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Novel urine-based DNA methylation biomarkers for urothelial bladder carcinoma detection in patients with hematuria

Novel urine-based DNA methylation biomarkers for urothelial bladder carcinoma detection in... ARAB JOURNAL OF UROLOGY https://doi.org/10.1080/2090598X.2023.2208492 ORIGINAL ARTICLE Novel urine-based DNA methylation biomarkers for urothelial bladder carcinoma detection in patients with hematuria a b b b,c d Hassan F. Abol-Elnazer , Amira Awadalla , Asmaa E. Ahmed , Hassan Abol-Enein , Munir Ali Al Ganzouri and Amr A. Elsawy a b Genetics Unit, Mansoura University Hospital, Mansoura University, Mansoura, Egypt; Center of Excellent for Genome and Cancer Research (CEG-CR), Urology and Nephrology Center, Mansoura University, Mansoura, Egypt; Urology department, Urology and Nephrology Center, Mansoura University, Mansoura, Egypt; Faculty of Science, Ain Shams University, Cairo, Egypt ABSTRACT ARTICLE HISTORY Received 28 February 2023 Background: Urothelial bladder carcinoma (UBC) is usually detected during work-up for Accepted 25 April 2023 hematuria. Cystoscopy and/or contrast-enhanced imaging are the gold standard tools for UBC diagnosis, despite limited by being invasive, expensive and low yield in small flat tumors. KEYWORDS Objectives: To assess the diagnostic performance of urine-based DNA methylation of six genes Urothelial bladder (GATA4, P16, P14, APC, CDH1 and CD99) for UBC detection in patients with hematuria. carcinoma; hematuria; Patients and methods: Voided urine was collected from consecutive patients presented with biomarkers; DNA hematuria for urine cytology and DNA methylation assay of the assigned genes using methyla- methylation; cystoscopy tion-specific Polymerase Chain Reaction (PCR). Further assessment by office cystoscopy and imaging with subsequent inpatient cystoscopic biopsy for positive findings was done. The diagnostic characteristics of DNA methylation and urine cytology were assessed based on its capability to predict UBC. Results: We included 246 patients in the study with identified macroscopic hematuria in 204 (82.9%) patients. Positive cytology was found in 78 (31.7%) patients. DNA methylation of GATA4, P16, P14, APC, CDH1 and CD99 genes was identified in 127 (51.6%), 52 (21.1%), 117 (47.6%), 106 (43.1%), 90 (36.6%) and 71 (28.9%) patients, respectively. The sensitivity of the assigned genes for UBC detection ranges from 35% (95%CI: 31–39) to 83% (95%CI: 79–87). Optimal specificity (SP) (100%) was noted for P16, APC and CDH1 genes. While for the other genes (GATA4, P14 and CD99), the SP was 95% (95%CI: 92–98), 96% (95%CI: 92–99) and 97% (95%CI: 93–99), respectively. On multivariate logistic regression analysis, all genes exclusively demonstrated independent prediction of UBC. On receiver operator characteristic (ROC) ana- lysis, all tested genes methylation showed superior area under the curve (AUC) when compared to urine cytology. Conclusions: We have developed a novel urine-based DNA methylation assay for detection of UBC in patients with hematuria with superior diagnostic performance and independent pre- dictive capacity over urine cytology. Introduction Currently, cystoscopy/cross sectional imaging is the gold standard tools for UBC diagnosis in patients with According to GLOBOCAN data, bladder cancer (BC) is hematuria [5]. Unfortunately, these costly, invasive considered a major health problem that represents 3% diagnostics could miss early, small/flat bladder lesions of all cancer diagnoses [1]. Urothelial bladder carci- [6]. Urine cytology has been proposed as a non- noma (UBC) accounts for the vast majority (>90%) invasive alternative test with high specificity; however, of BC cases with predominance of non-muscle invasive it lacks sensitivity for diagnosis of low grade (LG) disease (Ta, Tis or T1) in 75% of patients, while others tumors [7]. show muscle invasion (T2–4) [1]. Over the last decades, multiple researches have out- In refereed population, UBC is usually diagnosed as put different markers for UBC diagnosis. Based on their a result of work-up for hematuria at a rate of 2–5% target of assessment, these markers include screening following an evaluation of asymptomatic microscopic for soluble antigens (BTA-Stat, NMP-22, surviving, etc.), hematuria [2], and, up to 5–15% of patients with cell surface antigens (Cytokeratins and UroVysion), macroscopic hematuria [3]. Therefore, a timely prompt genomic markers (Cxbladder and Xpert) and urinary evaluation of hematuria can give to earlier diagnosis metabolomics (CRAT and SLC25A20) [8]. However, most and better survival of UBC [4]. CONTACT Amr A. Elsawy amrelsawy.unc@hotmail.com Urology department, Urology and Nephrology Center, Mansoura University, Mansoura, Egypt © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. 2 H. F. ABOL-ELNAZER ET AL. of these markers are limited by unsatisfying diagnostic obtained, while hyperplastic and negative samples were defined negative for malignancy. performance, high cost or lack of validation [7]. Several preliminary studies have shown that DNA Urine methylation tests methylation, a critical step in transcription regulation, Voided urine samples were obtained in a sterile fashion is chemically stable and can be precisely quantified, (40 ml per each patient). Collected samples were fro- making it an attractive marker for UBC detection [9]. zen to −25 C within 60 min of collection. In a central Both local and global DNA methylations in BC speci- laboratory, genomic DNA was isolated from the col- mens are usually associated with inactivation of tumor lected samples and analyzed using methylation- suppressor genes. These methylation changes could specific Polymerase Chain Reaction (PCR). The opera- be effectively identified in urine sediments as well as tors at the central laboratory were blinded to the tumor tissues [10]. cytology/cystoscopy results. In the current literature, multiple studies investi- DNA isolation was done using the standard gated the performance of DNA methylation on either method (Geneture, Luoyang, China). After that, individual or panel genes with reported sensitivity (SN) quantification of the samples was done using and specificity (SP) values that range from 40% to 95% DNA Quantitation PicoGreen Kit (Thermofisher, and 10% to 100%, respectively [9]. Most of these stu- Massachusetts, US). As directed by the manufac- dies were limited by tumor characteristic heterogene- turer, 1.5 mg of DNA was utilized for bisulphite ity (majority were ≥T2 and high grade (HG) disease) stabilization using the available kit (OptuIN, Ca, which did not reflect the daily practice, inclusion of USA). In this reaction, unmethylated cytosine fil - different BC histological variants, lack of external vali- trates are deaminated resulting in its conversion dation and small sample size [11]. to uracil, while methylated residues are kept The aim of our study is to assess the diagnostic unmodified. The analyte quantifications of the performance of novel urine-based DNA methylation modified DNA were processed using real-time of six genes (GATA4, P16, P14, APC, CDH1 and CD99) assays which included parallel amplification and for UBC detection in patients with hematuria. quantification cycles using specific probe and pri- Moreover, we investigated the methylation pattern of mer (Supplementary table) for each analyte. these genes in different stages and grades of UBC. The methylation results were defined using special generation software (Applied Biosystems), which is Study design, settings and participants expressed as cycle threshold values. Cell lines with Study participants identified methylation condition for each tested gene were applied in each cycle as positive and negative After Institutional Review Board approval, patients pre- status and included in the process at the extraction sented with macroscopic or microscopic hematuria phase. were evaluated for eligibility to our study. Eligible Validity of urine sample was ensured when at least patients were asked to participate in this study and ten copies of ACTB gene (Supplementary table) were were enrolled after signing an informed consent form. identified in the isolated DNA from the urine samples. Only valid samples were assessed for methylation sta- Inclusion and exclusion criteria tus of the tested six genes. The results were expressed as gene copy numbers. Inclusion criteria included patients with hematuria Methylated urine sample for each gene was defined (macroscopic or microscopic). On the other hand, when at least ten copies of this gene were measured. patients who had history of BC, pelvic irradiation, Unmethylated samples were defined when <10 copies bleeding diathesis or receiving anticoagulants and of assigned gene were measured. These cut-off values patients with upper urinary tract neoplasm or uro- were calculated during the performance analysis of the lithiasis detected by cross-sectional imaging were assigned gene sets. excluded. Upper tract assessment and office cystoscopy Voided urine cytology, urine-based DNA procedures methylation evaluation Within 2 weeks of urine sample collections, study par- Urine cytology evaluation ticipants were thoroughly evaluated by cross-sectional Prior to outpatient check cystoscopy, a voided urine imaging and outpatient check cystoscopy. sample was collected and assessed according to the Cross-sectional imaging in the form of multiphasic Paris classification system [12]. Positive results were computed tomography was done to exclude upper defined when suspicious or malignant results are ARAB JOURNAL OF UROLOGY 3 urinary tract malignancies or urinary stones (unless Results contraindicated; magnetic resonance imaging was Base line demographics done). All office cystoscopy procedures were done From February 2019 and August 2021, 306 enrolled using flexible white light cystoscopy. Precise scan- patients were assessed for study eligibility. After exclu- ning of the bladder was done. According to the sion of patients with invalid methylation or cytology findings obtained, patients were categorized into results, non-UBC and variant histology UBC, 246 three groups; (1) cystoscopy-positive (gross blad- patients (201 males, 45 females) with a median der lesion/s), (2) cystoscopy-suspicious (hyperemic/ (range) age of 58 (34–99) years were included in the suspicious mucosa) and (3) cystoscopy-free (no final analysis. The study flow chart is shown in Figure gross lesions). For cystoscopy-positive and suspi- (1) while baseline demographics of the study partici- cious patients, transurethral resection bladder pants are illustrated in Table (1). biopsy was done within 2 weeks from the check cystoscopy. Cystoscopy-free patients were followed-up accord- Voided urine cytology and DNA methylation assay ingly (by either urgent clinic visit for patients with As demonstrated in Table (1), negative and positive recurring hematuria or telephone call every 12 weeks cytology were found in 168 (68.3%) and 78 (31.7%) for asymptomatic cases). On the other hand, patients patients, respectively. DNA hypermethylation of with recurring hematuria and persistent/aggravated GATA4, P16, P14, APC, CDH1 and CD99 genes was iden- bladder symptoms were assigned to re-check cysto- tified in 127 (51.6%), 52 (21.1%), 117 (47.6%), 106 scopy and cytology. Positive/suspicious re-cystoscopy (43.1%), 90 (36.6%) and 71 (28.9%) patients, respec- (and/or cytology) was evaluated by biopsy to confirm tively. On the other hand, cystoscopy/biopsy showed or exclude malignancy. negative and positive findings for UBC in 100 (40.7%) and 146 (59.3%) patients, respectively. Outcome measures Outcome measures The primary outcome is to assess the diagnostic per- formance of urine cytology and evaluated genes The diagnostic performance characteristics of urine methylation (GATA4, P16, P14, APC, CDH1 and CD99) cytology and evaluated genes DNA methylation are for UBC diagnosis in patients with hematuria. clarified in Table (2). The SN of the assigned genes for Subsequently, SN, SP, positive and negative predictive UBC detection ranges from 35% (95%CI: 31–39) to 83% values (PPV, NPV) were calculated. In addition, further (95%CI: 79–87) for P16 and GATA4, respectively. On the capability of these genes hypermethylation for predic- other hand, optimal SP (100%) was noted for P16, APC tion of different stages and grades of UBC was and CDH1 genes. While for the other genes (GATA4, P14 assessed. and CD99), the SP was 95% (95%CI: 92–98), 96% (95% CI: 92–99) and 97% (95%CI: 93–99), respectively. Notably, the evaluated genes methylation showed Statistical analysis superiority to voided urine cytology as regard to SN (apart from P16), SP (all six genes), PPV (all six genes) We computed all data using IBM statistical software and NPV (apart from p16 and CD99) for UBC detection v. 19. Chi-square and Fisher exact tests were utilized (McNemar test; p < 0.001) Table (2). to assess the association between categorical vari- Table (3) shows the univariate and multivariate ables, while for the comparison of urine cytology logistic regression analysis of the independent predic- and genes hypermethylation as regard to cysto- tors of UBC in the study participants. All the six genes scopy/biopsy, McNemar test was utilized. Logistic demonstrated exclusively significant findings for pre- regression analysis was utilized for multivariate ana- diction of UBC (GATA4: HR 4.2 (95%CI 2.78–8.24) p = lysis of significant predictors of UBC in study partici- 0.001, P16: HR 2.1 (95%CI 1.72–3.12) p = 0.003, P14: HR pants. A receiver operator characteristic (ROC) curve 1.4 (95%CI 1.1–1.8) p = 0.007, APC: HR 2.1 (95%CI 1.8– was constructed for this logistic regression by plot- 2.5) p = 0.006, CDH1: HR 2.32 (95%CI 1.92–2.73) p = ting the true positive finding (SN) against the false- 0.002, CD99: HR 2.12 (1.83–2.53) p = 0.003). On ROC positive findings (100-SP). The area under the curve analysis, DNA methylation of all the study genes (AUC) was estimated. Boxplots were utilized to showed superior AUC when compared to urine cytol- demonstrate the genes methylation copies and its ogy (Figure 2). predictive capacity for UBC stage and grade. On sub-analysis of UBC cases, ≥T2 stage was identi- A critical two-sided P-value <0.05 was used for sta- fied in 115 patients, while HG tumors were observed in tistically significant differences. 4 H. F. ABOL-ELNAZER ET AL. Enrolled patients Excluded (n): (9): Decline to participate (10): Previously diagnosed bladder carcinoma (8): Patients on anticoagulants or with bleeding diathesis (6): Radiological features suggestive of upper tract urothelial carcinoma Eligible patients Urine-based methylation Voided urine cytology Cystoscopy biomarkers Excluded (n): (5): Invalid methylation or cytology results (8): Non urothelial bladder carcinoma (14): Urothelial carcinoma with variant histology Bladder Biopsy from cystoscopy- positive patients Included patients in final analysis Cystoscopy/Biopsy Voided urine cytology Urine-based methylation Positive for urothelial bladder Positive for malignancy 78 biomarkers carcinoma 146 Methylated Unmethylated Negative for malignancy 168 Negative for urothelial bladder carcinoma 100 GATA4 127 119 P16 52 194 P14 117 129 APC 106 140 CDH1 90 156 CD99 71 175 Figure 1. Study flowchart. 139 patients. As illustrated in Figure 3, GATA4 and P14 patients with macroscopic hematuria [13]. The recom- genes methylation were significantly associated with mendations of most available guidelines for the diag- ≥T2 stage disease (p = 0.002 and <0.001, respectively) nostic work-up in those patients include invasive tests and HG disease (p = 0.007 and 0.004, respectively). as diagnostic cystoscopy and/or contrast-enhanced The cystoscopy-free patients were followed-up for imaging despite its limitations, namely high cost, sub- a median (range) period of 18 (7–36) months according stantial patient burden (pain) and health-care to the predetermined protocol. Out of 100 patients, resources, associated adverse events and waiting sche- who completed the follow-up, persistent hematuria or dules somewhere [14,15]. aggravated bladder problems were reported in 12 Reliable non-invasive markers might provide patients. Re-cystoscopy and cytology were repeated a helpful tool for clinicians to overcome the drawbacks in those patients, with evidence of UBC in one patient. of cystoscopies/imaging and prioritize patients for Notably, DNA methylation was identified in the all six invasive diagnostic interventions [16]. Urine cytology evaluated genes in this patient. was one of the early utilized to complement cysto- scopy for detection of exfoliated tumor cells in urine despite its limitations, low sensitivity in LG tumors and Discussion inter-observer variability [17]. Thereafter, multiple urinary markers with different Hematuria is a significant clinical presentation that can targets and mechanisms have been proposed and imply an underlying serious pathology, especially in ARAB JOURNAL OF UROLOGY 5 Table 1. Patients’ demographics. Median age in years (range) 58 (34-99) Sex n (%) • Male 201 (81.7) • Female 45 (18.3) Hematuria type n (%) •Macroscopic 204 (82.9) •Microscopic 42 (17.1) Diabetes mellitus n (%) •No 184 (74.8) •Yes 62 (25.2) Hypertension n (%) •No 174 (70.7) •Yes 72 (29.3) Concomitant dysuria/suprapubic pain/urgency n (%) •No 184 (74.8) •Yes 62 (25.2) History of urological problems n (%) •No 194 (78.9) •Yes 52 (21.1) ○ Urolithiasis 16 (6.5) ○ Bladder outlet obstruction 16 (6.5) ○ Urinary tract infections 20 (8.1) Concomitant indwelling bladder foreign body n (%) •No 238 (96.7) •Yes 8 (3.3) ○ Catheter 3 (1.3) ○ Bladder stone 5 (2) Smoking history n (%) •Never 90 (36.6) •Former 75 (30.5) •Current 81(32.9) Cytology result n (%) •Negative 168 (68.3) •Positive 78 (31.7) Cystoscopy findings n (%) •Free 100 (40.7) •bladder lesion 146 (59.3) Urine methylation assay target n (%) •GATA4 Methylated 127 (51.6) Unmethylated 119 (48.4) •P16 Methylated 52 (21.1) Unmethylated 194 (78.9) •P14 Methylated 117 (47.6) Unmethylated 129 (52.4) •APC Methylated 106 (43.1) Unmethylated 140 (56.9) •CDH1 Methylated 90 (36.6) Unmethylated 156 (63.4) •CD99 Methylated 71 (28.9) Unmethylated 175 (71.1) Biopsy findings in positive cystoscopy (Urothelial carcinoma) n (%) Stage (TNM) ○ Ta, Tis, T1 31 (12.6) ○ ≥ T2 115 (46.7) Grade (WHO/ISUP 2004) ○ Low grade 7 (2.8) ○ High grade 139 (56.5) assessed over the last years [16]. One of the recent and SP between 20% and 100% [9]. The explanation of common markers to be introduced in this aspect is this variation is essentially attributed to the heteroge- the study of some urine-based DNA methylation neity of the evaluated gene/s, included heterogeneous genes. Methylation of some genes can silence corre- patients with different histopathological variants, sponding tumor suppressor gene/s [18]. stage and grades, as well as different presentation The available literature on the clinical performance patterns (primary vs. recurrent BC) [19]. of DNA methylation in urine of UBC patients showed In our study, we primarily included patients with high variability with ranging SN between 50% and 95% hematuria (macroscopic or microscopic) with 6 H. F. ABOL-ELNAZER ET AL. Table 2. Cross tables of different urine assay methylation targets and urine cytology in comparison to cystoscopy/biopsy results in study participants. Urothelial bladder carcinoma by cystoscopy and biopsy n % Diagnostic characteristics Positive Negative Total % (95%CI) GATA4 127 SN 83.6% (95%CI: 79–87) • Methylated 122 (83.6%) 24 (16.4%) 119 SP 95% (95%CI: 92–98) Unmethylated 5 (5%) 95 (95%) PPV 96.1% (95%CI: 92–99) Total Count 146 100 246 NPV 79.8% (95%CI: 75–84) P16 52 SN 35.6% (95%CI: 31–39) • Methylated 52 (35.6%) 0 194 SP 100% Unmethylated 94 (64.4%) 100 (100%) PPV 100% Total Count 146 100 246 NPV 51.5% (95%CI: 46–55) P14 117 SN 77.4% (95%CI: 72–83) • Methylated 113 (77.4%) 4 (4%) 129 SP 96% (95%CI: 92–99) Unmethylated 96 (96%) 96 (96%) PPV 96.6% (95%CI: 93–99) Total Count 146 100 246 NPV 74.4% (95%CI: 71–78) APC 106 SN 72.6% (95%CI: 67–79) • Methylated 106 (72.6%) 0 140 SP 100% Unmethylated 40 (27.4%) 100 (100%) PPV 100% Total Count 146 100 246 NPV 71.4% (95%CI: 66–75) CDH1 90 SN 61.6% (95%CI: 57–66) • Methylated 90 (61.6%) 0 156 SP 100% Unmethylated 56 (38.4%) 100 (100%) PPV 100% Total Count 146 100 246 NPV 64.1% (95%CI: 60–68) CD99 71 SN 46.6% (95%CI: 42–50) • Methylated 68 (46.6%) 3 (3%) 175 SP 97% (95%CI: 93–99) Unmethylated 78 (53.4%) 97 (97%) PPV 95.8% (95%CI: 91–99) Total Count 146 100 246 NPV 55.4% (95%CI: 51–59) Urine cytology 57 (39%) 21 (21%) 78 SN 39% (95%CI: 36–42) Positive Count 89 (61%) 79 (79%) 168 SP 79% (95%CI: 76–84) Negative Count PPV 84.5% (95%CI: 81–88) Total Count 146 100 246 NPV 61% (95%CI: 57–65) Note: SN: Sensitivity, SP: specificity, PPV: Positive predictive value, NPV: Negative predictive value. Table 3. Univariate and multivariate analyses for predictors of positive cystoscopy/biopsy for urothelial bladder carcinoma (UBC). UBC by cystoscopy and biopsy No Yes Univariate analysis Multivariate analysis Patient and tumor characteristics N (%) N (%) HR (95%CI) p value HR (95%CI) p value Median age in years (Range) 56 (36–75) 60 (34–99) 1.01 (0.9–1.03) 0.47 Sex n (%) 1.2 (0.9–1.5) 0.56 Male 80 (39.8) 121 (60.2) Female 20 (44.4) 25 (55.6) Hematuria n (%) 1.4 (0.95–1.85) 0.75 Macroscopic 82 (40.2) 122 (59.8) Microscopic 18 (42.9) 24 (57.1) Concomitant dysuria/suprapubic pain/urgency n (%) 1.2 (0.89–1.5) 0.59 No 73 (39.7) 111 (60.3) Yes 27 (43.5) 35 (56.5) History of urological problems n (%) 1.12 (0.8–1.4) 0.5 No 81 (41.8) 113 (58.2) Yes 19 (36.5) 33(63.5) Indwelling bladder foreign body n (%) 1.15 (0.88–1.34) 0.1 No 99 (41.6) 139 (58.4) Yes 1 (12.5) 7 (87.5) Smoking n (%) 0.92 (0.87–1.23) 0.17 Never 43 (47.8) 47 (52.2) Former 25 (33.3) 50 (66.7) Current 32 (39.5) 49 (60.5) Cytology result n (%) 1.17 (0.74–0.1.64) 0.07 Negative 83 (61) 53 (39) Positive 17 (15.5) 93 (84.5) Urine methylation assay target n (%) 2.58 (1.94–2.94) <0.001 4.2 (2.78–8.24) 0.001 GATA4 Methylated 5 (3.9) 122 (96.1) Unmethylated 95 (79.8) 24 (20.2) P16 3.42 (2.9–3.8) <0.001 2.1 (1.72–3.12) 0.003 Methylated 0 52 (100) Unmethylated 100 (51.5) 94 (48.5) P14 2.72 (2.34–2.98) <0.001 1.4 (1.1–1.8) 0.007 Methylated 4 (3.4) 113 (96.6) Unmethylated 96 (74.4) 33 (25.6) APC 4.9 (4.3–5.42) <0.001 2.1 (1.8–2.5) 0.006 Methylated 0 106 (100) Unmethylated 100 (71.4) 40 (28.6) CDH1 3.35 (2.97–3.86) <0.001 2.32 (1.92–2.73) 0.002 Methylated 0 90 (100) Unmethylated 100 (64.1) 56 (35.9) CD99 2.67 (2.32–2.91) <0.001 2.12 (1.83–2.53) 0.003 Methylated 3 (4.2) 68 (95.8) Unmethylated 97(55.4) 78 (44.6) ARAB JOURNAL OF UROLOGY 7 Figure 2. Receiver operator characteristic (ROC) curve for urine-based methylation biomarkers and urine cytology for urothelial bladder carcinoma detection in the study cohort. exclusion of patients with BC history. Most of the however, the nest reports which included gene previous studies included heterogeneous patients panels rather than a single gene [9]. Our higher SP (primary/recurrent/surveillance cases vs. healthy/ results could be attributed to the study design symptomatic controls) [9]. Moreover, as compared which included hematuria patients rather than to the previously published reports on DNA methyla- healthy controls and inclusion of UBC with exclu- tion markers for primary BC patients [20–22], our sion of other variant histologies [27]. study included the largest cohort (146 BC and 100 When compared to urine cytology, superior results symptomatic controls). of our study genes were obtained as regard to inde- Among the evaluated six genes, our study pendent diagnostic capacity in multivariate logistic assessed DNA methylation of two novel genes regression analysis, as well as, AUC on ROC curve (GATA4 and CD99) that were not previously investi- analysis. In addition, GATA4 and P14 genes methylation were gated in the literature for this purpose. On the significantly associated with ≥T2 stage disease (p = contrary, there is a paucity of the literature as regard to three genes (CDH1, P14 and P16) with 0.002 and <0.001, respectively) and HG disease (p = one report for each gene [23–25], these reports 0.007 and 0.004, respectively). This finding should add were limited by its small sample size (22–57 another diagnostic benefit of DNA methylation not patients) and heterogeneous presentation. only for UBC diagnosis but also for tumor characteriza- Our findings showed varying SN of evaluated tion (stage/grade). markers ranging from 35% (P16) to 83.5% (GATA4). Our study is advantaged by its prospective nat- As compared to SN of the previously investigated ure, inclusion of consecutive hematuria patients genes (APC, CDH1, P14 and P16), our findings were with no BC history (better reflection of target notably lower due to the nature of our study parti- population in daily practice than healthy controls), cipants with exclusion of patients under BC surveil- use of predefined threshold level of gene methyla- tion, exclusion of non-UBC or UBC with abnormal lance and lesser patients with ≥T2/HG disease as variants and study of diagnostic capacity of evalu- these tumors probably harbor more molecular ated genes for tumor stage and grade. On the alterations [26]. other hand, there are some limitations in our On the other hand, the SP of the evaluated study. First, our results need external validation in genes varies between 95% and 100%. The initial a larger cohort multicenter study. Second, our ana- reports in the literature about single-gene DNA lysis included only hypermethylation study with methylation showed compromised SP (0–60%), 8 H. F. ABOL-ELNAZER ET AL. Figure 3. Boxplots of urine-based DNA methylation biomarkers GATA4, P16, P14, APC, CDH1 and CD99 for detection of different urothelial bladder carcinoma stages (3A) and grade (3B). lack of other analyses that can optimize the diag- findings. Further longitudinal follow-up and external nostic capacity as mutation analysis [28]. Third, validation studies of our results are warranted before lack of further study of evaluated genes DNA future implementation in clinical practice. methylation on future tumor recurrence and/or progression is a considerable limitation. Disclosure statement No potential conflict of interest was reported by the authors. Conclusions We have developed a novel urine-based DNA methyla- tion assay for non-invasive detection of UBC in patients Funding with hematuria. Superior diagnostic performance and independent predictive capacity of the evaluated This work was funded by Science, Technology & Innovative Funding Authority (STDF); Project No.: 5236. genes methylation over urine cytology are promising ARAB JOURNAL OF UROLOGY 9 Clinical trials registration caring for this disease. Eur Urol. 2014;66(2):253–262. DOI:10.1016/j.eururo.2014.01.006 clinicaltrials.gov ID: NCT04714345 [7] Maas M, Bedke J, Stenzl A, et al. Can urinary biomarkers replace cystoscopy? World J Urol. 2019;37 (9):1741–1749. Descriptive key message [8] Elsawy AA, Awadalla A, Maher S, et al. Diagnostic Performance of Novel Urine-Based mRNA Tests The current study aimed to assess the diagnostic perfor- (Xpert and Urinary Metabolomics Markers Assay) for mance of novel urine-based DNA methylation of six genes Bladder Cancer Detection in Patients with Hematuria. (GATA4, P16, P14, APC, CDH1 and CD99) for UBC detection in Bladder Cancer. 2020;6(3):319–28. patients with hematuria. 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DOI:10.3389/fcell.2021.817086 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Arab Journal of Urology Taylor & Francis

Novel urine-based DNA methylation biomarkers for urothelial bladder carcinoma detection in patients with hematuria

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© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
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10.1080/2090598X.2023.2208492
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Abstract

ARAB JOURNAL OF UROLOGY https://doi.org/10.1080/2090598X.2023.2208492 ORIGINAL ARTICLE Novel urine-based DNA methylation biomarkers for urothelial bladder carcinoma detection in patients with hematuria a b b b,c d Hassan F. Abol-Elnazer , Amira Awadalla , Asmaa E. Ahmed , Hassan Abol-Enein , Munir Ali Al Ganzouri and Amr A. Elsawy a b Genetics Unit, Mansoura University Hospital, Mansoura University, Mansoura, Egypt; Center of Excellent for Genome and Cancer Research (CEG-CR), Urology and Nephrology Center, Mansoura University, Mansoura, Egypt; Urology department, Urology and Nephrology Center, Mansoura University, Mansoura, Egypt; Faculty of Science, Ain Shams University, Cairo, Egypt ABSTRACT ARTICLE HISTORY Received 28 February 2023 Background: Urothelial bladder carcinoma (UBC) is usually detected during work-up for Accepted 25 April 2023 hematuria. Cystoscopy and/or contrast-enhanced imaging are the gold standard tools for UBC diagnosis, despite limited by being invasive, expensive and low yield in small flat tumors. KEYWORDS Objectives: To assess the diagnostic performance of urine-based DNA methylation of six genes Urothelial bladder (GATA4, P16, P14, APC, CDH1 and CD99) for UBC detection in patients with hematuria. carcinoma; hematuria; Patients and methods: Voided urine was collected from consecutive patients presented with biomarkers; DNA hematuria for urine cytology and DNA methylation assay of the assigned genes using methyla- methylation; cystoscopy tion-specific Polymerase Chain Reaction (PCR). Further assessment by office cystoscopy and imaging with subsequent inpatient cystoscopic biopsy for positive findings was done. The diagnostic characteristics of DNA methylation and urine cytology were assessed based on its capability to predict UBC. Results: We included 246 patients in the study with identified macroscopic hematuria in 204 (82.9%) patients. Positive cytology was found in 78 (31.7%) patients. DNA methylation of GATA4, P16, P14, APC, CDH1 and CD99 genes was identified in 127 (51.6%), 52 (21.1%), 117 (47.6%), 106 (43.1%), 90 (36.6%) and 71 (28.9%) patients, respectively. The sensitivity of the assigned genes for UBC detection ranges from 35% (95%CI: 31–39) to 83% (95%CI: 79–87). Optimal specificity (SP) (100%) was noted for P16, APC and CDH1 genes. While for the other genes (GATA4, P14 and CD99), the SP was 95% (95%CI: 92–98), 96% (95%CI: 92–99) and 97% (95%CI: 93–99), respectively. On multivariate logistic regression analysis, all genes exclusively demonstrated independent prediction of UBC. On receiver operator characteristic (ROC) ana- lysis, all tested genes methylation showed superior area under the curve (AUC) when compared to urine cytology. Conclusions: We have developed a novel urine-based DNA methylation assay for detection of UBC in patients with hematuria with superior diagnostic performance and independent pre- dictive capacity over urine cytology. Introduction Currently, cystoscopy/cross sectional imaging is the gold standard tools for UBC diagnosis in patients with According to GLOBOCAN data, bladder cancer (BC) is hematuria [5]. Unfortunately, these costly, invasive considered a major health problem that represents 3% diagnostics could miss early, small/flat bladder lesions of all cancer diagnoses [1]. Urothelial bladder carci- [6]. Urine cytology has been proposed as a non- noma (UBC) accounts for the vast majority (>90%) invasive alternative test with high specificity; however, of BC cases with predominance of non-muscle invasive it lacks sensitivity for diagnosis of low grade (LG) disease (Ta, Tis or T1) in 75% of patients, while others tumors [7]. show muscle invasion (T2–4) [1]. Over the last decades, multiple researches have out- In refereed population, UBC is usually diagnosed as put different markers for UBC diagnosis. Based on their a result of work-up for hematuria at a rate of 2–5% target of assessment, these markers include screening following an evaluation of asymptomatic microscopic for soluble antigens (BTA-Stat, NMP-22, surviving, etc.), hematuria [2], and, up to 5–15% of patients with cell surface antigens (Cytokeratins and UroVysion), macroscopic hematuria [3]. Therefore, a timely prompt genomic markers (Cxbladder and Xpert) and urinary evaluation of hematuria can give to earlier diagnosis metabolomics (CRAT and SLC25A20) [8]. However, most and better survival of UBC [4]. CONTACT Amr A. Elsawy amrelsawy.unc@hotmail.com Urology department, Urology and Nephrology Center, Mansoura University, Mansoura, Egypt © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. 2 H. F. ABOL-ELNAZER ET AL. of these markers are limited by unsatisfying diagnostic obtained, while hyperplastic and negative samples were defined negative for malignancy. performance, high cost or lack of validation [7]. Several preliminary studies have shown that DNA Urine methylation tests methylation, a critical step in transcription regulation, Voided urine samples were obtained in a sterile fashion is chemically stable and can be precisely quantified, (40 ml per each patient). Collected samples were fro- making it an attractive marker for UBC detection [9]. zen to −25 C within 60 min of collection. In a central Both local and global DNA methylations in BC speci- laboratory, genomic DNA was isolated from the col- mens are usually associated with inactivation of tumor lected samples and analyzed using methylation- suppressor genes. These methylation changes could specific Polymerase Chain Reaction (PCR). The opera- be effectively identified in urine sediments as well as tors at the central laboratory were blinded to the tumor tissues [10]. cytology/cystoscopy results. In the current literature, multiple studies investi- DNA isolation was done using the standard gated the performance of DNA methylation on either method (Geneture, Luoyang, China). After that, individual or panel genes with reported sensitivity (SN) quantification of the samples was done using and specificity (SP) values that range from 40% to 95% DNA Quantitation PicoGreen Kit (Thermofisher, and 10% to 100%, respectively [9]. Most of these stu- Massachusetts, US). As directed by the manufac- dies were limited by tumor characteristic heterogene- turer, 1.5 mg of DNA was utilized for bisulphite ity (majority were ≥T2 and high grade (HG) disease) stabilization using the available kit (OptuIN, Ca, which did not reflect the daily practice, inclusion of USA). In this reaction, unmethylated cytosine fil - different BC histological variants, lack of external vali- trates are deaminated resulting in its conversion dation and small sample size [11]. to uracil, while methylated residues are kept The aim of our study is to assess the diagnostic unmodified. The analyte quantifications of the performance of novel urine-based DNA methylation modified DNA were processed using real-time of six genes (GATA4, P16, P14, APC, CDH1 and CD99) assays which included parallel amplification and for UBC detection in patients with hematuria. quantification cycles using specific probe and pri- Moreover, we investigated the methylation pattern of mer (Supplementary table) for each analyte. these genes in different stages and grades of UBC. The methylation results were defined using special generation software (Applied Biosystems), which is Study design, settings and participants expressed as cycle threshold values. Cell lines with Study participants identified methylation condition for each tested gene were applied in each cycle as positive and negative After Institutional Review Board approval, patients pre- status and included in the process at the extraction sented with macroscopic or microscopic hematuria phase. were evaluated for eligibility to our study. Eligible Validity of urine sample was ensured when at least patients were asked to participate in this study and ten copies of ACTB gene (Supplementary table) were were enrolled after signing an informed consent form. identified in the isolated DNA from the urine samples. Only valid samples were assessed for methylation sta- Inclusion and exclusion criteria tus of the tested six genes. The results were expressed as gene copy numbers. Inclusion criteria included patients with hematuria Methylated urine sample for each gene was defined (macroscopic or microscopic). On the other hand, when at least ten copies of this gene were measured. patients who had history of BC, pelvic irradiation, Unmethylated samples were defined when <10 copies bleeding diathesis or receiving anticoagulants and of assigned gene were measured. These cut-off values patients with upper urinary tract neoplasm or uro- were calculated during the performance analysis of the lithiasis detected by cross-sectional imaging were assigned gene sets. excluded. Upper tract assessment and office cystoscopy Voided urine cytology, urine-based DNA procedures methylation evaluation Within 2 weeks of urine sample collections, study par- Urine cytology evaluation ticipants were thoroughly evaluated by cross-sectional Prior to outpatient check cystoscopy, a voided urine imaging and outpatient check cystoscopy. sample was collected and assessed according to the Cross-sectional imaging in the form of multiphasic Paris classification system [12]. Positive results were computed tomography was done to exclude upper defined when suspicious or malignant results are ARAB JOURNAL OF UROLOGY 3 urinary tract malignancies or urinary stones (unless Results contraindicated; magnetic resonance imaging was Base line demographics done). All office cystoscopy procedures were done From February 2019 and August 2021, 306 enrolled using flexible white light cystoscopy. Precise scan- patients were assessed for study eligibility. After exclu- ning of the bladder was done. According to the sion of patients with invalid methylation or cytology findings obtained, patients were categorized into results, non-UBC and variant histology UBC, 246 three groups; (1) cystoscopy-positive (gross blad- patients (201 males, 45 females) with a median der lesion/s), (2) cystoscopy-suspicious (hyperemic/ (range) age of 58 (34–99) years were included in the suspicious mucosa) and (3) cystoscopy-free (no final analysis. The study flow chart is shown in Figure gross lesions). For cystoscopy-positive and suspi- (1) while baseline demographics of the study partici- cious patients, transurethral resection bladder pants are illustrated in Table (1). biopsy was done within 2 weeks from the check cystoscopy. Cystoscopy-free patients were followed-up accord- Voided urine cytology and DNA methylation assay ingly (by either urgent clinic visit for patients with As demonstrated in Table (1), negative and positive recurring hematuria or telephone call every 12 weeks cytology were found in 168 (68.3%) and 78 (31.7%) for asymptomatic cases). On the other hand, patients patients, respectively. DNA hypermethylation of with recurring hematuria and persistent/aggravated GATA4, P16, P14, APC, CDH1 and CD99 genes was iden- bladder symptoms were assigned to re-check cysto- tified in 127 (51.6%), 52 (21.1%), 117 (47.6%), 106 scopy and cytology. Positive/suspicious re-cystoscopy (43.1%), 90 (36.6%) and 71 (28.9%) patients, respec- (and/or cytology) was evaluated by biopsy to confirm tively. On the other hand, cystoscopy/biopsy showed or exclude malignancy. negative and positive findings for UBC in 100 (40.7%) and 146 (59.3%) patients, respectively. Outcome measures Outcome measures The primary outcome is to assess the diagnostic per- formance of urine cytology and evaluated genes The diagnostic performance characteristics of urine methylation (GATA4, P16, P14, APC, CDH1 and CD99) cytology and evaluated genes DNA methylation are for UBC diagnosis in patients with hematuria. clarified in Table (2). The SN of the assigned genes for Subsequently, SN, SP, positive and negative predictive UBC detection ranges from 35% (95%CI: 31–39) to 83% values (PPV, NPV) were calculated. In addition, further (95%CI: 79–87) for P16 and GATA4, respectively. On the capability of these genes hypermethylation for predic- other hand, optimal SP (100%) was noted for P16, APC tion of different stages and grades of UBC was and CDH1 genes. While for the other genes (GATA4, P14 assessed. and CD99), the SP was 95% (95%CI: 92–98), 96% (95% CI: 92–99) and 97% (95%CI: 93–99), respectively. Notably, the evaluated genes methylation showed Statistical analysis superiority to voided urine cytology as regard to SN (apart from P16), SP (all six genes), PPV (all six genes) We computed all data using IBM statistical software and NPV (apart from p16 and CD99) for UBC detection v. 19. Chi-square and Fisher exact tests were utilized (McNemar test; p < 0.001) Table (2). to assess the association between categorical vari- Table (3) shows the univariate and multivariate ables, while for the comparison of urine cytology logistic regression analysis of the independent predic- and genes hypermethylation as regard to cysto- tors of UBC in the study participants. All the six genes scopy/biopsy, McNemar test was utilized. Logistic demonstrated exclusively significant findings for pre- regression analysis was utilized for multivariate ana- diction of UBC (GATA4: HR 4.2 (95%CI 2.78–8.24) p = lysis of significant predictors of UBC in study partici- 0.001, P16: HR 2.1 (95%CI 1.72–3.12) p = 0.003, P14: HR pants. A receiver operator characteristic (ROC) curve 1.4 (95%CI 1.1–1.8) p = 0.007, APC: HR 2.1 (95%CI 1.8– was constructed for this logistic regression by plot- 2.5) p = 0.006, CDH1: HR 2.32 (95%CI 1.92–2.73) p = ting the true positive finding (SN) against the false- 0.002, CD99: HR 2.12 (1.83–2.53) p = 0.003). On ROC positive findings (100-SP). The area under the curve analysis, DNA methylation of all the study genes (AUC) was estimated. Boxplots were utilized to showed superior AUC when compared to urine cytol- demonstrate the genes methylation copies and its ogy (Figure 2). predictive capacity for UBC stage and grade. On sub-analysis of UBC cases, ≥T2 stage was identi- A critical two-sided P-value <0.05 was used for sta- fied in 115 patients, while HG tumors were observed in tistically significant differences. 4 H. F. ABOL-ELNAZER ET AL. Enrolled patients Excluded (n): (9): Decline to participate (10): Previously diagnosed bladder carcinoma (8): Patients on anticoagulants or with bleeding diathesis (6): Radiological features suggestive of upper tract urothelial carcinoma Eligible patients Urine-based methylation Voided urine cytology Cystoscopy biomarkers Excluded (n): (5): Invalid methylation or cytology results (8): Non urothelial bladder carcinoma (14): Urothelial carcinoma with variant histology Bladder Biopsy from cystoscopy- positive patients Included patients in final analysis Cystoscopy/Biopsy Voided urine cytology Urine-based methylation Positive for urothelial bladder Positive for malignancy 78 biomarkers carcinoma 146 Methylated Unmethylated Negative for malignancy 168 Negative for urothelial bladder carcinoma 100 GATA4 127 119 P16 52 194 P14 117 129 APC 106 140 CDH1 90 156 CD99 71 175 Figure 1. Study flowchart. 139 patients. As illustrated in Figure 3, GATA4 and P14 patients with macroscopic hematuria [13]. The recom- genes methylation were significantly associated with mendations of most available guidelines for the diag- ≥T2 stage disease (p = 0.002 and <0.001, respectively) nostic work-up in those patients include invasive tests and HG disease (p = 0.007 and 0.004, respectively). as diagnostic cystoscopy and/or contrast-enhanced The cystoscopy-free patients were followed-up for imaging despite its limitations, namely high cost, sub- a median (range) period of 18 (7–36) months according stantial patient burden (pain) and health-care to the predetermined protocol. Out of 100 patients, resources, associated adverse events and waiting sche- who completed the follow-up, persistent hematuria or dules somewhere [14,15]. aggravated bladder problems were reported in 12 Reliable non-invasive markers might provide patients. Re-cystoscopy and cytology were repeated a helpful tool for clinicians to overcome the drawbacks in those patients, with evidence of UBC in one patient. of cystoscopies/imaging and prioritize patients for Notably, DNA methylation was identified in the all six invasive diagnostic interventions [16]. Urine cytology evaluated genes in this patient. was one of the early utilized to complement cysto- scopy for detection of exfoliated tumor cells in urine despite its limitations, low sensitivity in LG tumors and Discussion inter-observer variability [17]. Thereafter, multiple urinary markers with different Hematuria is a significant clinical presentation that can targets and mechanisms have been proposed and imply an underlying serious pathology, especially in ARAB JOURNAL OF UROLOGY 5 Table 1. Patients’ demographics. Median age in years (range) 58 (34-99) Sex n (%) • Male 201 (81.7) • Female 45 (18.3) Hematuria type n (%) •Macroscopic 204 (82.9) •Microscopic 42 (17.1) Diabetes mellitus n (%) •No 184 (74.8) •Yes 62 (25.2) Hypertension n (%) •No 174 (70.7) •Yes 72 (29.3) Concomitant dysuria/suprapubic pain/urgency n (%) •No 184 (74.8) •Yes 62 (25.2) History of urological problems n (%) •No 194 (78.9) •Yes 52 (21.1) ○ Urolithiasis 16 (6.5) ○ Bladder outlet obstruction 16 (6.5) ○ Urinary tract infections 20 (8.1) Concomitant indwelling bladder foreign body n (%) •No 238 (96.7) •Yes 8 (3.3) ○ Catheter 3 (1.3) ○ Bladder stone 5 (2) Smoking history n (%) •Never 90 (36.6) •Former 75 (30.5) •Current 81(32.9) Cytology result n (%) •Negative 168 (68.3) •Positive 78 (31.7) Cystoscopy findings n (%) •Free 100 (40.7) •bladder lesion 146 (59.3) Urine methylation assay target n (%) •GATA4 Methylated 127 (51.6) Unmethylated 119 (48.4) •P16 Methylated 52 (21.1) Unmethylated 194 (78.9) •P14 Methylated 117 (47.6) Unmethylated 129 (52.4) •APC Methylated 106 (43.1) Unmethylated 140 (56.9) •CDH1 Methylated 90 (36.6) Unmethylated 156 (63.4) •CD99 Methylated 71 (28.9) Unmethylated 175 (71.1) Biopsy findings in positive cystoscopy (Urothelial carcinoma) n (%) Stage (TNM) ○ Ta, Tis, T1 31 (12.6) ○ ≥ T2 115 (46.7) Grade (WHO/ISUP 2004) ○ Low grade 7 (2.8) ○ High grade 139 (56.5) assessed over the last years [16]. One of the recent and SP between 20% and 100% [9]. The explanation of common markers to be introduced in this aspect is this variation is essentially attributed to the heteroge- the study of some urine-based DNA methylation neity of the evaluated gene/s, included heterogeneous genes. Methylation of some genes can silence corre- patients with different histopathological variants, sponding tumor suppressor gene/s [18]. stage and grades, as well as different presentation The available literature on the clinical performance patterns (primary vs. recurrent BC) [19]. of DNA methylation in urine of UBC patients showed In our study, we primarily included patients with high variability with ranging SN between 50% and 95% hematuria (macroscopic or microscopic) with 6 H. F. ABOL-ELNAZER ET AL. Table 2. Cross tables of different urine assay methylation targets and urine cytology in comparison to cystoscopy/biopsy results in study participants. Urothelial bladder carcinoma by cystoscopy and biopsy n % Diagnostic characteristics Positive Negative Total % (95%CI) GATA4 127 SN 83.6% (95%CI: 79–87) • Methylated 122 (83.6%) 24 (16.4%) 119 SP 95% (95%CI: 92–98) Unmethylated 5 (5%) 95 (95%) PPV 96.1% (95%CI: 92–99) Total Count 146 100 246 NPV 79.8% (95%CI: 75–84) P16 52 SN 35.6% (95%CI: 31–39) • Methylated 52 (35.6%) 0 194 SP 100% Unmethylated 94 (64.4%) 100 (100%) PPV 100% Total Count 146 100 246 NPV 51.5% (95%CI: 46–55) P14 117 SN 77.4% (95%CI: 72–83) • Methylated 113 (77.4%) 4 (4%) 129 SP 96% (95%CI: 92–99) Unmethylated 96 (96%) 96 (96%) PPV 96.6% (95%CI: 93–99) Total Count 146 100 246 NPV 74.4% (95%CI: 71–78) APC 106 SN 72.6% (95%CI: 67–79) • Methylated 106 (72.6%) 0 140 SP 100% Unmethylated 40 (27.4%) 100 (100%) PPV 100% Total Count 146 100 246 NPV 71.4% (95%CI: 66–75) CDH1 90 SN 61.6% (95%CI: 57–66) • Methylated 90 (61.6%) 0 156 SP 100% Unmethylated 56 (38.4%) 100 (100%) PPV 100% Total Count 146 100 246 NPV 64.1% (95%CI: 60–68) CD99 71 SN 46.6% (95%CI: 42–50) • Methylated 68 (46.6%) 3 (3%) 175 SP 97% (95%CI: 93–99) Unmethylated 78 (53.4%) 97 (97%) PPV 95.8% (95%CI: 91–99) Total Count 146 100 246 NPV 55.4% (95%CI: 51–59) Urine cytology 57 (39%) 21 (21%) 78 SN 39% (95%CI: 36–42) Positive Count 89 (61%) 79 (79%) 168 SP 79% (95%CI: 76–84) Negative Count PPV 84.5% (95%CI: 81–88) Total Count 146 100 246 NPV 61% (95%CI: 57–65) Note: SN: Sensitivity, SP: specificity, PPV: Positive predictive value, NPV: Negative predictive value. Table 3. Univariate and multivariate analyses for predictors of positive cystoscopy/biopsy for urothelial bladder carcinoma (UBC). UBC by cystoscopy and biopsy No Yes Univariate analysis Multivariate analysis Patient and tumor characteristics N (%) N (%) HR (95%CI) p value HR (95%CI) p value Median age in years (Range) 56 (36–75) 60 (34–99) 1.01 (0.9–1.03) 0.47 Sex n (%) 1.2 (0.9–1.5) 0.56 Male 80 (39.8) 121 (60.2) Female 20 (44.4) 25 (55.6) Hematuria n (%) 1.4 (0.95–1.85) 0.75 Macroscopic 82 (40.2) 122 (59.8) Microscopic 18 (42.9) 24 (57.1) Concomitant dysuria/suprapubic pain/urgency n (%) 1.2 (0.89–1.5) 0.59 No 73 (39.7) 111 (60.3) Yes 27 (43.5) 35 (56.5) History of urological problems n (%) 1.12 (0.8–1.4) 0.5 No 81 (41.8) 113 (58.2) Yes 19 (36.5) 33(63.5) Indwelling bladder foreign body n (%) 1.15 (0.88–1.34) 0.1 No 99 (41.6) 139 (58.4) Yes 1 (12.5) 7 (87.5) Smoking n (%) 0.92 (0.87–1.23) 0.17 Never 43 (47.8) 47 (52.2) Former 25 (33.3) 50 (66.7) Current 32 (39.5) 49 (60.5) Cytology result n (%) 1.17 (0.74–0.1.64) 0.07 Negative 83 (61) 53 (39) Positive 17 (15.5) 93 (84.5) Urine methylation assay target n (%) 2.58 (1.94–2.94) <0.001 4.2 (2.78–8.24) 0.001 GATA4 Methylated 5 (3.9) 122 (96.1) Unmethylated 95 (79.8) 24 (20.2) P16 3.42 (2.9–3.8) <0.001 2.1 (1.72–3.12) 0.003 Methylated 0 52 (100) Unmethylated 100 (51.5) 94 (48.5) P14 2.72 (2.34–2.98) <0.001 1.4 (1.1–1.8) 0.007 Methylated 4 (3.4) 113 (96.6) Unmethylated 96 (74.4) 33 (25.6) APC 4.9 (4.3–5.42) <0.001 2.1 (1.8–2.5) 0.006 Methylated 0 106 (100) Unmethylated 100 (71.4) 40 (28.6) CDH1 3.35 (2.97–3.86) <0.001 2.32 (1.92–2.73) 0.002 Methylated 0 90 (100) Unmethylated 100 (64.1) 56 (35.9) CD99 2.67 (2.32–2.91) <0.001 2.12 (1.83–2.53) 0.003 Methylated 3 (4.2) 68 (95.8) Unmethylated 97(55.4) 78 (44.6) ARAB JOURNAL OF UROLOGY 7 Figure 2. Receiver operator characteristic (ROC) curve for urine-based methylation biomarkers and urine cytology for urothelial bladder carcinoma detection in the study cohort. exclusion of patients with BC history. Most of the however, the nest reports which included gene previous studies included heterogeneous patients panels rather than a single gene [9]. Our higher SP (primary/recurrent/surveillance cases vs. healthy/ results could be attributed to the study design symptomatic controls) [9]. Moreover, as compared which included hematuria patients rather than to the previously published reports on DNA methyla- healthy controls and inclusion of UBC with exclu- tion markers for primary BC patients [20–22], our sion of other variant histologies [27]. study included the largest cohort (146 BC and 100 When compared to urine cytology, superior results symptomatic controls). of our study genes were obtained as regard to inde- Among the evaluated six genes, our study pendent diagnostic capacity in multivariate logistic assessed DNA methylation of two novel genes regression analysis, as well as, AUC on ROC curve (GATA4 and CD99) that were not previously investi- analysis. In addition, GATA4 and P14 genes methylation were gated in the literature for this purpose. On the significantly associated with ≥T2 stage disease (p = contrary, there is a paucity of the literature as regard to three genes (CDH1, P14 and P16) with 0.002 and <0.001, respectively) and HG disease (p = one report for each gene [23–25], these reports 0.007 and 0.004, respectively). This finding should add were limited by its small sample size (22–57 another diagnostic benefit of DNA methylation not patients) and heterogeneous presentation. only for UBC diagnosis but also for tumor characteriza- Our findings showed varying SN of evaluated tion (stage/grade). markers ranging from 35% (P16) to 83.5% (GATA4). Our study is advantaged by its prospective nat- As compared to SN of the previously investigated ure, inclusion of consecutive hematuria patients genes (APC, CDH1, P14 and P16), our findings were with no BC history (better reflection of target notably lower due to the nature of our study parti- population in daily practice than healthy controls), cipants with exclusion of patients under BC surveil- use of predefined threshold level of gene methyla- tion, exclusion of non-UBC or UBC with abnormal lance and lesser patients with ≥T2/HG disease as variants and study of diagnostic capacity of evalu- these tumors probably harbor more molecular ated genes for tumor stage and grade. On the alterations [26]. other hand, there are some limitations in our On the other hand, the SP of the evaluated study. First, our results need external validation in genes varies between 95% and 100%. The initial a larger cohort multicenter study. Second, our ana- reports in the literature about single-gene DNA lysis included only hypermethylation study with methylation showed compromised SP (0–60%), 8 H. F. ABOL-ELNAZER ET AL. Figure 3. Boxplots of urine-based DNA methylation biomarkers GATA4, P16, P14, APC, CDH1 and CD99 for detection of different urothelial bladder carcinoma stages (3A) and grade (3B). lack of other analyses that can optimize the diag- findings. Further longitudinal follow-up and external nostic capacity as mutation analysis [28]. Third, validation studies of our results are warranted before lack of further study of evaluated genes DNA future implementation in clinical practice. methylation on future tumor recurrence and/or progression is a considerable limitation. Disclosure statement No potential conflict of interest was reported by the authors. Conclusions We have developed a novel urine-based DNA methyla- tion assay for non-invasive detection of UBC in patients Funding with hematuria. Superior diagnostic performance and independent predictive capacity of the evaluated This work was funded by Science, Technology & Innovative Funding Authority (STDF); Project No.: 5236. genes methylation over urine cytology are promising ARAB JOURNAL OF UROLOGY 9 Clinical trials registration caring for this disease. Eur Urol. 2014;66(2):253–262. DOI:10.1016/j.eururo.2014.01.006 clinicaltrials.gov ID: NCT04714345 [7] Maas M, Bedke J, Stenzl A, et al. Can urinary biomarkers replace cystoscopy? World J Urol. 2019;37 (9):1741–1749. Descriptive key message [8] Elsawy AA, Awadalla A, Maher S, et al. Diagnostic Performance of Novel Urine-Based mRNA Tests The current study aimed to assess the diagnostic perfor- (Xpert and Urinary Metabolomics Markers Assay) for mance of novel urine-based DNA methylation of six genes Bladder Cancer Detection in Patients with Hematuria. (GATA4, P16, P14, APC, CDH1 and CD99) for UBC detection in Bladder Cancer. 2020;6(3):319–28. patients with hematuria. 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Journal

Arab Journal of UrologyTaylor & Francis

Published: Oct 2, 2023

Keywords: Urothelial bladder carcinoma; hematuria; biomarkers; DNA methylation; cystoscopy

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