladder EpiCheck methylation test for patients under surveillance for non–muscle-invasive bladder cancer: results of a multicenter, prospective, blinded clinical trial.
      They evaluated bladder Epicheck in comparison with the standard diagnostic tool (Cytology and cystoscopy) in a group 440 subjects during surveillance for NMIBC; more than 40% of patients had a low-grade disease and more than 50 % of patients had a primary tumor without any previous recurrence at the moment of the enrollment in the study.
      After excluding 87 patients in the final analysis, the authors found out 46 disease recurrences based on histology or only based on cystoscopy and cytology. The overall sensitivity for Epicheck was 68.2 % and overall specificity was 88% with a NPV of 95.1 %. When they excluded low grade recurrences the sensitivity and the NPV raised to 91.7 % and 99.3 %.
      They reported even a specificity of 91 % and a sensitivity of 93% among patients with previous endovesical treatment, without specifying the time between the treatment and the analysis.
      The AUC of the Epicheck excluding low grade recurrences was 0.94.
      The same population was analyzed in a second paper in which D'Andrea et al. reported a secondary, independent analysis, focusing on the clinical utility and influence on decision-making of Epicheck

      D'Andrea D, Soria F, Zehetmayer S, et al. Diagnostic accuracy, clinical utility and influence on decision making of a methylation urine biomarker test in the surveillance of non–muscle-invasive bladder cancer BJU Int. 2019;123:959-967. doi:10.1111/bju.14673.

      .
      Out of a total of 357 patients included and with 49 recurrences they reported a sensitivity of 67.3% (95% CI 52-80) and a specificity of 88% (95% CI 84-91) for the detection of any cancer (accuracy 85.2%, 95% CI 81-89; Table 2), and a sensitivity of 88.9% (95% CI 65-99) and a specificity of 88% (95% CI 84-91) for the detection of high-grade cancer (accuracy 88.1%, 95% CI 84–91). In logistic regression analyses, a positive Bladder EpiCheck result predicted the presence of any cancer with an odds ratio (OR) of 15.1 (95% CI 7.71-30.77; P < .001) and the presence of high-grade cancer with an OR of 58.6 (95% CI 15.8-380; P < .001).
      They conclude that EpiCheck could exclude the presence of high-grade BCa with an NPV of 99.3%, largely outperforming the other diagnostic tools used in the surveillance setting such as cytology or cystoscopy and with some results that are independence from previous or ongoing endovesical therapy.
      In a study by Trenti et al.
      • Trenti E
      • D'Elia C
      • Mian C
      • et al.
      Diagnostic predictive value of the bladder EpiCheck test in the follow-up of patients with non– muscle-invasive bladder cancer.
      with a population of 243 patients and with 69 recurrences (39 low grade and 30 high grade disease), sensitivity of Epicheck was 62.3 % compared to 33.3 % of cytology, whereas specificity was 98.6 % for cytology and 86.3 % for Epicheck. Furthermore, even in this population Epicheck showed to be a reliable marker for follow-up of NMIBC patients with a negative predictive value of 78.6 % and sensitivity reached 83.3 % when considering only high-grade disease.
      In another published experience from the same group
      • Trenti E
      • Pycha S
      • Mian C
      • et al.
      Comparison of 2 new real-time polymerase chain reaction-based urinary markers in the follow-up of patients with non-muscle-invasive bladder cancer.
      with a bigger group of patients (487) and a higher number of recurrences (92, of whom 54 low grade disease) negative predictive value showed to be 89.42 % for Epicheck compared to 83.56 %. Even in this study specificity for Epicheck was however considerably lower than cytology, leading the authors to conclude that this exam might be used in addiction to cytology to have a more detailed evaluation of these patients and to avoid more invasive exams in patients with both negative results.
      The slight differences in terms of sensitivity and specificity in our study compared to the literature could depend on the higher number of recurrences in our population, particularly regarding high grade recurrences that are prevalent in our cohort of patients compared to the previous studies.
      Such as have been shown in the previous studies
      • Witjes JA
      • Morote J
      • Cornel EB
      • et al.
      Performance of the Bladder EpiCheck methylation test for patients under surveillance for non–muscle-invasive bladder cancer: results of a multicenter, prospective, blinded clinical trial.
      ,

      D'Andrea D, Soria F, Zehetmayer S, et al. Diagnostic accuracy, clinical utility and influence on decision making of a methylation urine biomarker test in the surveillance of non–muscle-invasive bladder cancer BJU Int. 2019;123:959-967. doi:10.1111/bju.14673.

      the results of our analysis confirmed that Bladder Epicheck is not influenced by previous or ongoing endovesical therapy and this is a relevant point to keep in mind considering that other markers and moreover cytology are influenced by clinical features
      • Gopalakrishna A
      • Longo TA
      • Fantony JJ
      • et al.
      The diagnostic accuracy of urine-based tests for bladder cancer varies greatly by patient.
      .
      In our study we showed that the sensitivity and negative predictive value of Epicheck outperform cytology in patients with previous recent endovesical therapy and this is important considering that during these therapies there are some morphological alterations of the urothelium with some possible pattern of reactive atypia in urothelial cells that could mimic neoplastic cells.
      • Lopez-Beltran A
      • Paner GP
      • Montironi R
      • Raspollini MR
      • Cheng L.
      Iatrogenic changes in the urinary tract.
      ,
      • Pierconti F
      • Straccia P
      • Emilio S
      • et al.
      Cytological and histological changes in the urothelium produced by electromotive drug administration (EMDA) and by the combination of intravescical hyperthermia and chemotherapy (thermochemotherapy).
      In the Paris System for Reporting of Urine Cytology (TPS), the atypical urothelial cell category (AUC) is characterized by the presence of urothelial cells with an increase N/C ratio (> 0,5) with only one of the following features: nuclear hyperchromasia, irregular nuclear membranes, and irregular coarse clumped chromatin. It has been demonstrated that numerous “benign “conditions increase the number of AUC in the urinary samples and one of this condition is represented by BCG immunotherapy
      • Piaton E
      • Decaussin-Petrucci M
      • Mege-Lechevallier F
      • et al.
      Diagnostic terminology for urinary cytology reports including the new subcategories 'atypical urothelial cells of undetermined significance' (AUC-US) and 'cannot exclude high grade' (AUC-H).
      • Mokhtar GA
      • Al-Dousari M
      • Al-Ghamedi D
      Diagnostic significance of atypical category in the voided urine samples: A retrospective study in a tertiary care center.
      • Bhatia A
      • Dey P
      • Kakkar N
      • Srinivasan R
      • et al.
      Malignant atypical cell in urine cytology: a diagnostic dilemma.
      BCG produces cytological changes that can be mistaken for carcinoma.
      The reduced specificity of cytology in the early period after endovesical therapy therefore might be depended on the clusters of urothelial cells frequently observed in urinary specimen. Therefore, we hypothesized that also for expert pathologist could be more difficult to distinguish neoplastic “papillary” structures from aggregates of urothelial cells with dysplasia, increasing the rate of cytological false positive results.
      The possibility of having a test that is less dependent on simultaneous conditions such as endovesical therapy could be important.
      The study has some limitations that include the relatively small number of patients and the absence of a longer follow-up needed to understand how many patients would have a recurrence missed by all the diagnostic tests and even more to understand if patients with a positive Epicheck analysis and a negative histology might have a recurrence in the future.
      Indeed, it could be that methylation could play a role in identifying patients that will recur after therapy, with the possibility of recognize patients at high risk of recurrence and progression during endovesical therapy as it has already been shown with Fluorescence in situ hybridization (FISH)
      • Kim PH
      • Sukhu R
      • Cordon BH
      • et al.
      Reflex fluorescence in situ hybridization assay for suspicious urinary cytology in patients with bladder cancer with negative surveillance cystoscopy.
      .
      A detailed cost analysis is needed to understand if the actual cost of the bladder Epicheck, more expensive than cytology, giving a useful adjunctive clinical information's will reduce the economic burden of the follow-up of NMIBC.

      Conclusion

      Our study showed that the Bladder EpiCheck test could be a useful adjunctive tool in follow-up of patients with NMIBC due to the possibility to integrate results of cytology and outperform it in particular conditions such as during or after endovesical therapy.
      Larger studies and with longer follow-up are needed in order to confirm these preliminary results and include this test in the routine clinical assessment of patients with NMIBC.

      Clinical Practice Points

      The follow-up of patients with NMIBC is a crucial part of the management of patients with this cancer considering the risk of recurrence and progression.
      Non-invasive tests are an important part of this follow-up and the research in this field is of great interest considering the possibility of reducing costs and invasiveness for patients.
      Urinary cytology is a milestone in the management of patients with NMIBC but it has some limitations due to interobserver variability and to possible confounding factors such as inflammation and morphological alterations related to endovesical therapy.
      Methylation test such as Epicheck have been shown a good sensitivity and sensibility in detecting NMIBC particularly regarding high-risk disease.
      In our study Epicheck have results comparable to urinary cytology in terms of overall sensitivity and specificity for bladder cancer recurrence and could overcome cytology in particular conditions such as patients with recent endovesical therapy.
      This tool must be evaluated not for taking the role of urinary cytology but as an important adjunctive tool in patients with equivocal findings in cytology and in endoscopy. Therefore, if larger and randomized study will confirm our preliminary results, this tool must be integrated in the management of NMIBC patients.

      Authors’ Contributions

      M. Ragonese, L.D.: provided the conception and design of the study, acquisition of data, drafting the article and final approval of the manuscript. F.P., M.M., M.F.: supplied the acquisition of data, drafting of manuscript and final approval of the manuscript. M.R., P.B.: were responsible for the article critically for important intellectual content and final approval of the manuscript.

      Acknowledgments

      No funding to declare.

      Disclosure

      All authors have no conflict of interest to report.

      References

        • Babjuk M
        • Burger M
        Compérat EMet al European association of urology guidelines on non-muscle-invasive bladder cancer (TaT1 and Carcinoma In Situ) - 2019 update.
        Eur Urol. 2019; 76: 639-657https://doi.org/10.1016/j.eururo.2019.08.016
        • Leal J
        • Luengo-Fernandez R
        • Sullivan R
        • et al.
        Economic burden ofbladder cancer across the European Union.
        Eur Urol. 2016; 69: 438-447https://doi.org/10.1016/j.eururo.2015.10.024
        • Lotan Y
        • O'Sullivan P
        • Raman JD
        • et al.
        Clinical comparison of noninvasive urine tests for ruling out recurrent urothelial carcinoma.
        Urol Oncol. 2017; 35: e15-e22https://doi.org/10.1016/j.urolonc.2017.03.008
        • Gonzalo S
        Epigenetic alterations in aging.
        J Appl Physiol. 2010; 109: 586-597https://doi.org/10.1152/japplphysiol.00238.2010
        • Beukers W
        • Kandimalla R
        • Masius RG
        • et al.
        Stratification based on methylation of TBX2 and TBX3 into three molecular grades predicts progression in patients with pTa-bladder cancer.
        Mod Pathol. 2015; 28: 515-522https://doi.org/10.1038/modpathol.2014.145
        • Racioppi M
        • Di Gianfrancesco L
        • Ragonese M
        • et al.
        ElectroMotive drug administration (EMDA) of Mitomycin C as first-line salvage therapy in high risk "BCG failure" non muscle invasive bladder cancer: 3 years follow-up outcomes.
        BMC Cancer. 2018; 18: 1224https://doi.org/10.1186/s12885-018-5134-7
        • Rosenthal DL
        • Wojcik EM
        • Kurtycz DFI
        The Paris System for Reporting Urinary Cytology.
        Springer International Publishing AG, Cham, Switzerland2016
        • Daneshmand S
        • Patel S
        • Lotan Y
        • et al.
        Efficacy and safety of blue light flexible cystoscopy with hexaminolevulinate in the surveillance of bladder cancer: a phase III, comparative, multicenter study.
        J Urol. 2018; 199: 1158-1165https://doi.org/10.1016/j.juro.2017.11.096
        • Soria F
        • Droller MJ
        • Lotan Y
        • et al.
        An up-to-date catalog of available urinary biomarkers for the surveillance of non-muscle invasive bladder cancer.
        World J Urol. 2018; 36: 1981-1995https://doi.org/10.1007/s00345-018-2380-x
        • Schmitz-Dr€ager C
        • Bonberg N
        • Pesch B
        • et al.
        Replacing cystoscopy by urine markers in the follow-up of patients with low-risk non-muscleinvasive bladder cancer?-An International Bladder Cancer Network project.
        Urol Oncol. 2016; 34: 452-459https://doi.org/10.1016/j.urolonc.2016.06.001
        • Hurle R
        • Lazzeri M
        • Vanni E
        • et al.
        Active surveillance for low risk nonmuscle invasive bladder cancer: a confirmatory and resource consumption study from the BIAS project.
        J Urol. 2018; 199: 401-406https://doi.org/10.1016/j.juro.2017.08.091
        • Wasserstrom A
        • Frumkin D
        • Dotan ZA
        • et al.
        Molecular urine cytology—Bladder EpiCheck is a novel molecular diagnostic tool for monitoring of bladder cancer patients.
        J Urol. 2016; 195: e140
        • Witjes JA
        • Morote J
        • Cornel EB
        • et al.
        Performance of the Bladder EpiCheck methylation test for patients under surveillance for non–muscle-invasive bladder cancer: results of a multicenter, prospective, blinded clinical trial.
        Eur Urol Oncol. 2018; 1: 307-313https://doi.org/10.1016/j.euo.2018.06.011
      1. D'Andrea D, Soria F, Zehetmayer S, et al. Diagnostic accuracy, clinical utility and influence on decision making of a methylation urine biomarker test in the surveillance of non–muscle-invasive bladder cancer BJU Int. 2019;123:959-967. doi:10.1111/bju.14673.

        • Trenti E
        • D'Elia C
        • Mian C
        • et al.
        Diagnostic predictive value of the bladder EpiCheck test in the follow-up of patients with non– muscle-invasive bladder cancer.
        Cancer Cytopathol. 2019; 127: 465-469https://doi.org/10.1002/cncy.22152
        • Trenti E
        • Pycha S
        • Mian C
        • et al.
        Comparison of 2 new real-time polymerase chain reaction-based urinary markers in the follow-up of patients with non-muscle-invasive bladder cancer.
        Cancer Cytopathol. 2020; ([Epub ahead o)https://doi.org/10.1002/cncy.22246
        • Gopalakrishna A
        • Longo TA
        • Fantony JJ
        • et al.
        The diagnostic accuracy of urine-based tests for bladder cancer varies greatly by patient.
        BMC Urol. 2016; 16https://doi.org/10.1186/s12894-016-0147-5
        • Lopez-Beltran A
        • Paner GP
        • Montironi R
        • Raspollini MR
        • Cheng L.
        Iatrogenic changes in the urinary tract.
        Histopathology. 2017; 70: 10-25https://doi.org/10.1111/his.13090
        • Pierconti F
        • Straccia P
        • Emilio S
        • et al.
        Cytological and histological changes in the urothelium produced by electromotive drug administration (EMDA) and by the combination of intravescical hyperthermia and chemotherapy (thermochemotherapy).
        Pathol Res Pract. 2017; 213 (Epub 2017 Aug 1): 1078-1081https://doi.org/10.1016/j.prp.2017.07.026
        • Piaton E
        • Decaussin-Petrucci M
        • Mege-Lechevallier F
        • et al.
        Diagnostic terminology for urinary cytology reports including the new subcategories 'atypical urothelial cells of undetermined significance' (AUC-US) and 'cannot exclude high grade' (AUC-H).
        Cytopathology. 2014; 25 (Epub 2013 Mar 5): 27-38https://doi.org/10.1111/cyt.12050
        • Mokhtar GA
        • Al-Dousari M
        • Al-Ghamedi D
        Diagnostic significance of atypical category in the voided urine samples: A retrospective study in a tertiary care center.
        Urol Ann. 2010; 2: 100-106https://doi.org/10.4103/0974-7796.68857
        • Bhatia A
        • Dey P
        • Kakkar N
        • Srinivasan R
        • et al.
        Malignant atypical cell in urine cytology: a diagnostic dilemma.
        Cytojournal. 2006; 3 (15): 28https://doi.org/10.1186/1742-6413-3-28
        • Kim PH
        • Sukhu R
        • Cordon BH
        • et al.
        Reflex fluorescence in situ hybridization assay for suspicious urinary cytology in patients with bladder cancer with negative surveillance cystoscopy.
        BJU Int. 2014; 114 (Epub 2014 Feb 14): 354-359https://doi.org/10.1111/bju.12516